ARF Family GTPases

PROTEINS AND CELL REGULATION

Volume 1

Series Editors: Professor Anne Ridley

Ludwig Institute for Cancer Research
and Department of Biochemistry and Molecular Biology
University College London
London
United Kingdom

Dr. Jon Frampton

Institute for Biomedical Research,
Birmingham University Medical School,
Division of Immunity and Infection
Birmingham
United Kingdom

Aims and Scope

Our knowledge of the ways in which a cell communicates with its environment and how it responds to
information received has reached a level of almost bewildering complexity. The large diagrams of cells to
be found on the walls of many a biologist’s office are usually adorned with parallel and interconnecting
pathways linking the multitude of components and suggest a clear logic and understanding of the role
played by each protein. Of course this two-dimensional, albeit often colourful representation takes no
account of the three-dimensional structure of a cell, the nature of the external and internal milieu, the
dynamics of changes in protein levels and interactions, or the variations between cells in different tissues.

Each book in this series, entitled “Proteins and Cell Regulation”, will seek to explore specific protein
families or categories of proteins from the viewpoint of the general and specific functions they provide and
their involvement in the dynamic behaviour of a cell. Content will range from basic protein structure and
function to consideration of cell type-specific features and the consequences of disease-associated changes
and potential therapeutic intervention. So that the books represent the most up-to-date understanding,
contributors will be prominent researchers in each particular area. Although aimed at graduate, postgraduate
and principle investigators, the books will also be of use to science and medical undergraduates and to those
wishing to understand basic cellular processes and develop novel therapeutic interventions for specific
diseases.
 ARF Family GTPases

Edited by
RICHARD A. KAHN
Emory University School of Medicine,
Atlanta, Georgia, U.S.A.

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 Dedicated to the memory of Annette L. Boman, Ph.D.

Mother, scholar, wife, mentor, athlete, cook, and much more

She loved life and shared that joy with everyone

We were all made better by having known her

 TABLE OF CONTENTS

Preface: The Arf Family vii

Richard A. Kahn

List of Contributors xxv

1. The Arf Family Tree 1

John M. Logsdon Jr., Richard A. Kahn

2. The GDP/GTP Cycle of Arf Proteins 23

Sebastiano Pasqualato, Louis Renault, Jaqueline Cherfils

3. Arf and Phospholipids 49

Paula A. Randazzo, Zhongzhen Nie, Dianne S. Hirsch

4. The Sec7 Family of Arf Guanine Nucleotide Exchange 71

Factors
Catherine L. Jackson

5. Large Arf GEFs of the Golgi Complex 101

Paul Melançon, Xinhua Zhao, Troy K.R. Lasell

6. BIG1 and BIG2: Brefeldin A-Inhibited Exchange Factors 121

for Arfs
G. Pacheco-Rodriguez, J. Moss, M. Vaughan

7. Arf GTPase-Activating Protein 1 137

Dan Cassel

8. GIT Proteins: Arf Gaps and Signaling Scaffolds 159

Robert Schmalzigaug, Richard Premont

9. Paxillin-Associated Arf Gaps 185

Hisataka Sabe

10. Activation of Cholera Toxin and E. Coli Heat-Labile 209

Enterotoxin (LT) by Arf
G. Pacheco-Rodriguez, Naoko Morinaga, Masatoshi Noda,
J. Moss, M. Vaughan
vi

11. Phospholipase D, Arfaptins and Arfophilin 223

John H. Exton

12. Coat Proteins 241

Annette Boman, Tommy Nilsson

13. Heterotetrameric Coat Protein-Arf Interactions 259

M.L. Styers, V. Faundez

14. Arf6 283

James E. Casanova

15. A Role for Arf6 in G Protein-Coupled Receptor 305

Desensitization
Mary Hunzicker-Dunn

16. Arf-Like Proteins 325

Annette Schürmann, Hans-Georg Joost
Preface
THE ARF FAMILY

RICHARD A. KAHN
Department of Biochemistry, Emory University School of Medicine, Atlanta, USA

1. INTRODUCTION TO GTPASES

What questions drive biomedical research today? Much publicity has

surrounded the publication of the human genome in recent years. Certainly
this marks a milestone and a database from which a tremendous amount of
new insights will be learned. But the genome is linear, i.e., a one-
dimensional feature of all cells. I believe that what drives the majority of
research today is the goal of understanding the complete wiring diagram of
the eukaryotic cell. This will require much more complicated databases, in
large part because the solution is four-dimensional. We need to learn not
only all the pathways of metabolism and signaling, but how they are
integrated. How are nucleocytoplasmic transport, protein and lipid
metabolism, the cell cycle, organellogenesis, and the cytoskeleton all
regulated; individually and collectively? GTPases are at least one of the
keys to unraveling the layers of cell regulation required.
I remember my first biochemistry course and the expectation that the cell
is a bag of enzymes that we can decipher simply by knowing the
concentrations of reactants, their affinities for enzymes, and the rates of the
reactions. Perhaps for understanding intermediary metabolism this was
useful, though today even that is doubtful. Instead, as each metabolic or
signaling pathway is described we gain a better appreciation for the need to
understand the connections between pathways. All cells are changing,
modifying and responding to their environment and their neighboring cells,
be they friend or foe. A cell’s ability to mount an appropriate response to
whatever it encounters depends in large part on its ability to coordinate many
different systems or pathways and synchronize them in time. Thus, cell
vii
viii KAHN

regulation and the integration of signaling pathways can be viewed as a

second order system that increases a cell’s fitness. It is at these regulatory
points of intersection between critical cellular processes that we usually
encounter the regulatory GTPases.
They have been described as molecular switches. This is unfortunate
because they are so much more sophisticated and interesting than that. With
multiple inputs and outputs, they are never simply on or off. Instead, they sit
at junction points between essential cellular processes and direct the flow of
information that is required for all the parts to work together. Just like
computer software that became much more powerful when programmers
learned to introduce conditional points in a program, cells became much
more “fit” when they acquired the ability to use conditional regulators and
effectively integrate multiple essential processes.
This type of regulation is distinct from that used on a single enzyme or
pathway. The rate at which phosphofructokinase converts fructose 6-
phosphate to fructose 1,6-bisphosphate is subject to regulation by multiple
modulators (e.g., citrate, pH, ATP, ADP, fructose 2,6-bisphosphate) because
this is the commitment point in glycolysis and the substrate may be required
for other uses. But in this case, the product is known and only the rate is
varied. In contrast, several GTPases have more than a dozen different
effectors and may have a similar number of activators. Thus, not only is
their level of activity (percentage bound to GTP vs. GDP) varied but so also
is the response itself. It could be an alteration in lipid metabolism,
cytoskeletal organization, or macromolecular complex assembly. Thus, to
fully understand the functions of even one family of regulatory GTPases we
will likely need to understand the means of integration used by cells to
coordinate multiple aspects of cell biology.

2. A BRIEF INTRODUCTION TO ARFS

Although Ras was known to bind guanine nucleotides, it was the discovery
that Ras is a GTPase that heralded the start of what has grown into an
incredibly diverse and important area of cell regulation research (Gibbs et
al., 1984; Sweet et al., 1984). One is hard pressed to name an aspect of cell
biology that is not directly or indirectly affected by one or more regulatory
GTPase today. The Ras superfamily owes much to the heterotrimeric G
proteins. Many of the original models for GTPases as signal transducers and
techniques used in the field (e.g., nucleotide-binding, filter trapping assay,
GEF assays, and the use of slowly hydrolyzing GTP analogs) came from
work on G proteins. The three-component model (activator, G protein, and
effector) for cell signaling arose from studies of hormone- and light-
 THE ARF FAMILY OF REGULATORY GTPASES ix

activated second messenger systems, adenylyl cyclase and

phosphodiesterase, respectively. This debt to G protein research is nowhere
more evident than for the Arfs.
Before any G protein had been purified, the guanine nucleotide-binding
α subunit of Gs, the direct activator of adenylyl cyclase, had been identified
as a substrate for the ADP-ribosyltransferase activity of cholera toxin
(Cassel and Pfeuffer, 1978) (see Chapter 10). Membranes treated with
cholera toxin and [α-32P]NAD covalently incorporated radionucleotide
(ADP-ribose) into a band at 45 kDa (and sometimes 52 kDa) that was later
proven to be the α subunit. During the purification of Gs from mammalian
sources (Northup et al., 1980) it was discovered that it lost the ability to
serve as substrate for cholera toxin. It was soon realized that an essential
cofactor in the reaction (the ADP-ribosylation factor (Arf)) had been lost
during purification. Thus, the search was begun to identify Arf on the
grounds that it was likely to hold a key to other regulatory features of Gs and
adenylyl cyclase. A few years later the first Arf preparation was pure (Kahn
and Gilman, 1984, 1986), but 20 years later we still lack any evidence for a
role for Arf in the endogenous regulation of Gs or adenylyl cyclase. With
the discovery around that time that the human oncogene product, p21 Ras,
was a GTPase there was even a short period where we believed they could
be one and the same. They are not. And without the glamour of oncogenic
potential, Arf remained something of a curiosity without a clearly defined
cellular function for several years.
Arfs have been implicated in a large number of cellular processes,
including vesicular membrane traffic, Golgi morphology, lipid metabolism,
actin cytoskeleton, endocytosis, cytokinesis, and exocytosis. The situation
today, like other GTPase families, is that Arf has perhaps too many functions
and effectors (>12 so far). While working to describe actions of Arf on
specific signaling systems, we also hope to understand the broader question
of why nature has evolved to use these proteins in so many ways. It is my
premise that it is this diversity in functions that is the real key to Arfs, and
probably other families of GTPases. Because in sharing a common
regulatory factor these different cellular systems are provided with a way to
communicate with one another and systems integration is achieved (see
below).
Though Arf was identified, purified, and cloned in the 1980s (Kahn and
Gilman, 1984, 1986; Kahn et al., 1988; Sewell and Kahn, 1988) that work
would not have been possible without the groundwork performed earlier by
Dany Cassel and Thomas Pfeuffer (Cassel and Pfeuffer, 1978), whose work
established the mechanism of activation of adenylyl cyclase by cholera
toxin; by Joel Moss, Martha Vaughan, and colleagues (Moss et al., 1976;
Moss and Vaughan, 1977) who worked out the enzymology of cholera toxin
x KAHN

and the role of NAD as substrate and ADP-ribosyl donor; and of Mike Gill
and co-workers (Enomoto and Gill, 1979; Gill, 1975, 1976, 1977; Gill and
Meren, 1978; Gill and Rappaport, 1979) whose earlier work on ADP-
ribosylation by diphtheria toxin and movement into the cholera toxin field
contributed so much, along with his clever means of identifying the soluble
factor, later termed Arf. Mike died suddenly and far too prematurely. His
enthusiasm and inventiveness are still missed.
With the discovery of the conservation of Arf in eukaryotes (Kahn et al.,
1991; Sewell and Kahn, 1988) and its linkage both spatially and genetically
to the Golgi (Stearns et al., 1990), Arf functionality began to have a home.
The pace for modeling Arf functions quickened enormously with the
observation that Arfs, or more precisely the Arf activating proteins, were
targets of the drug brefeldin A, a fungal metabolite with profound effects on
Golgi morphology and membrane traffic (Donaldson et al., 1992a;
Donaldson et al., 1992b; Helms and Rothman, 1992; Lippincott-Schwartz et
al., 1989; Randazzo et al., 1993). A role for Arf in the initiation and
composition of vesicle coating became the first molecular mechanism for an
Arf action in mammalian cells (Orci et al., 1993; Rothman and Orci, 1992;
Serafini et al., 1991). However, with the identification of Arf as the
cytosolic factor that conferred sensitivity to in vitro assays of phospholipase
D (Brown et al., 1993; Cockcroft et al., 1994) and later phosphatidylinositol
4-phosphate 5-kinase (PI(4)P 5-kinase) (Honda et al., 1999), roles in lipid
metabolism became evident. Attempts to integrate the vesicle coating
actions of Arfs and those on lipid metabolisms are ongoing.
Though purified Arf preparations were heterogeneous (multiple bands in
SDS gels) it was molecular cloning techniques that defined the true
complexity of the Arf family, a process that is still continuing today. Work
from Joel Moss and co-workers (Bobak et al., 1989; Lee et al., 1992;
Monaco et al., 1990; Price et al., 1988; Tsuchiya et al., 1989) as well as
members of my own lab (Clark et al., 1993; Kahn et al., 1991; Sewell and
Kahn, 1988; Tamkun et al., 1991) and that of Hans Joost (Breiner et al.,
1996; Schurmann et al., 1994; Schurmann et al., 1995) described six
mammalian Arfs and most of the Arf-like (Arl) proteins. The high degree of
structural and functional conservation between Arf and Arl orthologs is
remarkable and has even led to the proposal that the Arf family may be the
predecessor of the Ras and perhaps also the heterotrimeric G protein families
(Lee et al., 1992; Murtagh et al., 1992). These and other aspects of the
evolution of the Arf family are considered in the chapter from John Logsdon
(see Chapter 1).
The overwhelming majority of studies to date have focused on roles and
activities of Arf1 and or Arf3. The most diverse Arf in sequence and
function is Arf6, which acts primarily at the plasma membrane and impacts
 THE ARF FAMILY OF REGULATORY GTPASES xi

both GPCR signaling (the long sought connection between Arf and G
proteins?) and the cytoskeleton. And we still lack functions for Arf4 and
Arf5. While the six Arfs are difficult to distinguish biochemically, recent
results reveal that each can be shown to possess distinctive functions in live
cells (K. Hill and R. A. Kahn, unpublished observations). Jim Casanova
(see Chapter 14) has undertaken the Herculean task of making the Arf6
literature comprehensible and he has succeeded beyond my wildest hopes.
Together with the exciting work and novel model presented by Mary
Hunzicker-Dunn (see Chapter 15), we all have a much better appreciation
for the roles of Arf6 in the plasma membrane.
The Arf family is divided into the Arfs, sharing very high percent
identities (>60%) and defining activities, and Arf-likes (Arls) of lesser but
still quite high (40-60%) identity and devoid of those activities1. It was Hans
Joost and Annette Schurmann (Breiner et al., 1996; Schurmann et al., 1994;
Schurmann et al., 1995) who continued to increase the number of Arf-like
proteins in the database through screens and their studies of differentiation
of 3T3-L1 cells and adipocytes. They describe their latest work, including
knockouts in mice, and provide the first substantive evidence for Arl
functions in development (see Chapter 16). The Arls are proving to be much
more diverse in function, as expected from their lower percent identities.
With further definition of Arl functions and their partners we will certainly
have a richer understanding of the Arf family as a whole.

3. THE GTPASE CYCLE

The reason these GTPases are often referred to as molecular switches is that
they exist in the cell in two forms, one bound to GDP and the other to GTP.
These are traditionally referred to as “off” and “on”, or “inactive” and
“active”, respectively, though this is clearly an over-simplification. It is true
that in the active conformation a GTPase may bind and allosterically activate
an enzymatic activity; as is the case with Arfs and phospholipase D (see
Chapter 11), PI(4)P 5-kinase, or the ADP-ribosyltransferase activity of
cholera toxin (see Chapter 10). However, many of the binding partners of
GTPases are not enzymes. In this case, the binding of the GTPase may serve
1
Because the original Arf assay (Kahn, 1991; Kahn and Gilman, 1984; Weiss et al., 1989)
is technically demanding and requires a source of purified Gs, other assays have been used
to characterize Arfs. These include the simple substitution of agmatine for Gs as ADP-
ribose acceptor (Tsai et al., 1987) to the completely independent assays of Arf dependent
PLD (Brown et al., 1993; Kuai and Kahn, 2002) or PI(4)P 5-kinase assays (Honda et al.,
1999). Perhaps the most stringent assay for Arf function, because it is a live cell assay and
tests for the essential activities of Arf in eukaryotes, is that of rescue of the double deletion
arf1- arf2- in the yeast S. cerevisiae (Kahn et al., 1991; Lee et al., 1992).
xii KAHN

to alter the cellular location of the effector, promote further protein

interactions and the assembly of a multisubunit complex, or perhaps prevent
the binding of another protein and thereby function indirectly. It is therefore
more accurate to simply state that the two forms of a GTPase interact with
distinct sets of binding partners. The best example of this paradigm is Ran,
which coordinates transport in and out of the nucleus depending on whether
it is liganded to GDP or GTP. Yet each form is active and must bind
specific partners, the functionally important difference is only that of
direction.
The two interconvertible states of any regulatory GTPase result from
nucleotide-sensitive conformational changes in two regions of the protein
referred to as switches I and II. Arf family members have two additional
regions that are important to their functions, referred to as the N-terminal
helix and the interswitch region. The story of how these are integrated in
signaling is beautifully told by Jacqueline Cherfils (see Chapter 2). The N-
terminal helix is central to the ability of Arfs to translocate from cytosol to
membranes in a fashion that coordinates activation with membrane binding
(Antonny et al., 1997; Franco et al., 1996; Paris et al., 1997). The presence
of a permanent, covalently attached, saturated, 14-carbon fatty acid
(myristate) to the N-terminal glycine of all Arfs (Kahn et al., 1988) (but
apparently not all Arls (Sharer et al., 2001)) is essential to both membrane
association and biological effects. The ability of Arfs to both directly bind
phospholipid membranes and modify their composition is expected to play
key roles in Arf biology, as summarized by Paul Randazzo (see Chapter 3).
The interconversion between the two states of a GTPase is promoted by
two types of activities: guanine nucleotide exchange factors (GEFs) increase
the level of activated (GTP-bound) GTPase and GTPase activating proteins
(GAPs) speed the intrinsic hydrolysis of the bound GTP. It is generally
assumed, but almost never documented, that the release of GDP is the rate-
limiting step in the GTPase cycle. The term GEF has tended to supersede
two more precise terms; guanine nucleotide dissociation inhibitor (GDI) or
stimulator (GDS). This is unfortunate because with so many different GEFs
for each GTPase there is ample opportunity for distinctive mechanisms of
activation that may prove to have important functional repercussions.
Unlike the heterotrimeric G proteins, which are activated by the G protein
coupled receptors (GPCRs) upon their binding ligand, we do not yet know
what the stimulus is for Arf GEFs to act or indeed if one exists. With many
of the processes effected by Arfs being constitutive in nature it is possible
that Arf GEFs function all the time and their activities are controlled not by
binding specific signaling ligands but perhaps by secondary factors such as
the presence of specific lipids or location in the cell. The diversity and
 THE ARF FAMILY OF REGULATORY GTPASES xiii

complexity in Arf activation is revealed clearly by the three chapters on the

Arf GEFs (see Chapters 4-6).
The importance of diversity in mechanism is even more evident for the
Arf GAPs. The elegant structure-based studies of Jonathan Goldberg
(Goldberg, 1998, 1999, 2000) have led him to propose a model in which an
Arf GAP and an effector (COPI) may bind the two switches of Arf•GTP
simultaneously. This model fits beautifully with a proofreading mechanism
for Arfs and Arf GAPs in cargo selection during vesicle biogenesis but is
unlikely to hold true for all Arf GAPs (Jacques et al., 2002; Nie et al., 2002).
The presence of the Arf GAP motif in >20 (predicted) human proteins
certainly holds the promise of great diversity in functions as well as
mechanisms. While many of these have yet to be shown to possess Arf GAP
activity they beg the question: does the presence of an Arf GAP in a
complex or pathway provide evidence for a role of an Arf? The first Arf
GAP discovered, by Dany Cassel (Cukierman et al., 1995; Makler et al.,
1995), now appears streamlined and specialized (see Chapter 7). In contrast,
many of the Arf GAPs contain multiple protein-interaction domains and the
promise of more complicated and diverse actions. Roles for Arf GAPs (and
Arfs?) in G-protein coupled receptor (see Chapter 8) and paxillin (see
Chapter 9) actions seem more than justified from the original work of
Richard Premont, Hisataka Sabe (respectively) and their colleagues.

4. NOMENCLATURE

After a section on GEFs and GAPs seems a very appropriate place to inject a
note on nomenclature. Like any area of signaling today, we are dealing in
each case with gene families and key discoveries coming from a number of
different labs, using model genetic systems, each using different systems of
naming genes and proteins. While it is important to honor priority in
discovery, names can quickly become outdated, confusing, or factually
incorrect. Change is happening so fast that it can even be the curators of
public databases, in well-intentioned efforts at consistency, which exacerbate
or perpetuate nomenclature problems. The discovery of the Arf GAP
domain (and to only a slightly lesser extent this is true of the Arf GEF/Sec7
domain as well) occurred at a time when genome sequence data was pouring
into databases around the world. Workers in several countries started
finding more proteins than we had functions. The presence of additional
protein interaction domains can help researchers identify biological roles for
a protein, but can also foster confusion in naming it. This has been just as
true in the Arf GAP and Arf GEF field as in any.
 xiv
Table I: Current and proposed names for human Arf GAPs and their orthologs

New
Sub-family Name #1 Name #2 Name #3 Name #4 KIAA# names Accession number (length) Domain organization

Arf GAP1 Arf GAP1 Arf1GAP Arf GAP Arf GAP1 NM_018209;NP_060679 (406aa) N-term. Arf GAP
Arf GAP3 ArfGAP1 Arf GAP3 NM_014570 (516aa) N-term. Arf GAP
Zfp289 Arf GAP4 NM_032389; NP_115765 (521 aa) N-term. Arf GAP

APAP Git1 Cat1 p95APP1 APAP1 NM_014030; NP_054749 (761 aa) N-term. Arf GAP, ankyrin repeats (3)
Git2 Cat2 p95APP2 p95PKL KIAA0148 APAP2 NP_476510 (759aa) N-term. Arf GAP, ankyrin repeats (3)

AMAP ASAP1 DEF1 DDEF1/SHAG1 Cent. Beta4 KIAA1249 AMAP1 XP_005169(483aa); BAA86563(949aa) PH, Arf GAP, ankyrin repeats (3), SH3
PAP(alpha) DDEF2 SHAG2/PAG3 Cent. Beta3 KIAA0400 AMAP2 NM_003887(1006aa) PH, Arf GAP, ankyrin repeats (3), SH3
UPLC1 Cent. Beta6 AMAP3 XP_031298 (EST only) PH, Arf GAP, ankyrin repeats (3)

ACAP ACAP1 Cent. Beta1 KIAA0050 ACAP1 NM_014716 (740aa) PH, Arf GAP, ankyrin repeats (2)
ACAP2 Cent. Beta2 KIAA0041 ACAP2 NM_012287 (778aa) PH, Arf GAP, ankyrin repeats (2)
ACAP3 Cent. Beta5 KIAA1716 ACAP3 NM_030649 (759aa) PH, Arf GAP, ankyrin repeats (2)
KAHN

AGAP AGAP1 Cent. Gamma1 PIKE KIAA0167 AGAP1 NM_014770/NM_023017(836aa) GTPase, PH, Arf GAP, ankyrin repeats (2)
AGAP2 Cent. Gamma2 KIAA1099 AGAP2 NM_014914 (804aa) GTPase, PH, Arf GAP, ankyrin repeats (2)
AGAP3 Cent. Gamma3 MRIP1 AGAP3 NM_031946; NP_114152 (875aa) GTPase, PH, Arf GAP, ankyrin repeats (2)

ARAP ARAP1 Cent. Delta2 ARAP1 NM_139181 (1204aa); NM_015242 (1210aa) PH, Arf GAP, PH (2), RhoGAP, PH
ARAP2 Cent. Delta1 FLJ13675 KIAA0580 ARAP2 NM_015230 (1704aa) PH, Arf GAP, PH (2), RhoGAP, PH
ARAP3 ARAP3 NM_022481; NP_071926 (1544aa)

AFAP Centaurin (alpha1) PIPBP p42IP4 AFAP1 NM_006869/NP_006860 (374aa) Arf GAP, ankyrin repeats (2), PH (2)
Centaurin (beta) Cent. Alpha2 AFAP2 NM_018404; NP_060874 (381aa) Arf GAP, ankyrin repeats (2), PH (2)

Arf GAP5 RIP/RAB/HRB Arf GAP5 NM_004504; NP_004495 (562aa) N-term Arf GAP, FG repeats
RAB-R/HRB1 Arf GAP6 NM_006076; NP_006067 (481aa) N-term Arf GAP, FG repeats

Arf GAP7 SMAP1a Arf GAP7 NM_021940; NP_068759 (440aa) N-term Arf GAP
SMAP1b Note: 48% identical to SMAP1a Arf GAP8 XM_029830; XP_029830 (429aa) N-term Arf GAP
List of acronyms
ACAP = Arf GAP, coiled-coil, ankyrin repeat, PH protein
AGAP = Arf GAP, GTPase, ankyrin repeat, PH protein
APP = Arf GAP putative, PIX1-interacting, paxillin bind
ARAP = Arf GAP, Rho GAP, ankyrin repeat, PH protein
ASAP = Arf GAP, SH3, ankyrin repeat, PH protein
CAT = Cool-associated tyrosine phosphorylated protein
DEF = Differentiation enhancing factor
GIT = G protein receptor kinase (GRK) i nteracting GT Pase activating protein
HRB = HIV Rev binding protein
PAG = paxillin associated Arf GAP
PAP = Pyk2 associated protein
PKL = paxillin-kinase linker
RAB = HIV Rev-associated binding protein
RIP = HIV Rev-interacting protein
SMAP = stromal membrane associated protein
List of new acronyms
APAP = Arf GAP with pix and paxillin binding
AMAP = A multi-domain Arf GAP protein
ACAP = Arf GAP, coiled-coil, ankyrin repeat, PH protein
AGAP = Arf GAP, GTPase, ankyrin repeat, PH protein
ARAP = Arf GAP, Rho GAP, ankyrin repeat, PH protein
AFAP = Arf GAP with phosphoinositide binding and PH domain
NOTES:
(1) Some human protein sequences are incomplete (based on the presence of orthologs in other mammals)
(2) Several messages are alternatively spliced; only the longest protein sequence is indicated
(3) Arf GAP 3 = ARG1 (Arf GAP1) locus in human
(4) APAP1 and APAP2 named GIT1 and GIT2 loci in mouse and human databases
THE ARF FAMILY OF REGULATORY GTPASES
xv
 xvi

Table 2: Verified and predicted human and other Arf GEFs

(PS50190) (PF01369) Protein Alternate

Species Name 1 Name 2 Name 3 Length SEC7 dom SEC7 dom Accession # Accession#
Human BIG1 Arf GEF1 p200 Arf GEP1 1843 709-840 695-882 XP_037726 XM_037726
Human BIG2 Arf GEF2 1785 654-785 640-827 NP_006411 NM_006420
Human BIG3 KIAA1244 1770 179-391 XP_050424 AAL04174
Human Cytohesin 1 398 73-202 59-244 NP_004753 NM_004762
Human ARNO Cytohesin 2 399 72-201 58-243 NP_059431 NM_017457
Human ARNO3 Cytohesin 3 GRP1 399 77-206 63-248 NP_004218 O43739
Human Cytohesin 4 394 72-201 58-243 NP_037517 NM_013385
Human GBF1 KIAA0248 1859 542-671/710-839 696-884 Q92538 NM_004194
Human EFA6B TIC 1056 578-696 549-738 O95621 NP_036587
Human EFA6A TYL PSD 645 173-287 142-329 Q15673 NP_002770
Human EFA6D HCA67 KIAA0942 534(F) 74-179 36-222 NP_056125 Q9NYI0
Human BRAG3 KIAA1110 740/701 /181-314 /167-358 BAA83062 Q9UPP2
Human Arf GEP100 BRAG2 KIA0763 841 413-546 399-590 O94863 NP_055684
Human BRAG1 KIAA0522 1560(F) O60275 T00080
KAHN

Human EFA6C DKFZ 771 317-422 277-464 CAB66494 NP_115665

Human FBS FBX8 319 146-276 133-313 NP_036312 Q9NRD0

D. melanogaster BIG1 DS00797.7 CG7578 1643/1653 /593-724 /579-767 AAF53331 Q9NKE8

D. melanogaster BRAG CG10577 1210/1082 /523-709 /520-711 AAF51675 Q95U36
D. melanogaster ARNO CG11628 348 26-155 12-197 AAF57230 Q9V9Q6
D. melanogaster CG11628 410 71-258 75-260 NP_610120 Q8SYT7
D. melanogaster CG8487 GBF1 2040 654-785 640-830 Q9V696
D. melanogaster EFA6 CG6941 900 421-533 399-575 AAF56027 Q9VCX8

C. elegans ARNO K06H7.4 CE26942 377/393 53-239 57-241 NP_498764 P34512

C. elegans BRAG M02B7.5 CE12328 614 209-343 195-391 NP_500420 Q94287
C. elegans EFA6 Y55D9A.1B CE19234 816 356-532 357-534 NP_502416 Q95Q14
C. elegans BIG1 Y6B3A.1 CAA21704 1628 524-655 510-697 NP_493386 Q9XWG5
C. elegans GBF1 C24H11.7 CE19701 1820 672-799 658-844 AL032627 Q9XTF0
 S. cerev. SEC7 YDR170C 2009 838-968 824-1010 P11075 AAB04031
S. cerev. Gea1 YJR031C 1408 552-706 538-748 P47102
S. cerev. Gea2 YEL022W 1459 570-714 556-756 P39993
S. cerev. Syt1 YPR095C 1226 477-578 426-622 Q06836 NP_015420
S. cerev. YBGO YBL060W 687 105-208 61-266 P34225 NP_009493

S. pombe YDG1 C26F1.01 928 263-420 252-422 Q10491

S. pombe YDYB C11E3.11C Arf GEF5 942 325-446 300-486 NP_594936 O13690
S. pombe YEE1 C19A8.01C 428 338-428 O13817 CAB11637.1
S. pombe YEP1 C23H3.01 660(F?) 1-46 1-84 O13937 CAB16232.1
S. pombe GEA1 BC211.03C Put. GEF 1462 559-688 545-732 NP_596613 Q9P7R8
S. pombe YE51 C4D7.01C 1571 710-840 696-882 O14168 CAB11286.1
S. pombe BIG1 C30.01C Put prot 1822 719-849 705-891 NP_594555 Q9P7V5
S. pombe BIG2 P8A3.15C 206.9 kDa Pr 1811 710-840 696-882 NP_594954 Q9UT02

A. thaliana GNOM1 EMB30 1451 572-703 558-745 Q42510 S65571

A. thaliana BIG1 F20D10.320 1698/1643 /561-690 /547-732 NP_195533 Q9SZM0
A. thaliana O65490 BIG4 1711 578-707 564-749 NP_195264 O65490
A. thaliana F22M8.9 BIG3 1750 619-748 605-790 NP_171698 Q9LPC5
A. thaliana F7K15.150 BIG5 1669 579-684 565-726 NP_189916 Q9LXK4
A. thaliana T4C21.270 BIG2 1793 628-757 614-799 NP_191645 Q9LZX8
A. thaliana GNL1 Patt. Form. 1443 572-703 558-745 NP_198766 Q9FLY5
A. thaliana GNL2 1375 NP_197462

Dictyostellum Discoideum Cytohesin3 931 252-441 256-443 AC115599.1 Q8T2K1

Pichia Pastoris SEC7 1772 672-860 676-862 AAK40234 Q96X17

Rickettsia prowazekii RP374 462 17-150 3-194 CAA14833.1 Q9ZDF5

THE ARF FAMILY OF REGULATORY GTPASES

Plasmodium falciparum Arf GEF 3384

Paramecium tetraurelia Sec7 1135 587-717 573-759 Q9NJQ4

Encephalitozoon cuniculi Arf GEF 1063 303-479 295-481 Q8SR27

F=fragment
xvii

InterPro#s used when others not available (e.g., Q9LZX8)

xviii KAHN

The occasion of the publication of this book led to an unusual effort by

researchers in the field to reach a consensus on nomenclature for the Arf
GAPs. The willingness of these people to put aside personal claims of
priority in the spirit of collegiality and to bring order to what is already a
complex field is laudable and is to the betterment of the field. The results of
this altruism are presented in Table 1. There you will find a compilation of
the names of the predicted human Arf GAPs, with accession numbers,
lengths of predicted proteins, domain structure, and the currently “preferred”
name settled on by workers in the field. While not all parties have adopted
this “system” of naming the Arf GAPs, a clear majority has and with
consistent use in the literature all have indicated their willingness to adopt
this system. It is hoped that as a result we can all spend more time on
documenting activities and functions for these proteins and less time
worrying about the names. Input from close to 20 investigators was
important to the construction of this Table.
A similar effort was undertaken with the Arf GEFs, though much less
exhaustive in terms of input from investigators. Here we tried to focus on
proteins containing Sec7 domains from multiple model organisms as help in
defining orthologous proteins. As a result, no effort was made to develop a
new system for naming these proteins/genes. I would like to thank Cathy
Jackson for her help in putting together Table 2.

5. ARF EFFECTORS

Despite the discussion above, that might lead one to believe Arfs do
everything, it now appears clear that at least a primary function of Arfs is the
regulation of aspects of vesicular membrane traffic. More specifically, Arfs
are involved in the formation of coated vesicles through the recruitment of
specific coat proteins and initiation of large protein complex assemblies.
Whether changes in phospholipid metabolism play an obligate or regulatory
role in this process remains uncertain. Whether other nucleotide-sensitive
binding partners of less well-defined function (including mitotic kinesin like
protein 1 (MKLP1), Arfaptin1 and Arfaptin2/POR1, and Arfophilin (see
Chapter 11)) are also involved in vesicle traffic is also not known. Two-
hybrid and genetic screens have identified other partners of Arfs, which lack
obvious ties to vesicle traffic (A. L. Boman, C. Zhang, and R. A. Kahn,
unpublished observations). Results with Arf6 strongly suggest actions
distinct from vesicle traffic, though changes in the cytoskeleton may be part
of the remodeling required for vesiculation. Whether all these effectors end
up being involved in vesicle biogenesis seems doubtful, but it is becoming
clear that it is there that we will first gain a clear understanding of a
 THE ARF FAMILY OF REGULATORY GTPASES xix

molecular model for a function of Arf and Arf GAPs in cells. For this
reason, we have two chapters devoted to aspects of coat protein biology,
those from Annette Boman and Tommy Nilsson (see Chapter 12) and Victor
Faundez (see Chapter 13).
Some people may be surprised to see a discussion of Arf GAPs in the
section on effectors. I believe this is because GAPs for several GTPases
were first discovered as negative regulators of the GTPase signaling.
However, GAPs share the biochemical feature of binding preferentially to
the GTP-bound form of the GTPase they act upon and necessarily localize to
the same place in cells. By using the same switch domains as effectors
GAPs have the potential to alter cell signaling both through the increased
hydrolysis of GTP bound to the GTPase but also by competing with
effectors for the binding of the GTP-bound GTPase. In addition, the
presence of other protein interaction domains on Arf GAPs is expected to
facilitate the assembly of multi-subunit complexes, perhaps analogous to the
action of Arfs to recruit coat proteins to budding vesicles. Whether a “pure”
GAP exists, whose function is solely to terminate GTPase signaling, remains
to be seen, but I doubt it. Rather, I believe that the vast majority of GAPs,
particularly the Arf GAPs with their multiple interaction domains, will prove
to be effectors in every sense of the word and transduce positive signals
between the GTPase and downstream effects. The fact that GTPase
stimulation is a part of their action lends some support to the basic
“proofreading” model proposed by Goldberg for Arf GAP1 (Goldberg,
1999, 2000), though modifications are required to accommodate many of the
other Arf GAPs. If we add Arf GAPs to the list of effectors, we currently
have >30 effectors mediating the actions of 6 mammalian Arfs, which were
activated by ≥16 Arf GEFs. And so far there appears to be little or no cross
reactivity of Arf GEFs and GAPs with the Arls. If Arfs are the earliest
regulatory GTPase they will have the most layers of complexity to untangle
as we develop models of their cellular functions. But they also offer the
most promise of teaching us some fundamental principles of cell regulation
and the evolution of cell regulation.

6. THE FUTURE OF ARF

In the next few years we will develop much more complete models of how
Arfs and their partners regulate vesicle biogenesis and integrate protein and
lipid signaling in eukaryotes. Efforts recently underway to investigate
global changes in lipid content of cells in response to agonist stimulation or
differentiation will give us a much more complete view of cell signaling and
xx KAHN

this will likely be very important in developing a broader view of Arf

biology.
Though we have plenty of work ahead to complete the molecular
description of Arf actions in eukaryotes, studies of Arls have begun to yield
answers that broaden the functions of Arf family members considerably (see
16). The percent identity between Arfs and Arls is comparable to that
between Gs and Gi or between Rac and Rho. Thus, we expect both
differences and overlap in functions of Arls and Arfs. There is still no Arl
GEF or Arl GAP described. I expect this will change soon and with that
change will come another quantum jump forward in our understanding of the
actions of this family; along with many more surprises and fascinating
features and functions for members of the Arf family and their
acquaintances. Perhaps one or more Arl will serve as a simpler model for
Arf actions and allow us to move faster in developing these models and in
furthering out understanding of cell regulation by members of the Arf
family.

7. ACKNOWLEDGEMENTS

I would like to thank Judith Karl for her assistance in putting together
and proof reading each of the chapters in this book, including this preface.
Her attention to detail and organizational skills made this a much easier task
for me. The patience and support of other members of the department of
Biochemistry at Emory University is also appreciated. I also thank Paul
Randazzo for his critical reading of this preface.

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 Contributors
Annette Boman, Ph.D./ Department of Biochemistry and Molecular
Biology/ University of Minnesota-Duluth School of Medicine/ 10
University Drive/Duluth, MN 55812/USA/Phone 218 726 7036/Fax
218 726 8014/Email aboman@d.umn.edu

James Casanova, Ph.D./Department of Cell Biology/Box

800732/University of Virginia Health Sciences Center/1300 Jefferson
Park Avenue/Charlottesville, VA 22908-0732/USA/Phone 434 243
4821/Fax 434 982 3912/Email jec9e@virginia.edu

Dan Cassel, Ph.D./Department of Biology/Technion-Israeli Institute

of Technology/Haifa 32000/Israel/Phone 972 4829 3408/Fax 972
4822 5153/Email danc@techunix.technion.ac.il

Jacqueline Cherfils/Laboratoire d'Enzymologie et Biochimie

Structurales/CNRS, Avenue de la Terrasse/91198 Gif-sur-Yvette
cedex/France/Phone 33 1 69 823 492/Fax 33 1 69 823 129/Email
cherfils@lebs.cnrs-gif.fr

John H. Exton, M.D., Ph.D./Department of Molecular

Physiology/Biophysics and Pharmacology/Howard Hughes Medical
Institute/Vanderbilt University/702 Light Hall/Nashville, TN 37232-
0615/USA/Phone 615 322 6494/Fax 615 322 4381/Email
john.exton@vanderbilt.edu

Victor Faundez, M.D., Ph.D./Department of Cell Biology and Center

for Neurodegenerative Diseases/446 Whitehead Biomedical Research
Building/615 Michael Street/Emory University School of
Medicine/Atlanta, GA 30322/USA/Phone 404 727 3900/Fax 404 727
6256/Email faundez@cellbio.emory.edu

Dianne S. Hirsch, Ph.D./Laboratory of Cellular Oncology

CCR, NCI/Bldg 37 Room 6032/Bethesda, MD 20892
USA/Phone 301 496 6808/Fax 301 480 1260/Email
hirschd@mail.nih.gov

Mary Hunzicker-Dunn/Department of Cell and Molecular

Biology/Northwestern University Medical School/Tarry Building 8-
xxv
xxvi

721/303 East Chicago Avenue/Chicago, IL 60611/USA/Phone 312

503 7459/Fax 312 503 0566 or 312 503 7912/Email
mhd@northwestern.edu

Catherine L. Jackson, Ph.D./Cell Biology and Metabolism

Branch/NICHD, NIH/Building 18T, Room 101/18 Library Drive
MSC 5430/Bethesda, MD 20892-5430/USA/Phone 301 594 3944
Fax 301 402 0078/Email cathyj@helix.nih.gov

Prof. Dr. H.-G. Joost/German Institute of Human Nutrition/Arthur-

Scheunert Allee 114-116D-14558 Potsdam-
Rehbrücke/Germany/Phone 49 33200 88216/Fax 49 33200
88555/Email joost@mail.dife.de

Mr. Troy Lasell/Department of Cell Biology/Medical Sciences

Building Room 5-14/University of Alberta/Edmonton AB/Canada
T6G 2H7/Phone 780 492 6309/Fax 780 492 0450/Email
tlasell@ualberta.ca

John Logsdon, Ph.D./Department of Biology/Emory University/1510

Clifton Road/Atlanta, GA 30322/USA/Phone 404 727 9516/Fax 404
727 2880/Email jlogsdon@biology.emory.edu

Paul Melancon, Ph.D./Department of Cell Biology/Medical Sciences

Building Room 5-14/University of Alberta/Edmonton AB/Canada
T6G 2H7/Phone 780 492 6183/Fax 780 492 0450/Email
paul.melancon@ualberta.ca

Naoko Morinaga, Ph.D./2nd Department of Microbiology/School of

Medicine/Chiba University/Chiba 280/Japan/Phone 81 043 226
2048/Fax 81 043 226 2049/Email morinaga@med.m.chiba-u.ac.jp

Joel Moss, M.D., Ph.D./Pulmonary-Critical Care Medicine

Branch/HLBI, NIH/Building 10, Room 6D03/9000 Rockville Pike, 10
Center Drive/Bethesda, MD 20892-1590/USA/Phone 301 496
1597/Fax 301 496 2363/Email mossj@nhlbi.nih.gov

Zhongzhen Nie, Ph.D./Laboratory of Cellular Oncology/CCR,

NCI/Bldg 37 Room 6032/Bethesda, MD 20892/USA/Phone 301 496
6833/Fax 301 480 1260/Email niezhong@mail.nih.gov
 xxvii

Professor Tommy Nilsson/Department of Medical Biochemistry/

Goteborg University/Medicinaregatan 9A/413 90 Gothenburg/Sweden
and until December 2003/Cell Biology and Biophysics Programe/
EMBL/Meyerhofstrasse 1/D-69017 Heidelberg/Germany/Phone 49
6221 387 294 (or 408)/Fax 49 6221 387 306 (or 512)/Email
nilsson@embl-heidelberg.de

Mastoshi Noda, Ph.D./2nd Department of Microbiology/School of

Medicine/Chiba University/Chiba 280/Japan/Phone 81 043 226
2046/Fax 81 043 226 2049/Email noda@med.m.chiba-u.ac.jp

Gustavo Pacheco-Rodriguez, Ph.D./Pulmonary-Critical Care

Medicine Branch/NHLBI, NIH/Building. 10, Room 5N314/9000
Rockville Pike, 10 Center Drive/Bethesda, MD 20892-1434/
USA/Phone 301 496 1454/Fax 301 402 1610/Email
pachecog@nhlbi.nih.gov

Sebastiano Pasqualato, Ph.D./Laboratoire d'Enzymologie et

Biochimie Structurales/CNRS, Avenue de la Terrasse/91198 Gif-sur-
Yvette cedex/France/Phone 33 1 69 823 492/Fax 33 1 69 823
129/Email pasquala@lebs.cnrs-gif.fr
Current Address: Dipartimento di Oncologia Sperimentale/Istituto
Europeo de Oncologia/Vai Ripamonti 435/20141 Milano, Italy

Richard T. Premont, Ph.D./Assistant Research Professor/Liver

Center - 329 Sands Building/Department of Medicine
(Gastroenterology)/Duke University Medical Center/Campus Box
3083/Durham, NC 27710/USA/Phone 919 684 5620/Fax 919 684
4983/Email richard.premont@duke.edu

Paul Randazzo, M.D., Ph.D./Laboratory of Cellular Oncology/CCR,

NCI/Bldg 37 Rm 6032/Bethesda, MD 20892/USA/Phone 301 496
3788/Fax 301 480 1260/Email randazzo@helix.nih.gov

Louis Renault, Ph.D./Laboratoire d'Enzymologie et Biochimie

Structurales/CNRS, Avenue de la Terrasse/91198 Gif-sur-Yvette
cedex/France/Phone 33 1 69 823 492/Fax 33 1 69 823 129/Email
renault@lebs.cnrs-gif.fr
xxviii

Hisataka Sabe, Ph.D./Department of Molecular Biology/Osaka

Bioscience Institute/6-2-4 Furuedai, Suita City/Osaka 565-
0874/Japan/Phone 81 6 6872 4814, 4850/Fax 81 6 6871 6686/Email
sabe@obi.or.jp

Robert Schmalzigaug, Ph.D./Liver Center - 334 Sands

Building/Department of Medicine (Gastroenterology)/Duke
University Medical Center/Campus Box 3083/Durham, NC 27710/
USA/Phone 919 684 8647/Fax 919 684 4983/Email
schma005@mc.duke.edu

Prof. Dr. Annette Schürmann/German Institute of Human

Nutrition/Department of Pharmacology/Arthur-Scheunert Allee 114-
116/D-14558 Potsdam-Rehbrücke/Germany/Phone 49 33200
88368/Fax 49 33200 88334/Email scheurman@mail.dife.de

M. L. Styers/Department of Cell Biology and theBiochemistry, Cell,

and Developmental Biology/Graduate Division of Biological and
Biomedical Sciences/455 Whitehead Biomedical Research
Building/615 Michael Street/Emory University School of
Medicine/Atlanta, GA 30322/USA/Phone 404 727 3945/Fax 404 727
6256/Email mstyers@emory.edu

Martha Vaughan, M.D./Pulmonary-Critical Care Medicine Branch/

NHLBI, NIH/Building. 10, Room 5N307/9000 Rockville Pike, 10
Center Drive/Bethesda, MD 20892-1434/USA/Phone 301 496 4554/
Fax 301 402 1610/Email vaughanm@nhlbi.nih.gov

Ms. Zinhua Zhao/Department of Cell Biology/Medical Sciences

Building Room 5-14/University of Alberta/Edmonton AB/Canada
T6G 2H7/Phone 780 492 6309/Fax 780 492 0450/Email
xinhua@gpu.srv.ualberta.ca
Chapter 1
THE ARF FAMILY TREE

JOHN M. LOGSDON, JR.,1 AND RICHARD A. KAHN2

Departments of Biology1 and Biochemistry2, Emory University School of Medicine, Atlanta,

USA

Abstract: A phylogeny of the Arf family of proteins has been constructed using
sequence data available from the complete (or nearly complete) genomes of
six model eukaryotic species; including three animals, two fungi and a protist.
Our analyses reveal distinct groupings of three Arf and ten Arl (Arf-like)
subfamilies. These Arf and Arl subfamilies have arisen by numerous gene
duplications that took place at different times during eukaryotic evolution.
Some apparently young subfamilies are comprised of proteins found only in
animals, while older families include members from all of the eukaryotic
lineages considered. Available functional information for these proteins is
considered in light of their evolutionary relationships.

1. INTRODUCTION

ADP-ribosylation factor(s) (Arf(s)) were first described as, and named after,
a biochemical activity: cofactor in the ADP-ribosylation of Gs by cholera
toxin (Kahn and Gilman, 1984, 1986; Schleifer et al., 1982). The original
cloned cDNAs and their proteins that possess that activity were named
accordingly (Sewell and Kahn, 1988). Unfortunately, that first Arf activity
is now understood to have little or nothing to do with the cellular functions
of Arfs. This disconnect between name and function is even more severe for
the Arf-like (Arl) proteins, which lack Arf activities and were named on the
basis of protein sequence homology (Cavenagh et al., 1994; Clark et al., 1993;
Tamkun et al., 1991). As the size of the Arf family expanded, so did the
number of activities and binding partners found associated with Arfs and
Arls. Other activities emerged that were predicted to be better
1
2 LOGSDON AND KAHN

discriminators between functionally different groups of proteins. These

included direct activation of phospholipase D (Brown et al., 1993; Cockcroft et
al., 1994) and the ability to rescue the lethality of the Saccharomyces
cerevisiae double null mutant arf1-arf2- (Kahn et al., 1991), activities that, in
fact, have correlated very well with the original Arf activity. Nonetheless,
with more than a dozen effectors now known for Arfs alone and evidence of
complicated networks of cross reactivity between Arf family GTPases and
effectors (e.g., see (Van Valkenburgh et al., 2001)), it is unlikely that a
single activity will be found with the ability to appropriately discriminate
between them to allow unambiguous classification on the basis of function.
Sequencing of entire genomes has allowed a complete, or very nearly so,
cataloging of Arf family members and allows the tracing of their
evolutionary relationships. Because functional redundancy is a common
complication in interpretation of genetic and biochemical data, this
genealogical framework of Arfs and Arls is important as we focus on
functions of these proteins.
In this chapter we present the results of recent efforts at identifying and
reconstructing the evolutionary relationships among all Arf family members
in a number of model eukaryotes: human (Homo sapiens), fly (Drosophila
melanogaster), worm (Caenorhabditis elegans), two fungi (the yeasts S.
cerevisiae, and Schizosaccharomyces pombe), and a protist (the diplomonad
Giardia lamblia). We describe evidence for the existence of three Arf
classes (the previously described Arf classes I-III) and ten Arls, including
Arls1-5, Arl8, Arl9, Arfrp and Sar1. A simplified nomenclature is proposed
based on these phylogenetic classifications. We then discuss unique features
of each group, their predicted evolutionary origins and functional
implications. A phylogenetic analysis of the human Arf family was
presented in Jacobs, et al. (Jacobs et al., 1999) and reached many of the same
conclusions regarding the naming and number of Arl groups.

2. THE FAMILY

An iterative bioinformatic process of probing both protein and nucleotide

databases with Arf and Arl sequences with BLAST (Altschul et al., 1990;
Altschul et al., 1997), followed by construction of protein alignments,
phylogenetic analyses, and re-probing for potentially “missing” members led
to a final collection of 64 Arf family members, listed in Table 1. These
include the protein-coding sequences of 23 human, 12 D. melanogaster, 12
C. elegans, 7 S. cerevisiae, 4 S. pombe, and 6 G. lamblia genes. Protein
sequences were aligned as shown in Figures 1 and 2. The alignment was
then analyzed by a variety of methods to construct phylogenetic trees,
 The ARF Family Tree 3

culminating in the Bayesian maximum likelihood analyses shown in Figure

3 and the simplifed view shown in Figure 4. The aligned and un-edited N-
terminal sequences are shown in Figure 1. This serves to illustrate the
editing process used and also reveals in many cases the conservation of
lengths in regions within groups. For example, note that all Arl2 and Arl3
group members are shorter at their N-termini than the other proteins. This is
also true for ArfIIIs vs Arf I or ArfIIs. The differences in lengths in the loop
that connects the N-terminal α-helix to the rest of the protein are also likely to
have functional significance as this essential domain has previously been shown
to be important in coupling nucleotide exchange to protein hydrophobicity
(and hence membrane association) and to interact with specific lipids.
We focused on these six completely sequenced genomes to compose a
list of predicted Arf family members, though we also made use of data from
other species to help resolve issues (most of which involved incorrect
assignment of intron/exon boundaries by gene prediction software). The
Arfs themselves are so well conserved (>60% identity) that there is little
ambiguity or difficulty in finding them with simple BLAST searches of
proteomes or genomes. Researchers have been inconsistent previously in
grouping Arfrp and Sar proteins in the Arf family, as they are the most
divergent. Our analysis clearly puts Arfrp in the Arf family and we argue
for inclusion of Sar on the basis of both sequence and functional
conservation. After Sar, the next proteins appearing on BLAST results
(using Arf or Arl sequence probes from any organism) were members of the Rab
family of GTPases. This suggests that by including Sar in the Arf family
we have found a clear demarcation between these two families of GTPases.
Arfs are small proteins at ~20 kDa and therefore smaller or similar in size
to several protein domains, including the Sec7 (Arf GEF) and PTB domains,
each ~200 residues. In fact, there exists at least one protein that uses the Arf
sequence as a domain within a larger context. These are the Arf-domain
proteins; e.g., the human 64 kDa Ard1 protein (569 residues) contains an Arf
domain at the C-terminus and an Arf GAP domain (Mishima et al., 1993;
Vitale et al., 1998). Rodents (rat and mouse; 97% identical) and C. elegans
(~37% identical) express orthologs of Hs Ard1. These proteins are not
included in our analyses, despite the fact that the Arf domain of Ard1 is
~60% identical to Arfs.

Table 1. Summary of Arf family members in H. sapiens (Hs), D. melanogaster (Dm), C.

elegans (Ce), S. pombe (Sp), S. cerevisiae (Sc), and G lamblia (Gl). Information from a
variety of sources is summarized here to help identify the different proteins. The names used
in this chapter are shown in the first column and common names from the different species
are shown in column two. Accession numbers are in column three to help locate full-length
cDNA and protein sequences. Locus identifiers are also included to aid in gene identification.
Information on the chromosomal location for each gene and the length of predicted proteins is
included, as are other names that appear in the literature or databases. Finally, the first 12
residues are shown as another means of resolving any confusion between protein names and
identity.
 Table I: Summary of Arf family members in H. sapiens , D. melanogaster , C. elegans , S. cerevisiae ,
S. pombe , and G. lamblia

4
Proposed Other Accession Locus Gene Length N-terminal
name name(s) number identifier Location (a.a.) Other names sequence
H. sapiens
HsArf1 Arf1 NM_001658/GI:6995997 LocusID: 375 1q42 181 MGNIFANLFKGL
(MmArf2) Arf2 NM_007477/GI:6671570 LocusID: 11841 11 E1(Mm) 181 MGNVFEKLFKSL
HsArf3 Arf3 NM_001659/GI:4502202 LocusID: 377 12q13 181 MGNIFGNLLKSL
HsArf4 Arf4 NM_001660/GI:6995998 LocusID: 378 3p21.2-p21.1 180 MGLTISSLFSRL
HsArf5 Arf5 NM_001662/GI:6995999 LocusID: 381 7q31.3 180 MGLTVSALFSRI
HsArf6 Arf6 NM_001663/GI:6996000 LocusID: 382 7q22.1 175 MGKVLSKIFGNK
HsArl1 Arl1 NM_001177/GI:4755126 LocusID: 400 12q23.3 181 Arfl1 MGGFFSSIFSSL
HsArl2 Arl2 NM_001667/GI:4502228 LocusID: 402 11q13 184 Arfl2 MGLLTILKKMKQ
HsArl3 Arl3 NM_004311/GI:4757773 LocusID: 403 10q23.3 182 Arfl3 MGLLSILRKLKS
HsArl4 Arl4 NM_005738/GI:5031602 LocusID: 10124 7p21-p15.3 200 MGNGLSDQTSILSN
HsArl4B Arl4B NT_033984/XP_166703.1 234 MGNGLSDQTSILSS
HsArl5 Arl5 NM_012097/GI:6912243 LocusID: 26225 2q23.3 179 MGILFTRIWRLF
HsArl5B Arl5B XM_167671/GI:20473688 LocusID: 221079 10p12.33 179 Sim. to Arl5 MGLIFAKLWSLF
HsArl6 Arl6 NM_032146/GI:21314750 LocusID: 84100 3q12.1 186 MGLLDRLSVLLG
HsArl7 Arl7 NM_005737/GI:6552295 LocusID: 10123 2q37.2 192 LAK MGNISSNISAFQ
HsArl8A Arl8A NM_138795/GI:15929963 LocusID: 127829 1q31.3 186 Hypo. Prot BC015408 MIALFNKLLDWF
HsArl8B Arl8B NM_018184/GI:8922600 LocusID: 55207 3p26.1 186 FLJ10702 MLALISRLLDWF
HsArl9 Arf4L/Arl6 NM_001661/GI:4502206 LocusID: 379 17q12-q21 201 Arl6 MGNHLTEMAPTA
HsArl10 Arl10 NM_025047/GI:13376573 LocusID: 80117 3q26.1 192 FLJ22595 MGSLGSKNPQTK
HsArl11 Arl11 NM_138450/GI:27764878 LocusID: 115761 13q14.11 196 MGC17429 MGSVNSRGHKAE
HsArfrp Arp NM_003224/GI:4507448 LocusID: 10139 20q13.3 201 Arfrp1 MYTLLSGLYKYM
HsSar1 Sar1 NM_020150/GI:21361614 LocusID: 56909 10q22.1 198 MSFIFEWIYNGF
HsSara Sara NM_016103/GI:7705826 LocusID: 51128 5q31.1 198 MSFIFDWIYSGF
LOGSDON AND KAHN

23
C. elegans
CeArf1 arf-1 NM_065834/GI:17551729 B0336.2 III 181 MGNVFGSLFKGL
CeArf3 arf-3 NM_068935/GI:17538183 F57H12.1 IV 180 arf-3 MGLTISSLFNRL
CeArf6 arf-6 NM_070610/GI:17538185 Y116A8C.12 IV 175 arf-6 MGKFLSKIFGKK
CeArl1 arl-1 NM_063415/GI:17531196 F54C9.10 II 180 arl-3 MGGVMSYFRGLF
CeArl2 evl-20/arl-2 NM_063378/GI:17532860 F22B5.1 II 184 evl-20 MGFLKILRKQRA
CeArl3 arl-3 NM_064636/GI:17531200 F19H8.3 II 184 arl-3C MGLIDVLKSFKS
CeArl5 arl-5 NM_066777/GI:17551731 ZK632.8 III 178 arl-2 MGLIMAKLFQSW
CeArl6 arl-6 NM_065592/GI:17551733 C38D4.8 III 190 arl-1, arl-query MGFFSSLSSLFG
CeArf arf NM_068841/GI:17538189 F45E4.1 IV 179 arl-6; arf-3 MGLFFSKISSFM
CeArl8 arl-8 NM_070390/GI:17543765 Y57G11C.13 IV 185 MLAMVNKVLDWI
CeArfrp arfrp NM_058693/GI:17510340 Y54E10BR.2 I 191 MYTLSAGIWEKF
CeSar1 sar-1 NM_068181/GI:17544539 ZK180.4 IV 193 MSFLWDWFNGVL
12
 Table I (cont.)

D. melanogaster
DmArf1 DmArf79F NM_057607/GI:24668765 Arf79F 3L 182 Darf1, dARFI, CG8385 MGNVFANLFKGL
DmArf2 DmArf102F NM_079892/GI:24638807 Arf102F 4 180 dARFII, ARFII MGLTISSLLTRL
DmArf3 DmArf51F NM_079027/GI:24653826 Arf51F 2R 175 dArf3, ARFIII MGKLLSKIFGNK
DmArl1 DmArf72A NP_524098/GI:24664933 Arf72A 3L 180 arflike, arl, dArl1 MGGVLSYFRGLL
DmArl2 DmArl84F NM_057538/GI:19549394 Arl84F 3R 184 arf-like2, arl2 MGFLTVLKKMRQ
DmArl3 DmCG6560 NM_142738/GI:24648859 CG6560 3R 179 CG6560 MGLLSLLRKLRP
DmArl4 DmCG2219 NP_651905/GI:24638544 CG2219 175 CG2219 MGATMVKPLVKN
DmArl5 DmCG7197 NM_139944/GI:24660780 CG7197 3L 179 CG7197 MGLLLSRLWRMF
DmArl6 DmArl6 NM_137577/GI:24655720 CG7735 2 202 CG7735 MGMLHNLADLIK
DmArl8 DmArl8 NP_649769/GI:21355085 CG7891 3 186 CG7891 MLALINRILEWF
DmArfrp DmArfrp NM_132298/GI:24640728 CG7039 1 200 CG7039 MYTLMHGFYKYM
DmSar1 DmSar1 NP_732717/GI:24648946 CG7073 3 193 CG7073, sar-1 MFIWDWFTGVLG
12
S. cerevisiae
ScArf1 Arf1 P11076/GI:114121 YDL192W IV 181 MGLFASKLFSNL
ScArf2 Arf2 NP_010144/GI:6320064 YDL137W IV 181 MGLYASKLFSNL
ScArf3 Arf3 NP_014737/GI:6324668 YOR094W XV 183 ARL2 MGNSISKVLGKL
ScArl1 Arl1 NP_009723/GI:6319641 YBR164C II 183 MGNIFSSMFDKL
ScArl2 Cin4 NP_013858/GI:6323787 YMR138W XIII 191 GTP1, UGX1 MGLLSIIRKQKL
ScArfrp Arl3 NP_015274/GI:6325206 YPL051W XVI 198 ARL3 MFHLVKGLYNNW
ScSar1 Sar1 NP_015106/GI:6325038 YPL218W XVI 190 MAGWDIFGWFRD
7
The ARF Family Tree

S. pombe
SpArf1 Arf1 P36579 /GI:543843 SPBC4F6.18c II 180 MGLSISKLFQSL
SpArf2 Arf2 Q9Y7Z2 /GI:20137584 SPBC1539.08 II 184 MGNSLFKGFSKP
SpArl2 alp41/Arl2 Q09767 /GI:1168499 SPAC22F3.05c II 186 alp41 MGLLTILRQQKL
SpSar1 Sar1 Q01475 /GI:266990 SPBC31F10.06c II 190 MFIINWFYDALA
4
G. lamblia
GlArf GlArf P26991 /GI:114131 191 MGQGASKIFGKL
GlArl2 Gl4192 197 MGFLSVLRAIKA
GlArl3 GlArlB BAB58895/GI:14275898 149 ArlB ...TILRKLCEEDTS
GlArl Gl7562 168 MGAASSRPARHK
GlArl8/Arfrp GlArlA BAB58894/GI:14275896 157 ArlA ...TTILHQMAYGMT
GlSar1 GlSar1 AY125726/GI:22035408 191 MGFGDWFKSALS
6
5
6 LOGSDON AND KAHN

2.1 The Arf sub-family

The Arf proteins have been previously divided into three classes (I, II, III),
based on their gene sequences and organization (Lee et al., 1994a). This
classification is further supported by our analyses. Flies and worms have
single representatives for each of these three classes, while mammals have
either five or six genes (see below), S. cerevisiae has three Arfs, S. pombe
has two and Giardia has only one. The class I and II Arfs, found only
among animals, apparently diverged from a common ancestor more recently
(after the animal-fungal split) than did class III Arfs, which are found in both
animals and fungi. The two yeasts have single representatives for class I/II
and class III (excepting that in S. cerevisiae, a very recent duplication of a
chromosome IV region resulted in two very similar Arf I/II genes, Arf1 and
Arf2). The fact that Arf classes I, II and III form a well-supported group
including representatives in animals, fungi and a single Arf gene from
Giardia supports our conclusion that all Arfs arose from a common ancestor.
The fact that the single Giardia Arf groups (albeit weakly) with class I/II
Arfs, suggests that it represents the class I/II ancestral type (as do Sc Arf 1
and 2). It is also possible that the single Giardia Arf represents the class
I/II/III ancestral type, which would indicate that the I/II vs. III duplication
occurred after the divergence of Giardia, but before the animal-fungal split.
In C. elegans a fourth Arf gene (Ce Arf) has been found. It is very likely a
pseudogene, based upon its proximity to another (highly expressed) gene
and lack of any evidence of its expression. This makes its phylogenetic
placement dubious due to its rapid evolutionary rate (and resulting long
branch on the phylogenetic trees).
The human Arf III class gene, Arf6, has activity as cofactor for cholera
toxin, activation of phospholipase D and rescue of the double arf- null in S.
cerevisiae (Lee et al., 1992; Massenburg et al., 1994). It will be interesting to
see if the class III representatives from flies, worms and yeasts share these
activities. Sc Arf3 has been reported to support cholera toxin activity, albeit
weakly, but it cannot substitute for Arf1 in yeast cells (Lee et al., 1994b).
 The ARF Family Tree 7

1 26 47 71
HsArf1 MGNIFANLFK-------------GLFGK-KEMRILMVGLDAAGKTTILYKLKLGEIV--------TTIPTIGFNVETVE-----YKNIS-FT--VWDVGGQDK
MmArf2 MGNVFEKLFK-------------SLFGK-KEMRILMVGLDAAGKTTILYKLKLGEIV--------TTIPTIGFNVETVE-----YKNIS-FT--VWDVGGQDK
HsArf3 MGNIFGNLLK-------------SLIGK-KEMRILMVGLDAAGKTTILYKLKLGEIV--------TTIPTIGFNVETVE-----YKNIS-FT--VWDVGGQDK
DmArf1 MGNVFANLFK-------------GLFGK-KEMRILMVGLDAAGKTTILYKLKLGEIV--------TTIPTIGFNVETVE-----YKNIS-FT--VWDVGGQDK
CeArf1 MGNVFGSLFK-------------GLFGK-REMRILMVGLDAAGKTTILYKLKLGEIV--------TTIPTIGFNVETVE-----YKNIS-FT--VWDVGGQDK
SpArf1 MGLSISKLFQ-------------SLFGK-REMRILMVGLDAAGKTTILYKLKLGEIV--------TTIPTIGFNVETVE-----YRNIS-FT--VWDVGGQDK
HsArf4 MGLTISSLFS-------------RLFGK-KQMRILMVGLDAAGKTTILYKLKLGEIV--------TTIPTIGFNVETVE-----YKNIC-FT--VWDVGGQDR
HsArf5 MGLTVSALFS-------------RIFGK-KQMRILMVGLDAAGKTTILYKLKLGEIV--------TTIPTIGFNVETVE-----YKNIC-FT--VWDVGGQDK
DmArf2 MGLTISSLLT-------------RLFGK-KQMRILMVGLDAAGKTTILYKLKLGEIV--------TTIPTIGFNVETVE-----YKNIC-FT--VWDVGGQDK
CeArf3 MGLTISSLFN-------------RLFGK-RQVRILMVGLDAAGKTTILYKLKLGEIV--------TTIPTIGFNVETVE-----YKNIS-FT--VWDVGGQDK
ScArf1 MGLFASKLFS-------------NLFGN-KEMRILMVGLDGAGKTTVLYKLKLGEVI--------TTIPTIGFNVETVQ-----YKNIS-FT--VWDVGGQDR
ScArf2 MGLYASKLFS-------------NLFGN-KEMRILMVGLDGAGKTTVLYKLKLGEVI--------TTIPTIGFNVETVQ-----YKNIS-FT--VWDVGGQDR
CeArf MGLFFSKISS-------------FMFPN-IECRTLMLGLDGAGKTTILYKLKLNETV--------NTIPTIGFNVETVT-----FQKIT-LT--VWDVGGQKK
GlArf MGQGASKIFG-------------KLFSK-KEVRILMVGLDAAGKTTILYKLMLGEVV--------TTVPTIGFNVETVE-----YKNIN-FT--VWDVGGQDS
HsArf6 MGKVLSKIF-----------------GN-KEMRILMLGLDAAGKTTILYKLKLGQSV--------TTIPTVGFNVETVT-----YKNVK-FN--VWDVGGQDK
DmArf3 MGKLLSKIF-----------------GN-KEMRILMLGLDAAGKTTILYKLKLGQSV--------TTIPTVGFNVETVT-----YKNVK-FN--VWDVGGQDK
CeArf6 MGKFLSKIF-----------------GK-KELRILMLGLDAAGKTTILYKLKLGQSV--------TTIPTVGFNVETVT-----YKNIK-FN--VWDVGGQDK
SpArf2 MGNSLFKGFSKPFS---------RLFSN-KEMRILMLGLDAAGKTTILYKLKLNQSV--------VTIPTVGFNVETVT-----YKNIK-FN--VWDVGGQDK
ScArf3 MGNSISKVLG-------------KLFGS-KEMKILMLGLDKAGKTTILYKLKLNKIK--------TSTPTVGFNVETVT-----YKNVK-FN--MWDVGGQQR
HsArl1 MGGFFSSIFS-------------SLFGT-REMRILILGLDGAGKTTILYRLQVGEVV--------TTIPTIGFNVETVT-----YKNLK-FQ--VWDLGGQTS
DmArl1 MGGVLSY-FR-------------GLLGS-REMRILILGLDGAGKTTILYRLQVGEVV--------TTIPTIGFNVEQVT-----YKNLK-FQ--VWDLGGQTS
CeArl1 MGGVMSY-FR-------------GLFGA-REMRILILGLDGAGKTTILYRLQVGEVV--------TTIPTIGFNVEQVE-----YKNLK-FQ--VWDLGGQTS
ScArl1 MGNIFSSMFD-------------KLWGSNKELRILILGLDGAGKTTILYRLQIGEVV--------TTKPTIGFNVETLS-----YKNLK-LN--VWDLGGQTS
HsArl5 MGILFTRIW--------------RLFNH-QEHKVIIVGLDNAGKTTILYQFSMNEVV--------HTSPTIGSNVEEIV-----INNTR-FL--MWDIGGQES
HsArl5B MGLIFAKLW--------------SLFCN-QEHKVIIVGLDNAGKTTILYQFLMNEVV--------HTSPTIGSNVEEIV-----VKNTH-FL--MWDIGGQES
DmArl5 MGLLLSRLW--------------RMFGN-EEHKLVMVGLDNAGKTTILYQFLMNEVV--------HTSPTIGSNVEEVV-----WRNIH-FL--VWDLGGQQS
CeArl5 MGLIMAKLFQ-------------SWWIG-KKYKIIVVGLDNAGKTTILYNYVTKDQV--------ETKPTIGSNVEEVS-----YRNLD-FV--IWDIGGQES
HsArl4 MGNGLSDQTSILS--------NLPSFQS---FHIVILGLDCAGKTTVLYRLQFNEFV--------NTVPTKGFNTEKIKVTLGNSKTVT-FH--FWDVGGQEK
HsArl4B MGNGLSDQTSILS--------SLPSFQS---FHIVMLGLDCAGKTTVLYRLQFNEFV--------NTVPTKAFNTEKIKVNLRNSKTVT-FH--FGDVGGQEK
HsArl7 MGNISS---------------NISAFQS---LHIVMLGLDSAGKTTVLYRLKFNEFV--------NTVPTIGFNTEKIKLSNGTAKGIS-CH--FWDVGGQEK
HsArl9 MGNHLTEMAPTASS-------FLPHFQA---LHVVVIGLDSAGKTSLLYRLKFKEFV--------QSVPTKGFNTEKIRVPLGGSRGIT-FQ--VWDVGGQEK
DmArl4 MGATMVKPLVKNGNI------LDALPSQ-ATLHVVMLGLDSAGKTTALYRLKFDQYL--------NTVPTIGFNCEKVQCTLGKAKGVH-FL--VWDVGGQEK
HsArl10 MGSLGSKNPQT------------------KQAQVLLLGLDSAGKSTLLYKLKLAKDI--------TTIPTIGFNVEMIEL----ERNLS-LT--VWDVGGQEK
HsArl11 MGSVNSRGHK-------------------AEAQVVMMGLDSAGKTTLLYKLKGHQLV--------ETLPTVGFNVEPLKA----PGHVS-LT--LWDVGGQAP
HsArl2 MGLLTILKKMKQ--------------KE-RELRLLMLGLDNAGKTTILKKFNGEDID--------TISPTLGFNIKTLE-----HRGFK-LN--IWDVGGQKS
DmArl2 MGFLTVLKKMRQ--------------KE-REMRILLLGLDNAGKTTILKRFNGEPID--------TISPTLGFNIKTLE-----HNGYT-LN--MWDVGGQKS
CeArl2 MGFLKILRKQRA--------------RE-REMRILILGLDNAGKTTLMKKFLDEPTD--------TIEPTLGFDIKTVH-----FKDFQ-LN--LWDVGGQKS
SpArl2 MGLLTILRQQKL--------------KE-REVRVLLLGLDNAGKTTILKCLLNEDVN--------EVSPTFGFQIRTLE-----VEGLR-FT--IWDIGGQKT
ScArl2 MGLLSIIRKQKL--------------RD-KEIRCLILGLDNSGKSTIVNKLLPKDEQ-----NNDGIMPTVGFQIHSLM-----IKDVT-IS--LWDIGGQRT
GlArl2 MGFLSVLRAIKA--------------SE-NEARVLVVGLDCSGKTTIVSSLLGKPLT--------EIAPTLGFKISTWR-----PQNTS-MAVALWDVGGQQT
HsArl3 MGLLSILRKLKSA-------------PD-QEVRILLLGLDNAGKTTLLKQLASEDIS--------HITPTQGFNIKSVQS-----QGFK-LN--VWDIGGQRK
DmArl3 MGLLSLLRKLRPN-------------PE-KEARILLLGLDNAGKTTILKQLASEDIT--------TVTPTAGFNIKSVAA-----DGFK-LN--VWDIGGQWK
CeArl3 MGLIDVLKSFKSP-------------SG-REIRILLLGLDNAGKTTILKQLSSEDVQ--------HVTPTKGFNVKTVAA----MGDIR-LN--VWDIGGQRS
GlArl3 TILRKLCEEDTS--------VIMPTQGFNAKTIKG----PKGVS-LN--CWDVGGQKA
HsArl8A MIALFNKLLDW----------FKALFWK-EEMELTLVGLQYSGKTTFVNVIASGQFN-------EDMIPTVGFNMRKIT-----KGNVT-IK--LWDIGGQPR
HsArl8B MLALISRLLDW----------FRSLFWK-EEMELTLVGLQYSGKTTFVNVIASGQFS-------EDMIPTVGFNMRKVT-----KGNVT-IK--IWDIGGQPR
DmArl8 MLALINRILEW----------FKSIFWK-EEMELTLVGLQFSGKTTFVNVIASGQFA-------EDMIPTVGFNMRKIT-----RGNVT-IK--VWDIGGQPR
CeArl8 MLAMVNKVLDW----------IRSLFWK-EEMELTLVGLQNSGKTTFVNVIASGQFT-------EDMIPTVGFNMRKIT-----KGNVT-IK--LWDIGGQPR
GlArfrp TTILHQMAYGMT--------VETIPTMGFTLQTVK-----KGKLE-LD--VWDIGGQSE
HsArfrp MYTLLSGL-------------YKYMFQK-DEYCILILGLDNAGKTTFLEQSKTRFNKNYKGMSLSKITTTVGLNIGTVD-----VGKAR-LM--FWDLGGQEE
DmArfrp MYTLMHGF-------------YKYMTQK-DDYCVVILGLDNAGKTTYLEAAKTTFTRNYKGLNPSKITTTVGLNIGTID-----VQGVR-LN--FWDLGGQQE
CeArfrp MYTLSAGI-------------WEKFFKK-KDYYVVIVGLDNAGKTTFLEQTKSHFVKDYGVLNPSKITATVGLNTGNVE-----LNNTC-LH--FWDLGGQES
ScArfrp MFHLVKGL-------------YNNWNKK-EQYSILILGLDNAGKTTFLETLKKEYSL--AFKALEKIQPTVGQNVATIP-----VDSKQILK--FWDVGGQES
HsArl6 MGLLDRLSV------------LLGL-KK-KEVHVLCLGLDNSGKTTIINKLKPSNAQ------SQNILPTIGFSIEKFK-----SSSLS-FT--VFDMSGQGR
DmArl6 MGMLHNLAD------------LIKI-KK-DKMTILVLGLNNSGKSSIINHFKKSSEQ------TSIVVPTVGFMVEQFYI---GMSGVS-IK--AIDMSGATR
CeArl6 MGFFSSLSS------------LFGL-GK-KDVNIVVVGLDNSGKTTILNQLKTPETR------SQQIVPTVGHVVTNFS-----TQNLS-FH--AFDMAGQMK
GlArl MGAASSRPA-----------------RH-KTAKLLILGRQFSGKSTLINYLKTNSFT--------VVNPTINFTAEKYN---------R-YE--VIDVGGSAE
HsSar1 MSFIFEWIYNG----FSSVLQFLGL-YK-KSGKLVFLGLDNAGKTTLLHMLKDDRLG--------QHVPTLHPTSEELT-----IAGMT-FT--TFDLGGHEQ
HsSara MSFIFDWIYSG----FSSVLQFLGL-YK-KTGKLVFLGLDNAGKTTLLHMLKDDRLG--------QHVPTLHPTSEELT-----IAGMT-FT--TFDLGGHVQ
CeSar1 MSFLWDW--------FNGVLNMLGL-AN-KKGKLVFLGLDNAGKTTLLHMLKDDRIA--------QHVPTLHPTSEQMS-----LGGIS-FT--TYDLGGHAQ
DmSar1 M-FIWDW--------FTGVLGYLGL-WK-KSGKLLFLGLDNAGKTTLLHMLKDDKLA--------QHVPTLHPTSEELS-----IGNMR-FT--TFDLGGHTQ
ScSar1 M-AGWDIFGW-----FRDVLASLGL-WN-KHGKLLFLGLDNAGKTTLLHMLKNDRLA--------TLQPTWHPTSEELA-----IGNIK-FT--TFDLGGHIQ
SpSar1 M-FIINW--------FYDALAMLGL-VN-KHAKMLFLGLDNAGKTTLLHMLKNDRLA--------VMQPTLHPTSEELA-----IGNVR-FT--TFDLGGHQQ
GlSar1 M-GFGDW--------FKSALSFLGL-YK-KKATIVFVGLDNAGKSTLLAMLKNSATT--------TVAPTQQPTSQELV-----MGSIR-FK--TFDLGGHEV
EXCLUDED XXXXXXXXXXXXXXXXXXX X XXXXXXXX XXXXX X XX

Figure 1. Protein alignment of the N-terminal region of the 64 members of the Arf family
described in this chapter. A dash (-) indicates a gap introduced for alignment; white space
indicates that two sequences are incomplete. Numbers above indicate residues in the un-
edited hArf1 and indicate critical residues and/or simply regular spacing. Positions excluded
from the alignment used for phylogenetic analyses (Figure 2) are indicated below.
 HsArf1
1 26 47 71 101 126 160 178
MGNIFANGKKEMRILMVGLDAAGKTTILYKLKLGEIVTTIPTIGFNVETVEYKNISFTVWDVGGQDKIRPLWRHYFQNTQGLIFVVDSNDRERVNEAREELMRMLAEDELRDAVLLVFANKQDLPNAMNAAEITDKLGLHSLRHRNWYIQATCATSGDGLYEGLDWLSNQLR
8
MmArf2 ...V.EK..............................................................................................T................V........................Q...........................K
HsArf3 .....G.................................................................................................................................................................A...K
DmArf1 ...V.........................................................................................IG.....................I..........................N...........................K
CeArf1 ...V.GS..R....................................................................................G..............................Q......V..........N.S.........................K
SpArf1 ..LSISK..R..........................................R............................I...........IS..H...Q...N.......L...............................Q..................E...TN.K
HsArf4 ..LTISS...Q............................................C..........R.....K....................IQ.VAD..QK..LV.........L...........AIS.M......Q...N.T..V......Q.T...........E.S
HsArf5 ..LTVSA...Q............................................C......................................Q.SAD..QK..Q.................M....PVS.L......QH..S.T..V......Q.T...D......HE.S
DmArf2 ..LTISS...Q............................................C...................................D.IT..ER..QN..Q......................T...L....R.NQ..N.H.F..S....Q.H..........AE.A
CeArf3 ..LTISS..RQV..............................................................................K..IE.S....HK..N.......T..............T...L.......N..S.Q.........Q.H.............S
ScArf1 ..LFASK.N...........G.....V........VI.............Q...............R..S.....YR..E.V.........S.IG....VMQ...N.....N.AW..........E..S.....E......I.N.P.F.........E......E....S.K
ScArf2 ..LYASK.N...........G.....V........VI.............Q...............R..S.....YR..E.V...I.....S.IG....VMQ...N.....N..W..........E..S.....E......I.N.P.F..S......E......E....N.K
CeArf ..LF.SKPNI.C.T..L...G............N.T.N............TFQK.TL........K...A..KY..P..TT.V.....S.I..IP..K...FSL...P..A.SH.........M...RSP..L.QL.D.G..KN.E.F.CG.N.H..Q......M.VKK.MK
GlArf ..QGASKS...V...................M...V...V...............N..........S........Y...DA..Y.I..A.L..IED.KN..HTL.G.......A...........K..STTDL.ER...QE.KK.D....P...R......Q......DYIF
HsArf6 ..KVLSK.N.......L.................QS......V.......T...VK.N.................YTG.........CA..D.ID...Q..H.IINDR.M...II.I........D..KPH..QE....TRI.D....V.PS............T..TSNYK
DmArf3 ..KLLSK.N.......L.................QS......V.......T...VK.N.................YTG.........CA..D.ID...T..H.IINDR.M...II.I........D..KPH..QE....TRI.D....V.PS........S...I..TSNHK
CeArf6 ..KFLSK....L....L.................QS......V.......T....K.N.................YTG..A....M.AA..D..D...M..H.IINDR.MKE.II.........AD..KPH..Q.....TRI.D....V.PS..ST....H...T...QNCK
SpArf2 ...SLFKSN.......L................NQS.V....V.......T....K.N..................TG.K........A.SN.IS...Q..H.IISDR.M..CL...L.......G.LSP.Q...V.Q.DK.KD.L.NV.P...LT....L...A...QNAK
ScArf3 ...SISK.S...K...L...K............NK.K.ST..V.......T...VK.NM......QRL........PA.TA....I..SA.N.ME..K...YSIIG.K.MENV....W......KD..KPQ.VS.F.E.EN.KNQP.CVIGSN.L..Q..V...S.I..NTN
HsArl1 ..GF.SS.TR.....IL...G........R.QV..V..............T...LK.Q...L...TS...Y..C.YS..DAV.Y....C..D.IGISKS..VA..E.E...K.I.V.......MEQ..TSS.MANS...PA.KD.K.Q.FK.S..K.T..D.AME..VET.K
DmArl1 ..GVLSY.SR.....IL...G........R.QV..V............Q.T...LK.Q...L...TS...Y..C.YS..DAI.Y....A..D.IGISKD..LY..R.E..AG.I.V.L.....MDGC.TV..VHHA...EN.KN.TFQ.FK.S..K.E..DQAM.....T.Q
CeArl1 ..GVMSY.AR.....IL...G........R.QV..V............Q.....LK.Q...L...TS...Y..C.YA..DAI.Y....A..D..GIS.Q..AT..Q....QG...A.L.....IAGCLTET.VYKA...DA..N.TIQ.FK.S.SK.E..DPAM...A...Q
ScArl1 .....SS.S..L...IL...G........R.QI..V...K.........LS...LKLN...L...TS...Y..C.YAD.AAV......T.KD.MST.SK..HL..Q.E..Q..A.........Q.G.LS.S.VSKE.N.VE.KD.S.S.V.SS.IK.E.IT......IDVIK
HsArl5 ..IL.TRNHQ.HKVII....N........QFSMN.V.H.S....S...EIVIN.TR.LM..I...ESL.SS.NT.YT..EFV.V....T....ISVT....YK...HED..K.G..I......VKEC.TV...SQF.K.T.IKDHQ.H...C..LT.E..CQ..E.MMSR.K
HsArl5B ..L...KCNQ.HKVII....N........QFLMN.V.H.S....S...EIVV..TH.LM..I...ESL.SS.NT.YS..EFI.L....I....LAITK...Y....HED..K.AV.I......MKGC.T....SKY.T.S.IKDHP.H..SC..LT.E..CQ..E.MTSRIG
DmArl5 ..LLLSR.NE.HKLV.....N........QFLMN.V.H.S....S...E.VWR..H.L...L...QSL.AA.ST.YT..ELV.M.I..T....LAVT....Y...QHED.SK.S...Y......KGS.S....SRQ.D.T.IKKHQ.H...C..LT.E...Q..E.IVQRIK
CeArl5 ..L.M.KIG.KYK.IV....N........NYVTKDQ.E.K....S...E.S.R.LD.VI..I...ESL.KS.ST.YVQ.DVV.V.I..S.TT.IPIMK.Q.HN..QHED.AR.HI..L.......G...P..VSTQ...QT..A.K.Q.NGC..VK.E..P.A.E.IA.N.-
HsArl4 ...GLSDQS--FH.VIL...C.....V..R.QFN.F.N.V..K...T.KIKS.TVT.HF......E.L....KS.TRC.D.IV.....V.V..ME..KT..HKITRIS.NQGVPV.IV......R.SLSLS..EKL.AMGE.SSTP.HL.P...II....K...EK.HDMII
HsArl4B ...GLSDQS--FH.V.L...C.....V..R.QFN.F.N.V..KA..T.KIKS.TVT.HFG.....E.LM...KS.TRC.D.IL.LM..V.I..ME..KT..HKITRLS.NQGVPV.TV......E.SLSLSG.EKL.ATGE.SSTP.HL.P...II....K...EK.HDMII
HsArl7 ....SS-QS--LH.V.L...S.....V..R..FN.F.N.V......T.KIKA.G..CHF......E.L....KS.SRC.D.I.Y....V.VD.LE..KT..HKVTKFA.NQGTP...I.......KSLPV...EKQ.A..E.IATTYHV.PA..II.E..T..M.K.YEMIL
HsArl9 ...HLTEQA--LHVVVI...S....SL..R..FK.F.QSV..K...T.KIRSRG.T.Q.......E.L.....S.NRR.D..V....AAEA..LE..KV..H.ISRASDNQGVPV..L.....Q.G.LS...VEKR.AVRE.AATLTHV.GCS.VD.L..QQ..ER.YEMIL
DmArl4 ..ATMVKSQATLHVV.L...S.....A..R..FDQYLN.V......C.K.QA.GVH.L.......E.L.....S.TRC.D.IL..I..V.T..ME..KM....TAKCPDNQGVPV.IL.........CG.M.LEKL...NE.--.G....P...IT.E..Q....A.YDMIL
HsArl10 ..SLGSK--.QAQV.LL...S...S.L......AKDI...........MI.ER.L.L........E.M.TV.GC.CE..D..VY....T.KQ.LE.SQRQFEHI.KNEHIKNVPVVLL.....M.G.LT.ED..RMFKVKK.-D....V.PC..LT.E..AQ.FRK.TGFVK
HsArl11 ..SVNSR--A.AQVV.M...S.....L.....GHQL.E.L..V.....PLKPGHV.L.L......APL.AS.KD.LEG.DI.VY.L..T.EA.LP.SAA..TEV.NDPNMAGVPF..L....EA.D.LPLLK.RNR.S.ERFQDHC.ELRGCS.LT.E..P.A.QS.WSL.K
HsArl2 ..LLTILKER.L.L..L...N.......K.FNGED.D.IS..L...IK.L.HRGFKLNI......KSL.SY..N..ES.D...W....A..Q.MQDCQR..QSL.V.ER.AG.T..I........G.LSSNA.REA.E.D.I.SHH.C..GCS.VT.EN.LP.I...LDDIS
DmArl2 ..FLTVLKER.....LL...N.......KRFNGEP.D.IS..L...IK.L.HNGYTLNM......KSL.SY..N..ES.D..VW....A..M.LESCGQ..QVL.Q.ER.AG.T...LC......G.LSSN..KEI.H.EDI-THH.LVAGVS.VT.EK.LSSM...IADIA
CeArl2 ..FLKILRER.....IL...N.....LMK.FLDEPTD.IE..L..DIK..HF.DFQLNL......KSL.SY.KN..ES.DA..W....S....LLQCS...KKL.G.ER.AG.S...L...S...G.IDVNS.AQV.D...I-SHH.K.FSC..L...R.VQAMT..CDDVG
SpArl2 ..LLTILKER.V.V.LL...N.......KC.LNEDVNEVS..F..QIR.L.VEGLR..I..I...KTL.NF.KN..ES.EAI.W....L.DL.LE.C.NT.QEL.V.EK.LFTSI..L...S.VSG.LSSE..SKI.NISKYKSSH.R.FSVS.LT.LNIKDAIS..A.D.K
ScArl2 ..LLSIIRD..I.C.IL...NS..S..VN..LPKDEQGIM..V..QIHSLMI.DVTISL..I...RTL..F.DN..DK..AM.WCI.VSLSM.FD.TLQ..KELINR..N.ECAVI.VL..I..VEDKSELH-RRC.LVEE.KDIRIELVKCSGVT.E.ID----N.RDR.V
GlArl2 ..FLSVLSEN.A.V.V....CS.....VSS.LGKPLTEIA..L..KIS.WRPQ.T.MAL......QT..TY..N..SS.DA..W....T..R.MDTC.KA.SEV.HAER.QGCPVMILC....I.S.LSVE..SAAIT.DA.-N.K.AVLPCS.M.RQ..D.AFS..LSE.L
HsArl3 ..LLSILPDQ.V...LL...N.....L.KQ.ASED.SHIT..Q...IKS.Q-QGFKLN...I...R....Y.KN..E..DI..Y.I..A..K.FE.TGQ..AEL.E.EK.SCVPV.I.......LT.AP.S..AEG.N..TI.D.V.Q..SCS.LT.E.VQD.MN.VCKNVN
DmArl3 ..LLSLLPE..A...LL...N.......KQ.ASED.T.VT..A...IKS.A-DGFKLN...I...W....Y.KN..A..DV..Y.I.CT..T.LP..GS..FE..MDNR.KQVPV.I......M.D..S...VAE.MS.VQ.QG.T.E.K.CT.VD.T..K..M..VCKNMK
CeArl3 ..L.DVLSGR.I...LL...N.......KQ.SSEDVQHVT..K....K..AMGD.RLN...I...RS...Y.SN.YE.IDT....I....KK.FD.MNI..GEL.D.EK..KVPV.I.......VT.ASSE...R..N.DL..D.T.H...CS.LKNE.IND.IT.VASN.K
GlArl3 -------------------------...R..CEEDTSVIM..Q...AK.IKP.GV.LNC......KA..TY.QN.YAG.TC..W...AA.VK.MD.VAL.VSLA.EDCR.AGVPI.........AT.LKPSDLC..I..WA..D.V.H..GCS.LT.E..E..IL.MVSKTD
HsArl8A .IAL.NKW.E..ELTL...QYS....FVNVIAS.QFNDM...V...MRKITKG.VTIKL..I...PRF.SM.ER.CRGVSAIVYM..AA.Q.KIEASKN..HNL.DKPQ.QGIPV..LG..R...G.LDEK.LIE.MN.SAIQD.EICCYSISCKEK.NIDIT.Q..IQHSK
LOGSDON AND KAHN

HsArl8B .LALISRW.E..ELTL...QYS....FVNVIAS.QFSDM...V...MRK.TKG.VTIKI..I...PRF.SM.ER.CRGVNAIVYMI.AA...KIEAS.N..HNL.DKPQ.QGIPV..LG..R.....LDEKQLIE.MN.SAIQD.EICCYSISCKEK.NIDIT.Q..IQHSK

sequence (human Arf1), while other conventions are as in Figure 1.

DmArl8 .LALINRW.E..ELTL...QFS....FVNVIAS.QFADM...V...MRKITRG.VTIK...I...PRF.SM.ER.CRGVNAIVYM..AA.LDKLEAS.N..HSL.DKPQ.AGIPV..LG..R...G.LDETGLIERMN.S.IQD.EICCYSISCKEK.NIDIT.Q..IQHSK
CeArl8 .LAMVNKW.E..ELTL...QNS....FVNVIAS.QFTDM...V...MRKITKG.VTIKL..I...PRF.SM.ER.CRGVNAIV.M..AA.E.KLEAS.N...QL.DKPQ.DAIPV..LG..K...G.LDERQLIERMN.S.IQN.EICCYSISCKEKENIDIT.Q..IDHSK
GlArfrp ------------------------....HQMAY.MT-E....M..TLQ..KKGKLELD...I...SEF.NI.V..YVDKHAA.....AA.KA.ME...TA.EGV.TAP..SGVPI.IL.....IDG..SGDAVASM.D.QNVKDHEVKVVE.VG.T.K..DDA.K...TAIE
HsArfrp .YTLLSGQ.D.YC..IL...N.....F.EQS.TRFNKKITT.V.L.IG..DVGKARLMF..L...EELQS..DK.YAECH.V.Y.I..T.E..LA.SKQAFEKVVTSEA.CGVPV..L.....VETCLSIPD.KTAFSDSKIGR.DCLT..CS.LT.K.VR..IE.MVKCVV
DmArfrp .YTLMHGQ.DDYCVVIL...N.....Y.EAA.TTFTRKITT.V.L.IG.IDVQGVRLNF..L...QELQS..DK.Y.ESH.V.Y.I.......ME.SKAIFDK.IKNEL.SGVP..IL.......DV.GVR..KPVFQQALIGR.DCLTIPVS.LH.E.VD..IK..VEAIK
CeArfrp .YTLS.GK..DYYVVI....N.....F.EQT.SHFVKKITA.V.L.TGN..LN.TCLHF..L...ESL.E..AT.YDDANAM.....ATRSDLFPIVASQFKEVM.NEIVQNIPV..AV..SEMEG.AA...VRML.EDDNH.-SDLAVLPVS.LE.TNIERCVH.IVRS.A
ScArfrp .FHLVKGK.EQYS..IL...N.....F.ET..KEYSLKIQ..V.Q..A.IPVDSKQLKF......ESL.SM.SE.YSLCH.I..I...S....LD.CSTT.QSVVMDE.IEGVPI.ML.....RQDR.EVQD.KEVFNKEHISA.DSRVLPIS.LT.E.VKDAIE.MIVR.E
HsArl6 ..LLDRLK...VHV.CL...NS.....IN...PSNAQNIL.....SI.KFKSSSL....F.MS..GRY.N..E..YKEG.AI...I..S..L.MVV.K...DTL.NHPDIKRIPI.F....M..RD.VTSVKVSQL.C.ENIKDKP.H.C.SD.IK.E..Q..V...QD.IQ
DmArl6 ..MLHNLK.DK.T..VL..NNS..SS.INHF.KSSEQIVV..V..M..QFYMSGV.IKAI.MS.ATRY.N..E.Q.K.CH.I.Y.I..S..M.FVVVKD..DLV.QHPD.CIVPI.FYG..M.MEDSLSSVK.AAA.R.ENIKDKP.H.CSSS.I..E..G..VQ..IQ.M.
CeArl6 ..FFSSL...DVN.VV....NS......NQ..TP.TRQIV..V.HV.TNFSTQ.L..HAF.MA..M.Y.ST.ES..HSS..V...L..S..L.MELLKD...MVMEHKDVVGIPIVIL...M.I.G..T.SD..VA...NLY.SGT.S.HS...LT....DKAMQQ..AEIT
GlArl ..AASSRRH.TAKL.IL.RQFS..S.LINY..TNSFTVVN...N.TA.KYN----RYE.I....SAEMMQ..SEHYSG..TCL....GTS--PLDQ..DLIKDI.GHTDM.ASRFA.VVT.I.Q.GVSQK.DYYRT-T.QLDKD.VQEVFMF.GLD.Q.CDKIAQ..--DVP
HsSar1 .SF..EWY..SGKLVFL...N.....L.HM..DDRLGQHV..LHPTS.ELTIAGMT..TF.L..HEQA.RV.KN.LPAIN.IV.L..CA.HS.LV.SKV..NALMTDETISNVPI.ILG..I.RTD.ISEEKLREIF..YGQTA.PMEVFMCSVLKRQ.YG..FR...QYID
HsSara .SF..DWY..TGKLVFL...N.....L.HM..DDRLGQHV..LHPTS.ELTIAGMT..TF.L..HVQA.RV.KN.LPAIN.IV.L..CA.H..LL.SK...DSLMTDETIANVPI.ILG..I.R.E.ISEERLREMF..YGQTA.PLEVFMCSVLKRQ.YG..FR.MAQYID
CeSar1 .SFLWDWAN.KGKLVFL...N.....L.HM..DDR.AQHV..LHPTS.QMSLGG....TY.L..HAQA.RV.KD..PAVDAVV.LI.VA.A..MQ.S.V..ESL.QDEQIASVPV.ILG..I.K.G.LSEDQLKWQ.NIQHMCS.PMEVFMCSVLQRQ.YG..IR..GQY.-
DmSar1 .-F.WDWW..SGKL.FL...N.....L.HM..DDKLAQHV..LHPTS.ELSIG.MR..TF.L..HTQA.RV.KD..PAVDAIV.LI.AW..G.FQ.SKN..DSL.TDEA.SNCPV.ILG..I.K.G.ASED.LRNVF..YQ.TG.PLELFMCSVLKRQ.YG..FR..AQYID
ScSar1 .-AGWDIWN.HGKL.FL...N.....L.HM..NDRLA.LQ..WHPTS.ELAIG..K..TF.L..HIQA.R..KD..PEVN.IV.L..AA.P..FD...V..DALFNIA..K.VPFVILG..I.A...VSE..LRSA...LNTTQ.PVEVFMCSVVMRN.YL.AFQ...QYI-
SpSar1 .-F.INWVN.HAKM.FL...N.....L.HM..NDRLAVMQ..LHPTS.ELAIG.VR..TF.L..HQQA.R...D..PEVN.IVYL..CC.F..LS.SKA..DAL..ME..ARVPF.ILG..I.A.G.ISED.LKAA...YQTTI.PIEVFMCSVVLRQ.YG..FK..AQYV-
GlSar1 .-GFGDWY..KAT.VF....N...S.L.AM..NSATT.VA..QQPTSQELVMGS.R.KTF.L..HEVA.Q..EQ.VT.SD.IV.L...A.PS.FE.S.RT.QEL.DNHD.ATTPI.ILS..V.IQT.VSMETMVQSF.IQH.LQ.PLEVFPCSVINRF.YTD.FK...KY.-

analyses. Protein sequences were aligned and edited to remove non-conserved insertions.
Figure 2. Protein alignment of the 64 members of the Arf family used for the phylogenetic

Lines are shown to delineate between sub-families. A dot (.) indicates identity with the top
 The ARF Family Tree 9

Figure 3. Phylogenetic tree of Arf family homologs from six model eukaryotic organisms.
The consensus Bayesian maximum likelihood tree is shown. The tree is based on the
alignment of Arf/Arl protein sequences (64 sequences, 172 aligned amino-acid sites) shown
in Figure 2. The analysis used MrBayes 2.01 (Huelsenbeck and Ronquist, 2001), using a JTT
model with nine gamma-distributed rates for 80400 generations (including 20,000 for burn-
in) and sampling frequency of 1000 generations. The resulting parameter estimates (and their
95% confidence intervals) are: LnL –13164.546 (80.502), alpha 1.585 (0.0261). The numbers
on the branches are the consensus percentages (clade confidences) among the 785 “good”
(post burn-in) trees; bullets represent 99-100%. The scale bar represents amino acid
substitutions per site.
10 LOGSDON AND KAHN

This further demonstrates the limitations of functional data as the sole

criterion of classification and naming these genes/proteins. Results from
studies of mammalian Arf6 support the conclusions that class III Arfs share
a degree of functional overlap with class I/II Arfs but have clearly distinctive
features as well (see Chapters 14 and 15 for details).
Despite the fact that cows, mice, and rats all have six Arf paralogs, the
Arf2 gene appears to be missing from humans. Given the current state of
completeness of human genome sequencing, combined with numerous
attempts to isolate Arf2 from humans (both experimentally and using
bioinformatics), it may be time to conclude that Arf2 has been lost from the
human lineage. Because mammalian Arf1-3 protein sequences are identical
between mammals, all are >96% identical to one another, and are
biochemically indistinguishable, there is no obvious need for this
redundancy. However, recent results (K. Hill and R. A. Kahn; unpublished
observations) establish functional differences in human cells between Arf1
and Arf3 and are supportive of distinct functions for each Arf. It is also very
likely that functional differences exist between the class I and II Arfs but
these have only begun to be described.

2.2 The Arl sub-families

In addition to the three Arf classes, our analysis helps to delineate ten
orthologous groups that collectively form the Arls: Arls1-5, Arl8, Arl9,
Arfrp, and Sar1. Each of these groups is discussed below. For more details
on the biology of Arl proteins, see Chapter 16 by Schurmann and Joost.

2.2.1 Arl1

Single representatives of the Arl1 proteins were found in human, fly, worm,
and S. cerevisiae proteomes. Mammalian Arl1s have previously been shown
to have slightly higher percent identity (~55%) with Arfs than do other Arls
and to share some binding partners (Van Valkenburgh et al., 2001). The
phylogenetic placement of Arl1 as the closest to the Arfs is consistent with
the idea that they are close relatives, both functionally and evolutionarily.
The first of these Arls described was the fly arflike, which is essential in
flies (Tamkun et al., 1991). The lack of Arf activities and essential nature of
the fly Arl1 revealed for the first time that Arls perform essential functions
that can be clearly distinguished from those of Arfs. Arl1 has been localized
to Golgi membranes (Lowe et al., 1996) and expression of dominant activating
or inactivating mutants in mammalian cells results in changes in Golgi
morphology that are highly reminiscent of, but of lesser magnitude than,
those resulting from expression of analogous Arf mutants (Lu et al., 2001;
 The ARF Family Tree 11

Van Valkenburgh et al., 2001). Studies of S. cerevisiae ARL1 are also

consistent with the idea that it regulates aspects of vesicular traffic but
cannot substitute for Arfs (Lee et al., 1997; Rosenwald et al., 2002).
All Arl1 proteins contain a glycine at position 2 and have either been
shown to be N-myristoylated or are predicted to be so modified (Lee et al.,
1997; Randazzo and Kahn, 1995). A structure has been solved for ScArl1
that revealed differences in both the electrostatics and in the structure of the
N-terminal region when compared to Arfs (Amor et al., 2001), though the
biological significance of these observations is uncertain.

2.2.2 Arl2

Arl2 proteins were identified in all six organisms included in this study,
including Giardia. This argues for their early origin in eukaryotic evolution.
In mammals, S. cerevisiae (Arl2/CIN4), S. pombe (Arl2/alp41), C. elegans
(Arl2/evl-20), and A. thaliana (Arl2/TITAN5), there is genetic evidence
supporting roles for Arl2 in tubulin assembly and microtubule dynamics
(Antoshechkin and Han, 2002; Bhamidipati et al., 2000; Hoyt et al., 1997;
Hoyt et al., 1990; McElver et al., 2000; Radcliffe et al., 2000). Arl2
mutations result in cytokinesis defects and death in most of these studies,
though Arl2/CIN4 is not essential in S. cerevisiae. A model for Arl2 acting
in tubulin folding has been proposed by Bhamidipati, et al. (Bhamidipati et al.,
2000), based on the evidence for the direct binding of the tubulin-specific
chaperone, cofactor D, by Arl2•GDP.
Based on all of the phylogenetic analyses we have carried out to date, the
Arl2 and Arl3 proteins group strongly together, making it very likely that
they diverged from a common ancestor. Indeed, this must have been an
early event in eukaryotic evolution as Giardia has representatives of each
group. In addition, each of the yeasts in our study appears to have lost one
of the Arl2/Arl3 group proteins. This appears to have been Arl3, though the
long branch lengths (e.g., see Sc Arl2 in Figure 3) make this conclusion
somewhat tenuous. There is also biochemical support for the conclusion that
Arl2 and Arl3 are more closely related to each other than to other Arls. In
particular, guanine nucleotide exchange is much more rapid, proceeds to
higher stoichiometries in vitro, and is independent of lipids or detergents;
properties that are distinct from those of Arfs and Arl1 (Cavenagh et al.,
1994; Clark et al., 1993; Hillig et al., 2000; Sharer and Kahn, 1999).
Structural data explain these differences in divalent metal and nucleotide
binding by Arl2 and Arl3, compared to other members of the Arf family
(Hillig et al., 2000), and further support the conclusion that Arl2 and Arl3 are
more closely related to each other than they are to other Arls.
12 LOGSDON AND KAHN

Figure 4. Schematic phylogenetic tree depicting the relationships between the three Arf and
ten Arl groups. The presence of orthologs of each group in animals (A, in triangle), fungi (F,
in box), or protists (P, in circle) is indicated on the right. In two cases, a Giardia protein
could not be unambiguously assigned between groups so arrows to each are shown with a
question mark.

These biochemical properties allowed the isolation of a specific

Arl2•GTP binding partner (binder of Arl2, BART) that does not bind Arfs or
Arl1 (Sharer and Kahn, 1999). Arl2•GTP also binds the delta subunit of
phosphodiesterase 6, and its homolog HRG4, and has been crystallized as a
co-complex (Hanzal-Bayer et al., 2002; Kobayashi et al., 2003; Renault et
 The ARF Family Tree 13

al., 2001; Van Valkenburgh et al., 2001). Arl2 was localized to

mitochondria (Sharer et al., 2001), cytosol, and plasma membranes (J. Shern
and R. A. Kahn; unpublished observations), suggesting roles for Arl2 in
addition to tubulin assembly. A future challenge is to define how these
different binding partners and localization patterns relate to the role for Arl2
in tubulin dynamics or other cellular functions.
The most prevalent covalent modification to members of the Arf family,
N-myristoylation, appears to be absent in both Arl2 and Arl3 (Randazzo and
Kahn, 1995; Sharer et al., 2001; Van Valkenburgh and Kahn, 2002).
Because of the central role of the N-myristate in the rapidly reversible
association of Arfs with membranes, we infer that Arl2 and Arl3 lack this
feature as well. This is consistent with the findings that a subset of Arl2 is
imported into mitochondria (Sharer et al., 2001) and that the majority of Arl2
is in a high molecular weight, cytosolic complex (J. Shern and R. A. Kahn;
unpublished observation).

2.2.3 Arl3

The presence of orthologs of Arl3 in Giardia and the three animals suggest
that the two yeasts have lost the Arl3 gene. Whether this is the result of the
lack of a particular pathway or specific function (e.g., cell motility) in yeast
cannot be determined without more functional data on Arl3. Like Arl2, Arl3
probably is not N-myristoylated. Despite sharing a number of binding
partners (Van Valkenburgh et al., 2001) and biochemical features (Hillig et
al., 2000) with Arl2, Arl3 appears to lack the inferred functions of Arl2 in
tubulin assembly and in mitochondrial localization (C. Snelson, J. Shern, and
R. A. Kahn, unpublished observations).

2.2.4 Arl4

The Arl4 group is unusual in having four paralogs in human, a single

ortholog in the fly, and none in any of the other genomes searched. Arl4,
Arl4B, and Arl7 appear to have diverged from a common ancestor more
recently than the divergence of this family from flies, and may be more
closely related functionally as well. This is supported by the observations
that these three Arls all exchange guanine nucleotides more rapidly than
does Arf1 and all localize to the nucleus (Jacobs et al., 1999). Mouse
knockouts of Arl4 and Arfrp (see below) are the first reported deletions for
any Arf family members in mammals. The impact of those deletions on
development of the animal and other aspects of Arl4 biology are described in
detail in Chapter 16. These roles in mammalian development are consistent
with the recent divergence and expansion of the Arl4 genes.
14 LOGSDON AND KAHN

2.2.5 Arl5

Representatives of the Arl5 group were identified in all three animals, with
two closely related paralogs in humans. Wormbase
(http://www.wormbase.org/) reports no phenotype resulting from RNAi of
Ce Arl5, and there is no functional information available on any of these
genes or proteins to date.

2.2.6 Arl6

The Arl6 proteins were found in each of the animal proteomes and that of
Giardia, but in neither fungus. Each of the sequences result from searches
of genomic, cDNA, or EST databases and nothing is known about the
activities or functions in cells of the proteins. The fact that the Giardia Arl6
has three (corresponding to Arf1 residues Asp26, Gln71, and Asn126) and
Drosophila Arl6 has two (Asp26 and Gln71) deviations in sequence at
critical, and otherwise very highly conserved, positions leads us to predict
that novel means of controlling guanine nucleotide binding and hydrolysis
are used by the Arl6 proteins.

2.2.7 Arl8

The human Arl8 sequences were only very recently reported (Pasqualato et
al., 2002) and our analysis identified single orthologs in the fly and worm
proteomes. One of the six Arf family members we have identified in
Giardia groups with Arl8, but in other analyses groups with the Arfrp.
Indeed, a weak, but consistently observed relationship is recovered between
the Arl8 and Arfrp subgroups. No functional or expression data are
available for any Arl8. However, we note (e.g., see Figures 1 and 2) that
each of the Arl8 proteins (and two of the Arl6’s) are the only members of the
Arf family that deviate from the aspartate at residue 26 of Arf1, and that
corresponds to glycine 12 of Ras proteins. This residue is critical to the
hydrolysis of GTP and so its change in Arl8’s may indicate a distinctive
means of hydrolysis or interaction with its GAP(s).

2.2.8 Arl 10/11

Human Arl10 and Arl11 are described here for the first time as members of
the Arf family, and specifically as possible close relatives of the Arl4 group.
Arl10 and Arl11 are their own closest relatives; their inferred protein
sequences are ~57% identical, and they also share N-terminal spacing (see
Figures 1 and 2). Arl10, Arl11, and DmArl4 were all found from genome
 The ARF Family Tree 15

sequence analyses and so no information is yet available on their functions

or biochemical properties.

2.2.9 Arfrp

Human and rat orthologs of the Arf-related protein, Arfrp1, were first
described in 1995 by Schurmann, et al. (Schurmann et al., 1995). Orthologs
are present also in D. melanogaster, C. elegans, S. cerevisiae, and, possibly
G. lamblia. This gene appears to have been lost from S. pombe. The genetic
loci and predicted proteins in human, rat, and mouse have been assigned the
name Arfrp1, though the protein has previously been named Arp. Arfrp
proteins all lack the glycine at position 2 and therefore cannot be N-
myristoylated. Arfrps are also unique in that three of the five proteins in our
analysis deviated from the conserved residue, corresponding to proline 41 in
Arf1 (see Figure 1). This proline is critical to the formation of the extra β-
sheet found in Arfs, in comparison to Ras family proteins, and its change
may indicate a novel structure in this critical domain of the GTPase. The
gene has been knocked out in mice and results in embryonic lethality during
gastrulation (Mueller et al., 2002). A much more detailed description of this
and other functional aspects of Arfrp biology are presented in Chapter 16.

2.2.10 Sar1

The Sar proteins share the lowest percent identity to Arfs (e.g. HsArf1 vs.
HsSar1, ~29% identity) or Arls and for that reason are often not included in
descriptions of the Arf family. Alignments with other families of GTPases
(e.g., Ras, Rab, Rho) reveal that Sar proteins are clearly more closely related
to Arfs than these other GTPases. The strongest argument in favor of
including Sar proteins in the Arf family is functional. Sar1 is thought to play
a highly analogous role to those of class I Arfs in the recruitment of coat
proteins to budding vesicles (Barlowe et al., 1993; Kimura et al., 1995; Kuge
et al., 1994; Nakano and Muramatsu, 1989; Nishikawa and Nakano, 1991;
Oka et al., 1991). But while Arfs act at the Golgi/TGN, Sar1 acts at the ER.
Interestingly, the Sar1 proteins have been highly conserved throughout
eukaryotes and Sar1 orthologs are present in each of the six organisms in our
study. Like the Arl8 and Arfrp proteins, Sar1’s lack an N-terminal glycine
and therefore are not N-myristoylated. Additional information on Sar1 can
be found in Chapter 16.
16 LOGSDON AND KAHN

3. NOMENCLATURE ISSUES IN THE ARF/ARL

FAMILY

Because this chapter focuses on the evolutionary relationships between

highly conserved proteins, we have adopted a simpler system of naming the
Arf family members in the different species that emphasizes the family
relationships. Any time you deal with gene families in multiple species
today you are likely to encounter issues regarding how proteins and genes
are named or referred to by researchers and the databases. And while it is
important to maintain the evidence of priority in discovery that may result
from deposition into databases, it does not make sense to continue using
names that are misleading or incorrect. Hence, Table 1 is included to help
with the correct identification of the different Arf family members and
includes other names found in databases or the literature. In some cases,
simultaneous discovery of cDNAs in two or more labs resulted in more than
one name for the same cDNA or protein. For example, two different
cDNAs/proteins were identified and published in 1999 under the name Arl6,
as the database at that time had Arl1-5. Rather than change both to new
names, we made the minimal changes in names that would be consistent
with the numbering of Arl groups, as shown in Figures 3 and 4. We have
introduced new names in the following cases:
1) The protein previously termed Arf4L/Arl6 has been named Arl4B
because it is 60% identical to Arl4, nearly identical in length, and because
another protein has also been named Arl6. In addition, the name Arf4-like is
misleading, as it is not more closely related to Arf4 than any other Arf. The
use of the A, B, C suffix is based upon the precedent from Rab proteins and
Arl8s. Extending such a system could have resulted in Arl7 and Arl9 being
re-named Arl4C and D but we chose not to do so, to minimize the numbers
of changes proposed.
2) One of the human proteins named Arl8 in at least one database has
been renamed Arl5B (see Table 1) because it is 80% identical to human
Arl5, the same length, and to prevent confusion with two other proteins,
called Arl8A and B (Pasqualato et al., 2002).
3) Human Arl8A and B were so named because they are 92% identical,
to avoid confusion with Arl8/Arl5B, and because they have appeared in print
under these names (Pasqualato et al., 2002).
4) Human Arl10 and Arl11 were named as the next open number, after
discovering these predicted proteins in database searches. They have not
been named previously to our knowledge (J. Logsdon and R. A. Kahn,
manuscript in preparation).
5) Several other proteins are referred to in the figures in this chapter by
their orthologs, despite the fact that “accepted” names exist in public
 The ARF Family Tree 17

databases. Whenever possible, both names are included; e.g., Sc Arl2/Cin4,

Sp Arl2/alp41, Sc Arfrp/Arl3, and Ce Arl2/evl-20.

3.1 Evolution of the Arf family

These analyses have revealed some key evolutionary features of the Arf
family. It is important to note that this family is—so far—unique to
eukaryotes, not being found in either Bacteria or Archaea. In contrast, the
fact that many of the Arf family members are found in Giardia, a divergent,
and likely early-diverging, protist (Roger, 1999), emphasizes that the these
proteins are quite ancient features of eukaryotic cells. Our phylogenetic
analyses indicate that the ages of the various subfamilies are quite varied
(Figure 4). The oldest families, which contain animal, fungal, and Giardia
orthologs, include Arf I/II, Arl2 and Sar (and possibly Arfrp). Some
apparently middle-aged families include only animal and fungal members,
such as ArfI/II, ArfIII and Arl1. Young sub-families, which are restricted to
animals, include Arf I, ArfII, Arl 4 and Arl5. The youngest family,
Arf10/11, is restricted only to mammals. These latter groups likely represent
functions unique to animals, including developmental roles (as is known for
Arl4). In addition to estimating the relative ages of each of the subfamilies
by their phylogenetic distribution, we can also make inferences about the
likely loss of particular orthologs from particular subfamilies (assuming that
the genomes in which these genes are not found are indeed complete and/or
that the genes are not so divergent as to not be recognized). These losses
include Arl 1 in S. pombe, Arl4 in C. elegans and Arfrp in S. pombe. The
apparent absence of these orthologs in particular organisms may provide
important clues as to their conserved/dispensable functions in other species.
Finally, we must point out that the phylogenetic sampling used here, while
useful for generally delineating the relationships among these proteins, does
not encompass the breadth of eukaryotic diversity. Additional analyses
including more eukaryotes, notably more protist species and plants, will be
important to further refine the family tree of Arfs.

4. SUMMARY

From the sequencing of genomes we now have a more clear idea of the size
of the Arf family. Members of this family regulate a wide range of essential
cellular processes and they have been performing those functions since the
emergence of eukaryotes. With 23 members of the Arf family in mammals,
and similar numbers of Arf effectors, Arf GEFs, and Arf GAPs (identified to
18 LOGSDON AND KAHN

date) we also have a minimal number to place on the complexity in signaling

that these GTPases are involved in. Specificity in signaling is required but
the mechanisms by which Arf family GTPases achieve this specificity is
currently uncertain. We will continue to benefit from each of the model
genetic systems used in our analysis, as well as others, to decipher the roles
and mechanisms of Arf family GTPases in cell regulation.

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factor domain. J. Biol. Chem., 268, 8801-8807.
Mueller, A. G., Moser, M., Kluge, R., Leder, S., Blum, M., Buttner, R., Joost, H. G., and
Schurmann, A. (2002). Embryonic lethality caused by apoptosis during gastrulation in
mice lacking the gene of the ADP-ribosylation factor-related protein 1. Mol. Cell Biol., 22,
1488-1494.
Nakano, A., and Muramatsu, M. (1989). A novel GTP-binding protein, Sar1p, is involved in
transport from the endoplasmic reticulum to the Golgi apparatus. J. Cell Biol., 109, 2677-
2691.
Nishikawa, S., and Nakano, A. (1991). The GTP-binding Sar1 protein is localized to the early
compartment of the yeast secretory pathway. Biochim. Biophys. Acta, 1093, 135-143.
Oka, T., Nishikawa, S., and Nakano, A. (1991). Reconstitution of GTP-binding Sar1 protein
function in ER to Golgi transport. J. Cell Biol., 114, 671-679.
Pasqualato, S., Renault, L., and Cherfils, J. (2002). Arf, Arl, Arp, and Sar proteins: a family
of GTP-binding proteins with a structural device for front-back communication. EMBO
Rep.
Radcliffe, P. A., Vardy, L., and Toda, T. (2000). A conserved small GTP-binding protein
Alp41 is essential for the cofactor-dependent biogenesis of microtubules in fission yeast.
FEBS Lett., 468, 84-88.
Randazzo, P. A., and Kahn, R. A. (1995). Myristoylation and ADP-ribosylation factor
function. Methods Enzymo.l, 250, 394-405.
Renault, L., Hanzal-Bayer, M., and Hillig, R. C. (2001). Coexpression, copurification,
crystallization and preliminary X-ray analysis of a complex of ARL2-GTP and PDE delta.
Acta Crystallogr. D.Biol. Crystallogr., 57, 1167-1170.
Roger, A.J. (1999). Reconstructing Early Events in Eukaryotic Evolution. American
Naturalist, 154, S146-S163.
Rosenwald, A. G., Rhodes, M. A., Van Valkenburgh, H., Palanivel, V., Chapman, G., Boman,
A., Zhang, C. J., and Kahn, R. A. (2002). ARL1 and membrane traffic in Saccharomyces
cerevisiae. Yeast, 19, 1039-1056.
Schleifer, L. S., Kahn, R. A., Hanski, E., Northup, J. K., Sternweis, P. C., and Gilman, A. G.
(1982). Requirements for cholera toxin-dependent ADP-ribosylation of the purified
regulatory component of adenylate cyclase. J. Biol. Chem., 257, 20-23.
Schurmann, A., Massmann, S., and Joost, H. G. (1995). ARP is a plasma membrane-
associated Ras-related GTPase with remote similarity to the family of ADP-ribosylation
factors. J. Biol. Chem., 270, 30657-30663.
Sewell, J. L., and Kahn, R. A. (1988). Sequences of the bovine and yeast ADP-ribosylation
factor and comparison to other GTP-binding proteins. Proc. Natl. Acad. Sci., USA, 85,
4620-4624.
Sharer, J. D., and Kahn, R. A. (1999). The ARF-like 2 (ARL2)-binding protein, BART.
Purification, cloning, and initial characterization. J. Biol. Chem., 274, 27553-27561.
Sharer, J. D., Shern, J. F., Van Valkenburgh, H., Wallace, D. C., and Kahn, R. A. (2001).
ARL2 and BART enter mitochondria and bind the adenine nucleotide transporter (ANT).
Mol. Biol. Cell, 2002, 71-83.
Tamkun, J. W., Kahn, R. A., Kissinger, M., Brizuela, B. J., Rulka, C., Scott, M. P., and
Kennison, J. A. (1991). The arflike gene encodes an essential GTP-binding protein in
Drosophila. Proc. Natl. Acad. Sci., USA, 88, 3120-3124.
 The ARF Family Tree 21

Van Valkenburgh, H., Shern, J. F., Sharer, J. D., Zhu, X., and Kahn, R. A. (2001). ADP-
ribosylation factors (ARFs) and ARF-like 1 (ARL1) have both specific and shared
effectors: characterizing ARL1-binding proteins. J. Biol. Chem., 276, 22826-22837.
Van Valkenburgh, H. A., and Kahn, R. A. (2002). Coexpression of proteins with methionine
aminopeptidase and/or N-myristoyltransferase in Escherichia coli to increase acylation and
homogeneity of protein preparations. Methods Enzymol., 344, 186-193.
Vitale, N., Moss, J., and Vaughan, M. (1998). Molecular characterization of the GTPase-
activating domain of ADP- ribosylation factor domain protein 1 (ARD1). J. Biol. Chem.,
273, 2553-2560.
Chapter 2
THE GDP/GTP CYCLE OF ARF PROTEINS
Structural and Biochemical Aspects

SEBASTIANO PASQUALATO, LOUIS RENAULT, AND JACQUELINE

CHERFILS
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Gif sur Yvette Cedex, France

Abstract: The structural GDP/GTP cycle of Arf proteins is implemented by switch

regions that change their conformations in response to the nature of their
interactions. They include the classical switch regions in the nucleotide-
binding site, and a unique switch region located on the opposite side of the
protein. These switch regions communicate with each other and couple
activation of Arfs by GTP to their recruitment to membranes. Variations in
this cycle determine the specificity and differences in Arf1 and Arf6 signaling.
The role of guanine nucleotide exchange factors (GEFs) as key players in the
implementation of the nucleotide/membrane coupling is also described. A
particular emphasis is given to the inhibition of GEF-activated nucleotide
exchange by the drug Brefeldin A and by a GEF point mutation, as tools to
trap intermediate states of the reaction. Taken together, structural and
biochemical studies yield a comprehensive model for one of the most
remarkable nucleotide cycle found in G proteins.

1. INTRODUCTION

Arf proteins are involved in a large and still expanding number of cellular
processes, all related to their pivotal role as regulators of protein traffic and
membrane dynamics (reviewed in Chavrier and Goud, 1999). These
functions are operated by their ability to switch between an inactive GDP-
bound and an active GTP-bound form, each able to bind selectively to
distinct sets of cellular regulators, while effectors bind only to their GTP-
bound form. In this chapter we will focus on the biochemical and structural
implementation of the GDP/GTP switch, which departs from the classical
switch of other small GTP-binding proteins.
23
24
2 PASQUALATO, RENAULT AND CHERFILS

Activation of Arf proteins by GTP is coupled to their recruitment to

membranes. The membrane- and nucleotide-binding sites are located on
opposite sides, where they feature switch regions that change their
conformation in response to the nature of their interactions and communicate
with each other. In the nucleotide binding site, these regions are the
classical switch 1 and switch 2 regions found in all members of the G protein
superfamily (reviewed in Vetter and Wittinghofer, 2001), while a unique N-
terminal extension that carries a myristoyl lipid changes its conformation
upon binding to membranes. Coupling of the GDP/GTP cycle to membrane
interactions was first characterized by biochemical studies, which provided
evidence for the role of the N-terminal helix and its lipid modification
(Antonny et al., 1997; Franco et al., 1993, 1995; Kahn et al., 1992; Randazzo
et al., 1995). How communication is established between the N-terminal
helix and the nucleotide binding site, and the consequences for the activation
mechanism, were revealed later by structural studies (Amor et al., 1994;
Goldberg 1998; Greasley et al., 1995; Menetrey et al., 2000; Pasqualato et
al., 2001) and recently proposed to extend to other small GTP-binding
proteins homologous to Arf, including Arf-like and Sar proteins (Pasqualato
et al., 2002).
This chapter will first describe the structural aspects of the GDP/GTP
cycle and its biochemical characteristics, and how variations in the structural
cycle provide a basis to understand how the closely related Arf1 and Arf6
have specific, non-overlapping functions. It will then discuss the role of
guanine nucleotide exchange factors (GEFs) as key players in the
implementation of the nucleotide/membrane coupling. A particular
emphasis will be given to the inhibition of GEF-activated nucleotide
exchange by the drug brefeldin A (BFA) and by a GEF point mutation, as
tools to trap intermediate states of the reaction. Taken together, structural
and biochemical studies yield a comprehensive model for one of the most
remarkable nucleotide cycle found in G proteins.

2. THE MEMBRANE/NUCLEOTIDE CYCLE OF

ARF PROTEINS

Although many biochemical studies have preceded the report of the first
crystal structure of Arf in 1994 (Amor et al., 1994), we have chosen to
introduce this chapter by a description of the structural aspects of the
mechanism, as they provide a convenient background for subsequent
discussions. The structure of human Arf1 bound to GDP (Amor et al.,
1994), followed by that of rat Arf1•GDP (identical in sequence) (Greasley et
al., 1995) were first reported. Structure of an activated Arf was then
 THE GDP/GTP CYCLE OF ARF PROTEINS 25
3

reported for a truncated mutant of human Arf1 lacking the N-terminus

extension (Arf1∆17) (Goldberg, 1998), together with the nucleotide-free
complex of Arf1∆17 with the Sec7 domain of the yeast GEF, Gea2
(Goldberg, 1998). More recently, the GDP/GTP cycle of full-length human
Arf6 (Menetrey et al., 2000; Pasqualato et al., 2001) and the GDP-bound
form of yeast Arf2 (Amor et al., 2001) have also been solved, as well as a
complex of the GTPase activating protein (GAP) domain of rat Arf GAP1
bound to Arf1∆17•GDP (that is, the product of the GTPase reaction)
(Goldberg, 1999).
Structures of Arf proteins display several unique features with respect to
the common fold of small GTP-binding proteins (Amor et al., 1994; Amor et
al., 2001; Greasley et al., 1995; Menetrey et al., 2000). They diverge most
in their GDP-bound forms, with an original conformation of switch 1, a
register shift of strands β2-β3 (a region we have coined the interswitch
(Pasqualato et al., 2001)) and an N-terminal helix that is anchored opposite
to the nucleotide-binding site (Figure 1). By contrast, active forms of Arf,
either truncated (Goldberg, 1999) or not (Pasqualato et al., 2001) of the N-
terminal extension, adopt conformations that are similar to those of other
small GTP-binding proteins. These distinctive features are at the heart of a
communication device that operates between the N-terminal helical
extension and the nucleotide-binding site.

2.1 Switch 1 forms an extra β-strand in Arf•GDP

The switch 1 region is located downstream from the P-loop (a conserved

signature of GTP-binding proteins that they use to bind the phosphates of
guanine nucleotides). It carries an invariant threonine residue that binds the
Mg2+ ion and the γ-phosphate of GTP, thereby loading a ‘spring’ that
contributes to organize the active conformation (reviewed in Vetter and
Wittinghofer, 2001). Unlike Ras, Rho and Rab proteins, where switch 1
interacts with the GDP nucleotide, switch 1 of Arf•GDP is remote from the
nucleotide and does not interact with it (Amor et al., 1994; Amor et al.,
2001; Greasley et al., 1995; Menetrey et al., 2000). Instead, it forms an
additional β-strand with strand β3, thus extending the central β-sheet
(Figure 1). The extra β-strand disappears in the active form as a large
conformational change restores the classical interactions, including those of
the invariant threonine (T48 in human Arf1, T44 in human Arf6) (Goldberg,
1998; Pasqualato et al., 2001) (Figure 1). This movement articulates on
hinge glycines, located at each end of switch 1 (G40/G50 in human Arf1,
G36/G46 in human Arf6), which provide the required flexibility. There are,
so far, two small GTP-binding proteins whose switch 1 forms an extra β-
strand remote from GDP, the other one being Ran (Scheffzek et al., 1995).
26
4 PASQUALATO, RENAULT AND CHERFILS

In the GDP-bound form of both proteins, interactions that wedge the guanine
base, which are provided by an aromatic residue of the switch 1 in Ras, Rho,

Figure 1. The GDP/GTP cycle and the interswitch toggle of full-length human Arf6.
Regions that change their conformation are in dark gray. Switch 1 and 2 regions are defined
here based on the conformational changes between the GDP- and GTP-bound Arf structures,
and therefore differ from their classical definitions in Ras. It should be noted that as the
switch 1, interswitch and switch 2 regions form a continuous switch peptide, their junctions
are arbitrary. In Arf6, switch 1 comprises residues 36-46, the interswitch residues 47-64 and
switch 2 residues 65-80. The flexible N-terminus in Arf6•GTPγS is shown as a dashed line.
D63 from the DxGGQ motif, which blocks the γ-phosphate binding-site in the GDP-bound
form, and the invariant T44 from switch 1, are shown. Hinge glycine residues that allow the
flexibility of switch 1 are indicated by their Cα. The DxGGQxxxRxxW signature is also
shown. Protein Data Bank entries are 1E0S (Arf6•GDP) and 1HFV (Arf6•GTPγS).

and Rab families, originate from a residue from the β6-α5 loop (T161 in
Arf1, K152 in Ran). However, only in Arf is this interaction maintained
throughout the GDP/GTP cycle, whereas in active Ran it is replaced by an
aromatic interaction from the switch 1 (Vetter et al., 1999) equivalent to that
of Ras, Rho and Rab. A +1 shift of the invariant threonine within the
 THE GDP/GTP CYCLE OF ARF PROTEINS 27
5

sequence of the switch 1 in Arf may explain this difference (Pasqualato et

al., 2001).
Edge-to-edge interaction of the switch 1 β-strand yields a symmetrical
dimer in the crystal of Arf1•GDP (interface area of ū1050 Å2), which could
not form with Arf1•GTP because of the entirely different conformation of
switch 1. Arf6•GDP also forms a dimer that is built from a crystal
symmetry. Its interface is different from that of the Arf1•GDP dimer and
somewhat larger (ū1400 Å2), and it is not compatible with the GTP-bound
conformation. On the other hand, yeast Arf2•GDP and the active forms of
Arf do not form crystalline dimers. An interesting question is whether the
observation of crystalline dimers could be relevant to the formation of
equivalent dimers at the membrane in the cell. Hints for a membrane
homodimerization of Arf1 have been obtained by cross-linking in the
presence of Golgi membranes and GTPγS (Zhao et al., 1999). With respect
to the crystal structures, an Arf1 dimer formed under these conditions must
either be different from the crystalline dimer, or be an Arf1•GDP dimer
despite the presence of GTPγS. Also, activation of Arf proteins by their
GEFs involves the unfolding of the switch 1 β-strand (Goldberg, 1998),
suggesting that, if an Arf•GDP dimer exists at the membrane, it must
dissociate in order to be activated by the GEF.

2.2 The interswitch toggle

The myristoylated N-terminal extension of Arf proteins is necessary for its

cellular activity, and has early been proposed to act as an additional switch
(Antonny et al., 1997; Kahn et al., 1992; Randazzo et al., 1995). Crystal
structures of non-myristoylated Arf proteins have provided striking insight
into this issue. In Arf•GDP, the N-terminus forms a helix that lies in a
hydrophobic groove comprising residues from the interswitch loop (the loop
that connects strands β2 and β3) and the tip of switch 1 (Amor et al., 1994;
Amor et al., 2001; Greasley et al., 1995; Menetrey et al., 2000) (Figure 1).
The existence of this binding pocket is made possible by the retraction of the
interswitch in the protein core, where it forms β-strand interactions with
strand β1 on one edge, and with the extra β-strand of switch 1 on the other
one. As a consequence, residues of switch 2 that are involved in binding the
γ-phosphate of GTP are displaced out of register such that they are
incompatible with the binding of GTP. Furthermore, an invariant aspartate
at the interswitch-switch 2 junction (sequence DxGGQ, D67 in Arf1, D63 in
Arf6) is displaced so as to occlude the binding site for the γ-phosphate
(Pasqualato et al., 2002) (Figure 2).
These large differences, as compared to other small GTP-binding
proteins, vanish in structures of Arf proteins bound to slowly hydrolysable
28
6 PASQUALATO, RENAULT AND CHERFILS

Figure 2. Conformational changes of the nucleotide-binding site along the GDP/GTP cycle.
From top to bottom: Arf1•GDP (PDB entry 1HUR), nucleotide-free Arf1∆17-Gea2-Sec7
complex (gift from J. Goldberg), Arf1∆17•GDP(NH)P (gift from J. Goldberg). Hydrogen
bonds are shown as dotted lines. The carbonyls of A28 and A29 and the Cα of G69 and G70
are shown.
 THE GDP/GTP CYCLE OF ARF PROTEINS 29
7

GTP analogs, as a consequence of a large amplitude movement that affects

switch 1, the interswitch, switch 2, and the N-terminal helix (Figure 1).
These rearrangements were first observed in the structure of Arf1∆17 bound
to GDP(NH)P (Goldberg, 1998), and subsequently in full-length human
Arf6 bound to GTPγS (Pasqualato et al., 2001), the latter structure ruling out
an artifact of the N-terminal truncation. In these structures, the interswitch
unzips its interactions with its two neighboring β-strands, and reforms the
interswitch-β1 interaction with a two-residue register shift while switch 1
has moved away, reaching the GTP nucleotide. These concerted
conformational changes restore a classical conformation of switch 2 that is
then able to bind GTP. In particular, the aspartate from the DxGGQ motif is
back in register and interacts with the P-loop (T31 in Arf1) and indirectly
through a water molecule with Mg2+, both interactions found in other small
GTP-binding proteins. It also interacts with a conserved asparagine (N52 in
Arf1) whose mutation to arginine in Arf1 impairs activation of
phospholipase D (Skippen et al., 2002). The rearrangement of the DxGGQ
motif also positions the second glycine to bind the γ-phosphate of GTP, thus
providing, together with the invariant threonine from switch 1, the second
‘loaded spring’ of the classical GDP/GTP switch (Figure 2). On the
opposite side, the movement pushes the interswitch out of the protein core
by about 7Å, thereby destroying the hydrophobic binding site for the N-
terminal helix. In turn, in full-length Arf6 (the helix is truncated in the
Arf1∆17 structures) this helix is displaced into the solvent and not visible
because of its mobility in the crystal. Thus, the conformational changes of
the N-terminus, the interswitch and the switch regions are strongly coupled:
either the N-terminus interacts with the hydrophobic pocket and fastens the
retracted interswitch, hence a conformation of the switch regions that is
incompatible with the binding of GTP; or the N-terminus is displaced,
allowing the interswitch to be pushed out of the protein core and restoring
the ability of switch 1 and 2 to bind GTP.
Conformational changes that occur upon replacement of GDP by GTP in
Arf are the largest ever observed within the core G domain of a small GTP-
binding protein. They are all the more spectacular that they are grafted onto
a remarkably conserved architecture found in tens of GTP-binding proteins
which undergo a GDP/GTP conformational cycle but lack this extra
movement. As we will see below, these movements reflect a front-back
communication device that we have termed the “interswitch toggle”
(Pasqualato et al., 2002), coupling interactions of the N-terminus with
membranes with the GDP/GTP nucleotide status of the protein.
Comparison of the GDP/GTP cycle of Arfs to that of small GTP-binding
proteins that have no interswitch toggle reveals two potential determinants
for this structural mechanism (Pasqualato et al., 2002). One is the length of
30
8 PASQUALATO, RENAULT AND CHERFILS

the interswitch, which is shorter in Arf proteins than in other small GTP-
binding proteins, allowing it to be eclipsed in the protein core and form the
hydrophobic binding site for the N-terminal helix in the inactive form. The
second is a conserved sequence signature, wDvGGQxxxRxxW (residues 66-
78 in Arf1, 62-74 in Arf6) that spans the β3 strand of the interswitch and
switch 2. In Arf•GDP, the interswitch and switch 2 are mutually stabilized
by interaction of the tryptophan (W78 in Arf1) with surrounding aromatic
residues, including another tryptophan from the interswitch (W66 in Arf1).
Reorganization of this peptide upon binding of GTP creates a novel network

Figure 3. The GTP-bound conformation of the DxGGQxxxRxxW signature. The carbonyls

of G69 and G70 and the N of G70 are shown. The hydrogen bond network is shown as dotted
lines. This close-up view is from Arf1∆17•GDP(NH)P.

of hydrogen bonds involving the glycine pair, the arginine and the
tryptophan (G69, G70, R75 and W78 in Arf1) (Figure 3). This structural
transition is probably made possible by the flexibility provided by the
glycine pair, the first one being found only in the Arf family. The large
change in environment of the tryptophan accounts for the increase in
intrinsic fluorescence when GDP is exchanged for GTP (Kahn and Gilman,
1986), which provides a built-in reporter to monitor the kinetics of
spontaneous as well as GEF-catalyzed nucleotide exchange (Antonny et al.,
1997).
 THE GDP/GTP CYCLE OF ARF PROTEINS 31
9

2.3 Arf6 vs Arf1: a structural basis for their functional

specificity

We have focused so far on the features that are common to the GDP/GTP
cycles of Arf1 and Arf6, and likely to other Arf proteins as well. This
knowledge is essential to go a step further and identify the specific structural
features beyond the ‘air de famille’ that may explain why Arf1 and Arf6
have distinct cellular functions (reviewed in Chavrier and Goud, 1999, see
Chapter 14) despite having 67% sequence identity and very similar switch 1
and 2 regions. For instance, there is a single V/I change between Arf1 and
Arf6 in the binding interface with the Sec7 domain as established from the
nucleotide-free Arf1/Gea2-Sec7 complex (Goldberg, 1998). However, Arf6
has a family of specific exchange factors, termed EFA6, that are inactive on
Arf1 in vivo and in vitro (Franco et al., 1999; Macia et al., 2001). We have

Figure 4. Arf1 and Arf6 have different GDP-bound conformations. Arf6•GDP is shown in
light gray. Regions of Arf1•GDP that have significant conformational differences to
Arf6•GDP are superimposed in dark gray. The flexible switch 2 region in Arf1•GDP is also
shown.
32
10 PASQUALATO, RENAULT AND CHERFILS

investigated the structural basis for this specificity with the crystallographic
study of the GDP/GTP cycle of human Arf6 and its comparison to that of
Arf1 (Menetrey et al., 2000; Pasqualato et al., 2001).
While the sequence identity between Arf1 and Arf6 predicts that they
should be extremely similar, this study revealed unexpected differences
affecting their GDP-bound forms (Menetrey et al., 2000) (Figure 4). First, if
the N-terminus of Arf6 does form an amphipathic helix that fastens the
retracted interswitch despite being shorter by four residues, the structure
shows that it is the linker to the G protein core that is shortened and has a
different conformation. More surprisingly, switch 1 of Arf6, although
forming the extra β-sheet, is displaced by up to 3-4Å relative to Arf1, and its
switch 2 is less flexible than that of Arf1. Where do these differences come
from, given that Arf1 and Arf6 have almost identical switch regions?
Comparison of Arf6•GDP with Arf1•GDP identifies residues located either
on the remote ends of the switch regions, or in contact with the switch
regions, that are different between Arf1 and Arf6 and could account for their
biochemical and functional specificities. The contribution of these residues
was tested by mutational studies. The crystal structure of Arf6•GDP pointed
to a sequence difference in the vicinity of the nucleotide-binding site (I42 in
Arf1, S38 in Arf6), which positions a conserved glutamate (E54 in Arf1,
E50 in Arf6) in or out of the active site, respectively (Figure 5). Permutation
of these residues was sufficient to switch the kinetics of spontaneous
nucleotide dissociation between N-terminally truncated Arf1 and Arf6
(Menetrey et al., 2000), which is about 15 times faster in Arf6 than in Arf1
(Macia et al., 2001), thus supporting its proposed role in the nucleotide
binding site. A functional role for this residue is further supported by the
inability of the double Arf6-Q37I,S38I mutant to form the actin protrusions
upon treatment with aluminum fluorides in HeLa cells that are characteristic
of wild type Arf6 (Al-Awar et al., 2000). Other residues potentially
involved in stabilizing Arf6•GDP in a conformation different from that of
Arf1•GDP were introduced in Arf1 and tested for their activation by an
unspecific GEF, ARNO, and an Arf6-specific GEF, EFA6 (Macia et al.,
2001). This study showed that ARNO is very robust at activating these
various Arf1 mutants, while mutations targeting separately either switch 1,
the interswitch or switch 2 are not sufficient on their own to gain activation
by EFA6. Thus, although specificity is demonstrated to lie within the G
protein core of Arf and to be fully defined by the Sec7 domain of the GEF,
its structural implementation is likely to result from the combination of
subtle and localized effects. It should be noted that chimeras that paste
entire sub-regions of Arf1 and Arf6 together may, from that point of view,
induce structural effects that are larger than the differences between Arf1
and Arf6 that specify their interactions.
 THE GDP/GTP CYCLE OF ARF PROTEINS 33
11

On the other hand, the active structures of Arf1∆17 and Arf6 are
surprisingly similar (Pasqualato et al., 2001). In particular, the switch 1 and
switch 2 regions do not show the discrepancies of the inactive GDP-bound
forms. Altogether, the conformations of the switch regions fully identify the

Figure 5. A structural basis for the different nucleotide-binding properties of Arf1 and Arf6.
Replacement of S38 in Arf6 by I42 in Arf1 modifies the interactions of a conserved glutamate
residue in the nucleotide-binding site.

inactive from the active form of an individual Arf isoform, and their
different conformations in Arf1•GDP and Arf6•GDP can be discriminated
by partners of the inactive form, in particular their GEFs (Menetrey et al.,
2000). On the contrary, as the switch regions have the same conformation
and virtually identical sequences in the active forms of Arf1 and Arf6, they
are expected to be poor contributors to their specific interactions with
effectors (Pasqualato et al., 2001). As a consequence, specific interactions
established by activated Arf must originate from contributions other than the
sole recognition of the switch regions by protein partners. This can be
accounted for by several, non-exclusive hypotheses. One is that regions
other than the switch regions, which feature sequence differences between
Arf1 and Arf6, are necessary for establishing specific interactions. Another
is that specificity is initiated while Arf is still bound to GDP and
subsequently propagates to Arf•GTP-effector complexes. This could be
accomplished by the formation of transient Arf-GEF-effector complexes,
where the identity of the effector would be specified by interactions with
both Arf and the GEF (Pasqualato et al., 2001). Such a concept of
GEF/effector interactions is indeed emerging for another family of small
34
12 PASQUALATO, RENAULT AND CHERFILS

GTP-binding proteins involved in intracellular traffic, the Rab family

(reviewed in Zerial and McBride, 2001).

2.4 The coupled membrane-nucleotide switch

Full appreciation of the communication device in Arf comes from the

combination of these structural data with the biochemical studies, many of
which had been carried out before the structures were known. The first
biochemical studies on Arfs purified from bovine brain showed that they are
present in both the cytosol and membrane fractions (Kahn et al., 1988).
Subsequent experiments showed that binding of GTP to Arf is required for
its membrane association (Kahn, 1991; Walker et al., 1992) and vice versa
Arf1 cannot be activated in the absence of membranes (Randazzo et al.,
1995). This supported a model in which Arf activation, mediated by a
membrane-associated GEF, is concomitant with its recruitment to
membranes (Serafini et al., 1991).
Myristoylation of Arf proteins is necessary for their GTP-dependent
binding to membranes (Haun et al., 1993; Kahn et al., 1992). The relative
contributions of the myristate and the N-terminal helix in binding to
membranes were studied by Franco and co-workers by in vitro experiments
in the presence of vesicles (Franco et al., 1993, 1995). Unmyristoylated
(unmyr) Arf1•GTPγS, but not unmyr-Arf1•GDP, binds to phospholipid
vesicles or phospholipid-cholate micelles, and the interaction of
myristoylated (myr)-Arf1•GTPγS with membranes is stronger than that of
myr-Arf•GDP (Franco et al., 1993, 1995). These observations suggested
that Arf interacts with membranes through two components: the myristate,
that allows a basal interaction regardless of the nucleotide state; a protein
region, that becomes available for binding when Arf switches to the GTP-
bound form, which is the N-terminal helix that has been described in the
previous section (Antonny et al., 1997; Kahn et al., 1992; Randazzo et al.,
1995). This amphipathic N-terminal α-helix buries hydrophobic residues in
a pocket of the G protein core, thereby fastening the inactive conformation
in the absence of membrane (Amor et al., 1994; Greasley et al., 1995;
Menetrey et al., 2000). Mutation of these residues to alanine, which is
expected to lower the hydrophobic interactions, also decreased the affinity of
mutant Arf proteins for phospholipids (Antonny et al., 1997). Thus, in the
GTP-bound conformation, the N-terminal helix was predicted to insert its
hydrophobic residues’ side chains at the level of phospholipid acyl chains
(Antonny et al., 1997). This model is supported by NMR experiments that
showed that the N-terminal Arf1 peptide, either myristoylated or not, is
predominantly helical in a lipid environment, with the helix axis nearly
parallel to the bilayer surface (Losonczi and Prestegard, 1998; Losonczi et
 THE GDP/GTP CYCLE OF ARF PROTEINS 35
13

al., 2000). Moreover, myristoylation, while enhancing lipid binding, does

not affect peptide structure or behavior to a significant degree.
Altogether, biochemical and structural data are fully accounted for by a
model where the N-terminal amphipathic helix buries its hydrophobic face
either in a pocket of the G protein core in cytoplasmic Arf•GDP (the
conformation observed in crystal structures), or in interaction with the
membrane in Arf•GTP. Its conformation in Arf•GDP serves as a ‘hasp’ to
fasten the retracted conformation of the interswitch, and must be released to
allow nucleotide exchange. This is facilitated by interaction of the N-
terminal myristate with membranes, which occurs regardless of the nature of
the bound nucleotide. This interaction of the myristate with membranes
possibly also favors a dynamic equilibrium between the Arf- and membrane-
bound conformations of the N-terminus. After the N-terminal hasp is
unfastened, binding of GTP pushes and/or stabilizes the interswitch in the
conformation where it destroys the hydrophobic pocket, such that the N-
terminal helix is now stably bound to the membrane. An important
consequence is that kinetics of nucleotide exchange of Arf are correctly
measured only in the presence of membranes using myr-Arf, or in solution
using mutants truncated of their N-terminal ‘hasp’, while all other
combinations are likely to yield artifactual exchange rates. Therefore,
Arf1∆17 provides a convenient alternative to measure exchange activities in
solution (Antonny et al., 1997; Paris et al., 1997).

2.5 A different GTPase mechanism in Arf proteins?

Like other small GTP-binding proteins, the active state of Arfs is terminated
by hydrolysis of the bound GTP. However, spontaneous GTPase activity is
not measurable in Arf proteins (Kahn and Gilman, 1986), for which the
appellation of “GTPases” would therefore be illegitimate. Structural studies
allow us to address this difference with other small GTP-binding proteins,
which have intrinsic GTPase activity. Surprisingly, in the active forms, the
nucleotide binding site and GTP have the classical arrangement found in
other small GTP-binding proteins, including the localization of the catalytic
glutamine (DxGGQ motif, Q71 in Arf1) in the vicinity of the γ-phosphate of
GTP with which it could interact with only minor conformational changes
(Figure 4). Moreover, the wDvGGQxxxRxxW signature, which is also
found in the Gα subunit of heterotrimeric proteins, has the same
conformation in activated Arf1 and Arf6 and in the Gαi•GDP•AlF4 complex
(Coleman et al., 1994). The latter structure mimics the transition state of the
GTPase reaction. One essential difference, however, lies in the replacement
of G42 in the P-loop of Gα, by an aspartate (D26 in Arf1) (Figures 2 and 4).
Superposition of activated Gαi•GDP•AlF4 with structures of activated Arfs
36
14 PASQUALATO, RENAULT AND CHERFILS

suggests that the side chain of this aspartate may hinder the catalytic
glutamine from interacting with and stabilizing the leaving phosphate group.
This aspartate is in fact equivalent to the G12 position of Ras, whose
mutation to valine results in impairment in both spontaneous and GAP-
stimulated GTPase activity (Scheffzek et al., 1997). It is as yet unclear how
the GAP releases this unfavorable contact in Arf, but this observation opens
the possibility that the contribution of the catalytic glutamine may differ
from other small GTP-binding proteins. The crystal structure of the complex
of Arf1∆17•GDP with the catalytic domain of rat Arf GAP1 does not
provide insight into this issue, as the complex features an interface that is
remote from the nucleotide binding site (Goldberg, 1999). This study
proposes that the actual GAP is composed of the GAP domain and the
coatomer, which may provide the interactions that are missing in the GTP
binding site (Goldberg, 1999). At the moment this question is the subject of
an on-going debate as to the existence or not of a classical GAP mechanism
involving the complementation of the active site with an ‘arginine finger’
provided by the Arf GAP (Click et al., 2002; Goldberg, 1999; Szafer et al.,
2000; Szafer et al., 2001).

3. ROLE OF GEFS IN COUPLING THE

MEMBRANE AND NUCLEOTIDE SWITCHES

Activation of Arf by their guanine nucleotide exchange factors, which are

characterized by a conserved Sec7 domain of §200 amino acids, is the
subject of several chapters in this book (see Chapters 4-6). Here we will
focus on the catalytic mechanism of Sec7-domain-stimulated nucleotide
exchange and how GEF activity is grafted onto the basic
membrane/nucleotide switch, emphasizing the role of membranes in this
process. Further insight into this mechanism was gained from use of a GEF
point mutant and BFA as tools to trap otherwise transient steps of the
reaction. A comprehensive model for the GDP/GTP cycle can be formalized
from this ensemble of studies.

3.1 The nucleotide-free Arf-Sec7 complex and the

glutamic finger

Crystal structures of the Sec7 domain of human ARNO (ARNO-Sec7)

(Cherfils et al., 1998; Mossessova et al., 1998) and the NMR structure of
cytohesin1-Sec7 (Betz et al., 1998) have shown that this domain is a
superhelix of helices, which form a hydrophobic groove lined with
conserved amino acids, on the rim of which stands an invariant glutamate
 THE GDP/GTP CYCLE OF ARF PROTEINS 37
15

(the ‘glutamic finger’, E156 in ARNO) (see Figure 6). Site-directed

mutagenesis identified this groove as the binding site for Arf (Betz et al.,
1998; Cherfils et al., 1998; Mossessova et al., 1998). The functional
importance of the invariant glutamate was first demonstrated in a protein
from A. thaliana, EMB30 (also known as GNOM and recently demonstrated
to be an Arf GEF (Steinmann et al., 1999)), where its mutation to lysine
resulted in various defects in embryogenesis (Shevell et al., 1994). Mutation
of the glutamic finger to lysine in the Sec7 domain of ARNO (ARNO-K156)
produces the most severe decrease in catalysis of nucleotide exchange as
compared to other mutations within the active site (Beraud-Dufour et al.,
1998; Cherfils et al., 1998). Despite its crucial catalytic defect, ARNO-
K156 forms an abortive complex with Arf1∆17•GDP at low Mg2+
concentration (Beraud-Dufour et al., 1998). No abortive complex is formed
with ARNO-D156, suggesting that the positive charge of the lysine mutant
mimics Mg2+ in the nucleotide binding site of Arf1∆17•GDP (Beraud-
Dufour et al., 1998). From these studies it was predicted that the ‘glutamic
finger’ would act in the vicinity of Mg2+ and the phosphates of the
nucleotide, where its negative charges may, in a first step, destabilize the
phosphates of GDP by electrostatic repulsion, before they mimic the β-
phosphate to stabilize the empty nucleotide-binding site (Beraud-Dufour et
al., 1998). On the other hand, the surface of Arf interacting with the Sec7
groove was mapped to the switch 1 and 2 regions by mutagenesis (Beraud-
Dufour et al., 1998) and hydroxyl-radical footprinting (Mossessova et al.,
1998). These data were combined by docking simulation to predict the
structure of the nucleotide-free complex before its crystal structure was
solved (Beraud-Dufour et al., 1998). Unambiguous orientation was achieved
by charge permutation between ARNO-Sec7-D183 and K73 in switch 2 of
Arf. Individually, each mutant failed to support catalyzed nucleotide
exchange, but significant activity was restored when they were assayed
together, suggesting that these two residues would form a salt bridge in the
complex (Beraud-Dufour et al., 1998). Taken together, these data provided
sufficient information to dock switch 1 of Arf1•GDP into the hydrophobic
groove of ARNO-Sec7, with the nucleotide-binding site open to the solvent.
These ‘low resolution’ predictions were essentially confirmed by the
crystal structure of the nucleotide-free complex of human Arf1∆17 with the
Sec7 domain of yeast Gea2 (Goldberg, 1998) (Figure 6). In this complex,
the glutamic finger binds to the invariant lysine of the P-loop (which is a
ligand of the β- and γ- phosphates of guanine nucleotides) (Figure 2). It is
interesting to note that electrostatic stabilization of the empty nucleotide
binding site by a negatively charged residue has turned out to be a general
feature of the GEFs of the small GTP-binding proteins (Renault et al., 2001).
Unexpectedly though, the ‘glutamic finger’ is contributed by the GEF only
38
16 PASQUALATO, RENAULT AND CHERFILS

in the case of the Sec7 domain, while in other systems it is provided by the
G protein itself. In addition, the complex revealed that the P-loop is
distorted such that carbonyls of its main chain (A27 and A28) would conflict
with binding of the α- and β-phosphates of a nucleotide

Figure 6. The nucleotide-free Arf∆17-Gea2-Sec7 complex. Coordinates of the complex

were a gift from J. Goldberg.

(Figure 2). Although this may be a simple consequence of the absence of

nucleotide (Goldberg, 1998), this conformation is stabilized by an elaborate
network of hydrogen bonds supported by D26 in the P-loop (Figure 2),
suggesting that residue may be critical for the alternative conformations of
the P-loop (Pasqualato et al., 2001).
Besides these aspects, the most spectacular and unpredictable finding
from the nucleotide-free Arf1∆17-Gea2 complex was that nucleotide-free
Arf has already undertaken the interswitch toggle that is observed in
activated Arf•GTP (Goldberg, 1998). This finding has been pivotal in
formalizing the model for the membrane/nucleotide switch and its regulation
by exchange factors as will be discussed below.
 THE GDP/GTP CYCLE OF ARF PROTEINS 39
17

3.2 Regulation of Arf GEF activity by membranes

In the nucleotide-free complex, the interswitch is pushed out of the protein

core as in activated Arf, thus destroying the hydrophobic pocket that forms
the binding site of the N-terminal helix in Arf•GDP (Goldberg, 1998). This
is made possible in the crystal by the use of the truncated Arf1∆17 mutant,
but in the cell this implies that the N-terminal hasp must be displaced by
membranes to permit Sec7-catalysed nucleotide exchange. The role of
membranes in assisting the mechanism of nucleotide exchange had been
shown for a partially purified Arf GEF (Franco et al., 1996) and later for
ARNO, which is inactive on full-length myr-Arf1 in the absence of
membranes (Beraud-Dufour et al., 1999) and for its Sec7 domain under
various experimental set-ups (Paris et al., 1997). In particular, the Sec7
domain of ARNO was shown to activate Arf1∆17•GDP in the absence of
phospholipids (Paris et al., 1997). On the contrary, the Sec7 domain of
ARNO did not readily activate the equally soluble unmyr-Arf1•GDP in
solution. Thus, activation of exchange stimulated by Sec7 domains occurs
either in solution with the truncated Arf mutant, where the interswitch is not
fastened by the N-terminus, or in the presence of lipids if myr-Arf is used
instead, in which case membranes are required to displace the N-terminal
hasp. Other conditions used in exchange assays yield artifactual exchange
rates.
This role of membranes in Sec7-stimulated nucleotide exchange reflects
the general requirement of Arfs for the unfastening of the N-terminal hasp in
order to create a binding site for GTP. On the other hand, the Sec7 domain
is found in multidomain proteins with different architectures, including two
families, the ARNO/cytohesin and EFA6 families, that carry phospholipid-
interacting pleckstrin homology (PH) domains and a novel Arf GEF, p100,
with a PH domain yet to be characterized as such (Someya et al., 2001).
Phospholipids stimulate the nucleotide exchange activity of GEFs of the
ARNO/cytohesin family by interacting with their PH domains, hence
recruiting the GEF at the membrane where it finds its substrate (see also
Chapters 4-6). For instance, nucleotide exchange activity stimulated by
ARNO is enhanced when increasing amounts of phosphatidylinositol 4,5-
bisphosphate (PI(4,5)P2) are added to phospholipid vesicles (Chardin et al.,
1996). More detailed studies on the role of PI(4,5)P2 demonstrated that
binding of the PH domain to PI(4,5)P2-containing vesicles does not affect
the catalytic activity of ARNO, but simply promotes membrane recruitment
of the exchange factor (Paris et al., 1997). Furthermore, this effect is
combined in ARNO with interaction of a polybasic C-terminus with
membranes (Macia et al., 2000). It should be noted that these latter
experiments were done with the triglycine splice variant of ARNO (S. Paris,
40
18 PASQUALATO, RENAULT AND CHERFILS

personal communication). This variant carries three consecutive glycines in

the PH domain and has a high affinity for PI(4,5)P2 and low discrimination
between PI(4,5)P2 and PI(3,4,5)P3, unlike the diglycine variant (reviewed in
Cullen and Chardin, 2000). It is not known yet how other families of large
Arf GEFs interact with membranes, but it is likely that uncharacterized non-
catalytic domains fulfill such a function, either directly or through binding to
a membrane-located partner. Thus, membranes have two essentially
independent functions: as a co-factor of the Sec7 domain that acts directly on
Arf and unfastens its N-terminal hasp; and in recruiting the exchange factor
by domains other than the catalytic Sec7 domain, thereby contributing to
localizing the GEF at the compartment where Arf is to be activated.

3.3 The intermediate steps of the exchange reaction

studied by inhibition by BFA and by the glutamic
finger mutation

The basic GEF-catalyzed exchange reaction common to all small GTP-

binding proteins involves an initial ternary complex comprising GDP and the
GEF. This complex isomerizes to yield the high affinity nucleotide-free
complex (reviewed in Cherfils and Chardin, 1999). The ternary complexes
are usually difficult to study because their affinity is low. In Arf, the
cytosol/membrane switch superimposes onto that scheme, but luckily the
ternary complex can be trapped under two circumstances. One is found in
nature in the form of the fungal metabolite BFA, which has been widely
used over the years as a powerful inhibitor of Golgi traffic (reviewed in
Klausner et al., 1992). The other one is the above-mentioned mutation of the
glutamic finger to a lysine. Both interferences yield complexes with distinct
partitioning to membranes, which further refine our understanding of the
discrete steps of the exchange reaction.
Investigation of the biochemical mechanism of action of BFA has been
made possible by the identification of the amino acids that determine the
sensitivity to BFA in yeast Arf GEFs using random mutagenesis, which were
then introduced in the otherwise BFA-insensitive ARNO-Sec7 (Peyroche et
al., 1999). Inhibition by BFA increases with the addition of Arf1•GDP,
ruling out a competition between Arf and the drug for binding the Sec7
domain. Using gel filtration experiments and radiolabeled BFA and
nucleotides, the target of BFA was shown to be the complex between
Arf•GDP and the Sec7 domain, which it traps in an abortive complex
(Peyroche et al., 1999; Robineau et al., 2000). BFA efficiently traps both
Arf1∆17 and myr-Arf1•GDP, and in the latter case the trapped complex has
a low affinity for membranes comparable to that of myr-Arf1•GDP (Beraud-
Dufour et al., 1999; Robineau et al., 2000). Thus, BFA is proposed to act as
 THE GDP/GTP CYCLE OF ARF PROTEINS 41
19

a double-face adhesive tape, gluing the Sec7 domain to Arf•GDP which has
not yet undertaken the interswitch toggle (Beraud-Dufour et al., 1999).
These studies also suggest that BFA has no direct effect on the
membrane/cytosol partitioning of the trapped Sec7-Arf•GDP complex. This
non-competitive mechanism was also proposed for members of the
p200/BIG family of Arf GEFs, which are likely cellular targets of BFA in
mammalian cells (Mansour et al., 1999). It should be noted that this non-
competitive inhibition mechanism is all the more remarkable in that it targets
a low-affinity complex, yet it is very efficient in the cell (Peyroche et al.,
1999; reviewed in Chardin and McCormick, 1999). It is also very specific,
as it does not target Arf6 in vitro (our unpublished data) nor in vivo (Peters
et al., 1995), and Arf1 is a target for BFA only with certain exchange factors.
These observations make BFA a model inhibitor for GEF-catalyzed
activation of small GTP-binding proteins. Differences in the structures and
flexibility of Arf1•GDP and Arf6•GDP (Menetrey et al., 2000), and in the
flexibility of the Sec7 domains at the residues that determine BFA-
sensitivity (Renault et al., 2002) may contribute to this specificity.
In contrast, the complex of ARNO-K156-Sec7 with Arf•GDP forms only
at low Mg2+ concentration (µM), either in solution with Arf1∆17 or in the
presence of membranes with myr-Arf1, in which case its affinity for
membranes is comparable to that of the nucleotide-free complex (Beraud-
Dufour et al., 1999; Beraud-Dufour et al., 1998). Thus, the E156K mutation
probably traps still another discrete step of the reaction, where the N-
terminal hasp binds to membranes where it is stabilized by the interswitch
toggle, while only the final nucleotide dissociation step is inhibited.
Mutation of the glutamic finger does not interfere with the formation of the
BFA complex (Robineau et al., 2000), reinforcing the idea that the glutamic
finger is not involved until late in the course of the exchange reaction. The
mechanism of the Sec7 domain can thus be separated in (at least) two steps:
a docking step where the Sec7 domain binds to Arf•GDP; and a catalytic
step where the glutamate has a crucial role in dissociating the GDP
nucleotide. Comparison of yeast Gea2-Sec7 in complex with nucleotide-free
human Arf1∆17 (Goldberg, 1998) with its unbound structure (Renault et al.,
2002) shows that a subdomain motion closes the hydrophobic groove upon
binding to Arf, suggesting that Arf•GDP may bind onto the side of the
groove opposite to the glutamic finger at the docking step, while closure of
Sec7 subdomains brings the glutamic finger into the active site at the
catalytic step (Renault et al., 2002).
42
20 PASQUALATO, RENAULT AND CHERFILS

3.4 A comprehensive model for biochemical and

structural front-back communication

Biochemical and structural data together can be formalized into a

remarkably consistent and detailed model for the activation reaction of Arfs
by their GEFs, which also provides valuable insight into the mechanisms
that take place in the cell. This model is summarized in Figure 7.
1) Myr-Arf•GDP is mainly cytosolic, but it can associate weakly with
membranes by its N-terminal myristate. In its cytosolic form, Arf•GDP has
a retracted interswitch that is fastened by the amphipathic N-terminal helix.
The small fraction of membrane-bound Arf•GDP is probably in equilibrium
between a form where only the myristate is inserted in the membrane, and a
form where both the myristate and the N-terminal helix interact with the
membrane. In either case, the interswitch remains retracted, such that the
hydrophobic pocket for the N-terminal hasp is available for reversible
unbinding. The retracted interswitch prevents the binding of GTP by
inserting an aspartate in the γ-phosphate binding-site.
2) The Arf GEF is recruited in the vicinity of a membrane by PH
domains or by yet to be identified domains, where it encounters myr-
Arf•GDP. It acts either on the fraction of Arf•GDP where the hasp has
already been unfastened, or promotes the unfastening of the N-terminal hasp,
which is facilitated by its proximity to membranes; two possibilities that are
currently not resolved. In solution, interaction of myr-Arf•GDP with the
Sec7 domain of soluble Arf GEFs is also likely to occur, but it is
unproductive and dissociates readily. The glutamic finger is not involved at
that early docking stage, which, together with the localization of residues
that determine the sensitivity to BFA, suggests that the docking site of myr-
Arf•GDP onto Sec7 domains is remote from the nucleotide-binding site,
possibly centered on the switch 2 region of Arf and the C-terminal
subdomain of the Sec7 domain. This step is the target of BFA in the cell.
3) The Sec7 domain then induces the interswitch toggle, which destroys
the binding pocket for the N-terminal hasp and thereby anchors Arf to
membranes. However, this complex is still able to bind GDP, indicating that
conformational changes such as the distortion of the P-loop may not have
taken place yet. Mutation of the glutamic finger traps this intermediate
complex.
4) Closure of the subdomains of the Sec7 domain, as well as changes in
the switch regions and possibly the orientation of Arf, eventually yield a
large protein-protein interface and position the glutamic finger into the
active site where it distorts the P-loop and dissociates the GDP phosphates
by charge repulsion. The glutamic finger then proceeds to stabilize the
 THE GDP/GTP CYCLE OF ARF PROTEINS 43
21

nucleotide-binding site by mimicking the interactions of the β-phosphate

with the P-loop.

Figure 7. The model for the nucleotide/membrane switch. See text for details of steps 1 to 5.
Gua=guanine base, myr=myristate, E=glutamic finger, D=aspartate from the DxGGQ motif,
Sec7Nt= Sec7 N-terminal domain; Sec7Ct= Sec7 C-terminal domain.
44
22 PASQUALATO, RENAULT AND CHERFILS

5) Finally, entry of GTP, binding first by the guanine base whose binding
site is readily accessible in the nucleotide-free complex, dissociates the GEF
and provides alternative interactions that stabilize the active conformation of
the switch/interswitch machinery. As the interswitch is now firmly
stabilized, the interaction of the N-terminal hasp with membranes becomes
irreversible as long as Arf is bound to GTP.
The model and results discussed so far argue clearly for the need of both
the guanine nucleotide exchange factor and the membrane to perform an
efficient activation of Arf proteins. Notably, both membranes and the Sec7
domain induce conformational changes that drive the activation process. For
these reasons, catalyzed nucleotide exchange towards Arf is composed of
two factors acting in concert, but mostly inactive on their own: a classical
GEF that changes the conformation of the switch regions and builds up the
GTP-binding site, and a membrane co-factor unfastening the N-terminal
hasp. We have recently proposed that this mechanism of front-back
communication by the interswitch toggle is also found in most Arf-like
proteins as well as in Sar1, suggesting that these proteins will also be
activated by a “dual” exchange factor (Pasqualato et al., 2002).

4. ACKNOWLEDGEMENTS

We thank Sonia Paris (CNRS, Valbonne) for critical reading of the

manuscript. This work was supported by grants from the Association pour
la Recherche contre le Cancer, and from the CNRS, INSERM and French
Research Ministry.

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characterization of the 21-kDa ADP-ribosylation factor of adenylate cyclase. J. Biol.
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Kahn, R. A., Randazzo, P., Serafini, T., Weiss, O., Rulka, C., Clark, J., Amherdt, M., Roller,
P., Orci, L., and Rothman, J. E. (1992). The amino terminus of ADP-ribosylation factor
(ARF) is a critical determinant of ARF activities and is a potent and specific inhibitor of
protein transport. J. Biol. Chem., 267, 13039-13046.
Klausner, R. D., Donaldson, J. G., and Lippincott-Schwartz, J. (1992). Brefeldin A: insights
into the control of membrane traffic and organelle structure. J. Cell Biol., 116, 1071-1080.
Losonczi, J. A., and Prestegard, J. H. (1998). Nuclear magnetic resonance characterization of
the myristoylated, N-terminal fragment of ADP-ribosylation factor 1 in a magnetically
oriented membrane array. Biochemistry, 37, 706-716.
Losonczi, J. A., Tian, F., and Prestegard, J. H. (2000). Nuclear magnetic resonance studies of
the N-terminal fragment of adenosine diphosphate ribosylation factor 1 in micelles and
bicelles: influence of N-myristoylation. Biochemistry, 39, 3804-3816.
Macia, E., Chabre, M., and Franco, M. (2001). Specificities for the small G proteins ARF1
and ARF6 of the guanine nucleotide exchange factors ARNO and EFA6. J. Biol. Chem.,
276, 24925-24930.
Macia, E., Paris, S., and Chabre, M. (2000). Binding of the PH and polybasic C-terminal
domains of ARNO to phosphoinositides and to acidic lipids. Biochemistry, 39, 5893-5901.
Mansour, S. J., Skaug, J., Zhao, X. H., Giordano, J., Scherer, S. W., and Melancon, P. (1999).
p200 ARF-GEP1: a Golgi-localized guanine nucleotide exchange protein whose Sec7
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Menetrey, J., Macia, E., Pasqualato, S., Franco, M., and Cherfils, J. (2000). Structure of Arf6-
GDP suggests a basis for guanine nucleotide exchange factors specificity. Nat. Struct.
Biol., 7, 466-469.
Mossessova, E., Gulbis, J. M., and Goldberg, J. (1998). Structure of the guanine nucleotide
exchange factor Sec7 domain of human arno and analysis of the interaction with ARF
GTPase. Cell, 92, 415-423.
Paris, S., Beraud-Dufour, S., Robineau, S., Bigay, J., Antonny, B., Chabre, M., and Chardin,
P. (1997). Role of protein-phospholipid interactions in the activation of ARF1 by the
guanine nucleotide exchange factor Arno. J. Biol. Chem., 272, 22221-22226.
Pasqualato, S., Menetrey, J., Franco, M., and Cherfils, J. (2001). The structural GDP/GTP
cycle of human Arf6. EMBO Rep., 2, 234-238.
Pasqualato, S., Renault, L., and Cherfils, J. (2002). Arf, Arl, Arp and Sar proteins: a family of
GTP-binding proteins with a structural device for front-back communication. EMBO Rep.,
3, 1035-1041.
Peters, P. J., Hsu, V. W., Ooi, C. E., Finazzi, D., Teal, S. B., Oorschot, V., Donaldson, J. G.,
and Klausner, R. D. (1995). Overexpression of wild-type and mutant ARF1 and ARF6:
distinct perturbations of nonoverlapping membrane compartments. J. Cell. Biol., 128,
1003-1017.
Peyroche, A., Antonny, B., Robineau, S., Acker, J., Cherfils, J., and Jackson, C. L. (1999).
Brefeldin A acts to stabilize an abortive ARF-GDP-Sec7 domain protein complex:
involvement of specific residues of the Sec7 domain. Mol. Cell, 3, 275-285.
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25

Randazzo, P. A., Terui, T., Sturch, S., Fales, H. M., Ferrige, A. G., and Kahn, R. A. (1995).
The myristoylated amino terminus of ADP-ribosylation factor 1 is a phospholipid- and
GTP-sensitive switch. J. Biol. Chem., 270, 14809-14815.
Renault, L., Christova, P., Guibert, B., Pasqualato, S., and Cherfils, J. (2002). Mechanism of
domain closure of Sec7 domains and role in BFA sensitivity. Biochemistry, 41, 3605-
3612.
Renault, L., Kuhlmann, J., Henkel, A., and Wittinghofer, A. (2001). Structural basis for
guanine nucleotide exchange on Ran by the regulator of chromosome condensation
(RCC1). Cell, 105, 245-255.
Robineau, S., Chabre, M., and Antonny, B. (2000). Binding site of brefeldin A at the interface
between the small G protein ADP-ribosylation factor 1 (ARF1) and the nucleotide-
exchange factor Sec7 domain. Proc. Natl. Acad. Sci., USA, 97, 9913-9918.
Scheffzek, K., Ahmadian, M. R., Kabsch, W., Wiesmuller, L., Lautwein, A., Schmitz, F., and
Wittinghofer, A. (1997). The Ras-RasGAP complex: structural basis for GTPase
activation and its loss in oncogenic Ras mutants. Science, 277, 333-338.
Scheffzek, K., Klebe, C., Fritz-Wolf, K., Kabsch, W., and Wittinghofer, A. (1995). Crystal
structure of the nuclear Ras-related protein Ran in its GDP-bound form. Nature, 374, 378-
381.
Serafini, T., Orci, L., Amherdt, M., Brunner, M., Kahn, R. A., and Rothman, J. E. (1991).
ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles: a
novel role for a GTP-binding protein. Cell, 67, 239-253.
Shevell, D. E., Leu, W. M., Gillmor, C. S., Xia, G., Feldmann, K. A., and Chua, N. H. (1994).
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Skippen, A., Jones, D. H., Morgan, C. P., Li, M., and Cockcroft, S. (2002). Mechanism of
ADP ribosylation factor-stimulated phosphatidylinositol 4,5-bisphosphate synthesis in
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Someya, A., Sata, M., Takeda, K., Pacheco-Rodriguez, G., Ferrans, V. J., Moss, J., and
Vaughan, M. (2001). ARF-GEP(100), a guanine nucleotide-exchange protein for ADP-
ribosylation factor 6. Proc. Natl. Acad. Sci., USA, 98, 2413-2418.
Steinmann, T., Geldner, N., Grebe, M., Mangold, S., Jackson, C. L., Paris, S., Galweiler, L.,
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and phospholipids in GTPase-activating protein-dependent hydrolysis of GTP by ADP-
ribosylation factor-1. J. Biol. Chem., 275, 23615-23619.
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dimensions. Science, 294, 1299-1304.
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GDP analogues promote association of ADP-ribosylation factors, 20-kDa protein
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Cell Biol., 2, 107-117.
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Zhao, L., Helms, J. B., Brunner, J., and Wieland, F. T. (1999). GTP-dependent binding of
ADP-ribosylation factor to coatomer in close proximity to the binding site for dilysine
retrieval motifs and p23. J. Biol. Chem., 274, 14198-14203.
Chapter 3
ARF AND PHOSPHOLIPIDS

PAUL A. RANDAZZO, ZHONGZHEN NIE, AND DIANNE S. HIRSCH

Laboratory of Cellular Oncology, CCR, NCI, Bethesda, USA

Abstract: Arfs have been implicated in remodeling of both biological membranes and
the actin cytoskeleton. In each case, Arf function appears to be integrally
related to lipid metabolism. As regulators of membrane traffic, Arfs bind to
membrane surfaces and affect membrane structure. Effects of Arf on the actin
cytoskeleton may be mediated through acid phospholipids. Examination of
the interactions of Arfs and lipids has revealed complex relationships. Arfs
directly bind lipids. Arfs also bind and activate two enzymes that metabolize
lipids, phospholipase D and phosphatidylinositol 4-phosphate 5-kinase. The
products of these enzymes, phosphatidic acid and phosphatidylinositol 4,5-
bisphosphate, affect both positive and negative regulators of Arfs. Although
precise mechanisms have yet to be defined, regulation of the concentrations of
these signaling lipids appears to mediate at least some effects of Arfs.

1. INTRODUCTION

Membrane traffic is the transport of membrane and proteins between

membrane bound organelles in eukaryotes. The actin cytoskeleton is
integrally associated with biological membranes. It likely is involved in
membrane transport and actin, in turn, is affected by lipid components of
biological membranes. Given Arfs’ regulatory roles in both membrane
traffic and actin remodeling, the importance of lipids for Arf function was
anticipated. Not anticipated was the number of relationships identified
between Arfs and lipids.
The role of lipids in Arf function was first appreciated with the initial
identification of Arf as a cofactor for cholera toxin catalyzed ADP-
ribosylation of the heterotrimeric G-protein Gs (Kahn and Gilman, 1986;
Kahn and Gilman, 1984). Mixed micelles of dimyristoyl
phosphatidylcholine and cholate increased the efficiency of ADP-
ribosylation in a reaction mixture containing GTP, NAD, cholera toxin,
49
50
2 RANDAZZO, NIE, AND HIRSCH

Gs and Arf. At least part of the effect of the micelles in this reaction is
related to the properties of Arf. Arf•GTP is the cofactor for cholera toxin;
however, Arf•GDP is purified from mammalian tissues. For activity in the
assay, Arf has to exchange GTP for GDP. Exchange of nucleotide on native
Arf is greatly enhanced by lipids. Once exchange does occur, Arf•GTP
binds tightly to and is stabilized by lipid interfaces, where it can function as
a cofactor for cholera toxin (see Chapter 10 for additional details). The basis
of the lipid requirement for nucleotide exchange was uncovered shortly after
the cloning of Arf and identification of N-terminal myristate as a
modification. Arf bound to GTP was found to directly associate with
hydrophobic surfaces through Arf’s N-terminal myristate and -helix.
Arf also interacts with specific phospholipids, the phosphoinositides,
within biological membranes. A cluster of basic amino acids mediates the
binding to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). This specific
interaction, together with the general hydrophobic association of Arf with
lipids, directs Arf binding to target proteins, by both recruiting Arf to
specific surfaces and by affecting the affinity of Arf for effectors. For
instance, acid phospholipids have been found to influence the binding of two
Arf effectors, GGA and Arfaptin, to Arf. Phosphatidic acid (PA), PI(4,5)P2,
and their metabolites have also been found to influence the activity Arf-
directed guanine nucleotide exchange factors (GEFs) and Arf GTPase
activating proteins (GAPs).
Adding complexity to the regulation of Arf by lipids is the ability of Arf
to directly influence the lipid composition of the membranes to which it
binds. Arf activates both phospholipase D (PLD), which hydrolyzes
phosphatidylcholine (PC) to PA, and phosphatidylinositol 4-phosphate 5-
kinase (PIP kinase I), which transfers a phosphate from ATP to
phosphatidylinositol 4-phosphate (PI(4)P) to form PI(4,5)P2 (also see
Chapter 11). The physiological relevance of many of these reactions and
their relationships to one another is yet to be determined.
In this chapter, the literature examining the impact of phospholipids on
Arf function will be discussed followed by some limited speculation about
how these reactions may be related to one another and how the lipids
themselves may mediate the effects of Arf on membrane traffic and the actin
cytoskeleton.

2. STRUCTURAL DETERMINANTS OF ARF

INTERACTIONS WITH LIPIDS

Several characteristics of Arf proteins contribute to their unique nucleotide-

dependent association with membranes. The most critical are the amino
 ARF AND PHOSPHOLIPIDS 51
3

terminal myristate and amphipathic -helix. Although crystal structures of

the N-myristoylated Arfs (myrArf) have not been determined, crystal
structures of the non-acylated proteins have provided insights into the
potential role of the myristate. The crystal structure of Arf•GDP (Greasley
et al., 1995; Amor et al., 1994) reveals a hydrophobic cleft in which the N-
terminal myristate likely inserts. In the Arf•GTP structure, the cleft is
rearranged and the myristate is predicted to be free to interact with a
hydrophobic surface (Goldberg, 1999; Goldberg, 1998). Lipids would,
therefore, bind to myrArf•GTP more tightly than to myrArf•GDP,
stabilizing the GTP bound form of the protein. Consistent with this
interpretation of the role of myristate, GTPS dissociation from myrArf was
slowed at least seven-fold by mixed micelles of dimyristoyl
phosphatidylcholine and cholate and GDP dissociation was accelerated 3-
fold. In contrast, mixed micelles had no effect on GDP dissociation from
nonmyristoylated Arf1 and slowed GTPS dissociation by only 2-fold
(Randazzo et al., 1995). Thus, the proposed structural differences in the
myristate between the GTP and GDP structures can explain two biochemical
results: (i) lipids promote GTP binding to myrArf to a much greater extent
than to nonmyrArf1 (Randazzo et al., 1995; Randazzo and Kahn, 1995) and;
(ii) myrArf1•GTP binds to lipid micelles much more tightly than does either
myrArf1•GDP or nonmyrArf1•GTP (Randazzo et al., 1993).
The amino terminal -helix of Arf1 also contributes to association with
lipid surfaces. This domain of Arf1 was not present in the Arf1•GTP crystal
structure as an N-terminal truncation mutant was used to obtain crystals.
However, based on other differences between the GTP and GDP bound
structures of Arf1, changes in the N-terminal -helix were inferred
(Goldberg, 1999; Goldberg, 1998). In Arf1•GTP, the amphipathic helix is
thought to be free to bind hydrophobic surfaces. Consistent with this
prediction, GTPS dissociation from Arf1 is slowed by lipids. In addition,
nonmyristoylated Arf1•GTPS binds to phospholipids, although with a
much lower affinity than myristoylated Arf1 (Franco et al., 1993; Randazzo
et al., 1993; Antonny et al., 1997a), so that detection of the interaction
required the presence of 30 mM phospholipid, in contrast to 100 M to 3
mM total phospholipid used to study binding to myristoylated Arf (Franco et
al., 1993; Randazzo et al., 1993; Antonny et al., 1997a; Paris et al., 1997a;
Franco et al., 1995). Further supporting the role of the -helix in lipid
association, deletion of either the first 13 or 17 amino acids of Arf1 resulted
in proteins (Arf1-13, Arf1-17) that did not detectably associate with lipid
vesicles. Nucleotide binding to Arf1-13 and Arf1-17 was not affected by
phospholipids (Hong et al., 1995; Hong et al., 1994; Randazzo et al., 1995;
Randazzo et al., 1994; Kam et al., 2000).
52
4 RANDAZZO, NIE, AND HIRSCH

A third structural feature of Arfs that contributes to membrane binding is

a patch of solvent-exposed basic residues (Greasley et al., 1995; Amor et al.,
1994). These residues interact electrostatically with acidic phospholipids that
are rich in the cytoplasmic face of biological membranes. Prominent among
these residues are those contributed from the N-terminal -helix and the C-
terminus (K10, K15, K16, K59, R178 and K181). Consistent with a specific
interaction of PI(4,5)P2 with Arf, PI(4,5)P2 stimulated GDP dissociation
from Arf under a specific set of conditions (Terui et al., 1994), while other
acid phospholipids were not effective. GTP dissociation was not affected by
PI(4,5)P2. Although these data did not address the potential physiologic
function of PI(4,5)P2 binding, they did demonstrate the specificity of the
interaction between Arf and PI(4,5)P2. Subsequent studies directly
examined binding (Kam et al., 2000; Randazzo, 1997a). By using
nonmyristoylated protein, the role of the residues comprising this basic patch
in PI(4,5)P2 binding could be examined independently of the general
hydrophobic interactions with nonspecific lipids. Arf1•GTPS was found to
bind PI(4,5)P2 with a Kd of approximately 40 M. Similar results were
obtained whether lipids were presented in the form of large unilamellar
vesicles or mixed micelles with Triton X-100. Arf1•GTPS also bound to
PA and phosphatidylserine, but with lower affinity. Mutation of K15, K16,
R178 or K181 to leucine reduced the affinity for PI(4,5)P2.

3. ROLE OF LIPID BINDING IN ARF FUNCTION

3.1 General hydrophobic interaction

The importance of Arf-lipid binding in biochemical and biological functions

of Arfs has been established from a number of studies. N-myristoylation of
Arfs is co-translational and stable so no non-myristoylated Arf is thought to
be present in cells. Although bacterially expressed nonmyristoylated Arf has
been useful for dissecting the role of lipid interactions with other parts of
Arf, a description of the role of the critical myristoyl group is necessary for
understanding the biological function of Arf. The nonspecific association of
Arf with phospholipids mediated by the N-terminal myristate has been found
to have at least two distinct roles.

3.2 Nucleotide exchange

The N-terminal myristate increases the rate of nucleotide exchange on Arf

catalyzed by the ARNO family of GEFs. Two mechanisms explaining the
effect of the myristate have been considered. Although the membrane
 ARF AND PHOSPHOLIPIDS 53
5

association is greatest for the GTP bound form of the Arf, myrArf•GDP also
associates with membranes (Franco et al., 1996; Beraud-Dufour et al., 1999),
while non-myristoylated Arf•GDP does not. Targeting myrArf•GDP to the
membrane is thought to facilitate interaction with ARNO family exchange
factors that are recruited to membranes through their PH domains (described
below and in Chapters 4-6). A second effect of myristate in GEF-catalyzed
nucleotide exchange is facilitation of the binding of lysines 10, 15 and 16 to
acid phospholipids such as PI(4,5)P2. Chardin and colleagues (Paris et al.,
1997b) found that ARNO catalyzed nucleotide exchange on both
myristoylated and nonmyristoylated Arf1 in the presence of PI(4,5)P2.
Under physiologic conditions of Mg2+ and ionic strength, myristoylated Arf1
was the preferred substrate. Lowering Mg2+ and ionic strength greatly
enhanced the rate of exchange using nonmyristoylated Arf1. The authors
noted that low Mg2+ and salt concentrations favor electrostatic interactions
such as those between acid phospholipids and basic amino acid residues.
Based on these observations, the authors concluded that the electrostatic
binding between acid phospholipids and lysines 10, 15 and 16 of Arf1,
though weak under physiologic conditions, was necessary for activity. The
myristate, by providing a second binding site, would increase the affinity of
Arf for the membrane, thereby driving these otherwise weak electrostatic
interactions. Note that these results are consistent with the reported (Terui et
al., 1994) effect of PI(4,5)P2 to increase nucleotide exchange on Arf1.

3.3 Coat proteins

The high affinity of Arf1•GTP for membranes, imparted by the myristate on

Arf, is also critical for recruiting coat proteins to membranes (see Chapters
12 and 13 for details). Vesicle coat protein recruitment to Golgi membranes
has been studied in reconstitution systems (Donaldson et al., 1992; Rothman,
1994; Serafini et al., 1991; Orci et al., 1993; Palmer et al., 1993; Stamnes
and Rothman, 1993; Ooi et al., 1998; Dell'Angelica et al., 2000; Zhu et al.,
1999; Traub et al., 1993). Coat proteins, purified away from Arfs, were
incubated with recombinant Arf proteins and either isolated Golgi
membranes or lipid vesicles. In these experiments, nonmyristoylated Arfs
did not recruit coat proteins (including coatomer, AP-1, and GGA) to lipid
vesicles or to isolated Golgi membranes, whereas myrArf1 did. The
inability to recruit the coat proteins is not due to lack of protein – protein
interaction. Nonmyristoylated Arf1 binds GGA as well as myristoylated
Arf1 (Jacques et al., 2002). Instead, the myristate increases the affinity of
Arf for the membrane surface where Arf can function as a membrane
binding site for the coat proteins.
54
6 RANDAZZO, NIE, AND HIRSCH

The amino terminal -helix has a role in directing protein interaction.

Although myristate is not required for interaction of Arf with GGA and
GAP, the N-terminal -helix does contribute to binding. Arf1-13 has a
lower affinity for both GGA and GAP than does nonmyristoylated Arf1
(Jacques et al., 2002). Furthermore, although nonmyristoylated Arf1 can
activate PLD to some extent, Arf1-17 has no detectable effect on PLD
(Jones et al., 1999). Further studies are needed to determine the basis for the
decreased affinity for GGA and GAP, and decreased ability to activated
PLD. Two possibilities appear most likely and are considered. First, the
effects of deleting the amino terminus may be due to loss of its membrane
binding and/or a specific orientation of Arf at the membrane surface. The
other possibility is that, in addition to binding lipids, the N-terminal -helix
of Arf directly interacts with target proteins.

4. INTERACTION OF SIGNALING
PHOSPHOLIPIDS WITH ARF AND ITS
REGULATORS

As is true for nonspecific hydrophobic interactions, the binding of specific

lipids to Arf also controls Arf function and regulation. In this section, the
influence of phosphoinositide binding to Arf, Arf GEFs, and Arf GAPs is
discussed.

4.1 Acid phospholipid binding to Arf

Acidic phospholipid binding to the cluster of basic amino acids on the

surface of Arf has a role beyond membrane targeting. A role in GEF-
catalyzed nucleotide exchange is discussed above. PI(4,5)P2 binding to Arf1
also has a role in the function of the GAP, ASAP1 (Kam et al., 2000;
Randazzo, 1997a). PI(4,5)P2 stimulation of GAP activity correlated with
PI(4,5)P2 binding to Arf1. Blocking PI(4,5)P2 binding, either by
sequestering PI(4,5)P2 or by introducing mutations into Arf that reduced
PI(4,5)P2 binding, also reduced PI(4,5)P2-dependent GAP activity.
Similarly, PI(4,5)P2 may direct Arf interaction with PLD. Arf1 with
mutations in two residues involved in PI(4,5)P2 binding, lysines 15 and 16,
did not stimulate PLD activity (Jones et al., 1999).
The effect of acid phospholipid binding to Arf on protein interactions is
selective. PI(4,5)P2 did not have an effect on Arf binding to GGA and
mutation of lysines 15 and 16 did not affect Arf binding to GGA. Instead of
PI(4,5)P2, PA enhanced the interaction of GGA with Arf•GTP. Interaction
of Arf with Arfaptin and Arf activity as a cofactor for cholera toxin was
 ARF AND PHOSPHOLIPIDS 55
7

inhibited by acid phospholipids, including PI(4,5)P2 (K. Jacques, Z. Nie, K.

Stanley and P. A. Randazzo, unpublished observations). Taken together,
these results indicate that the specific phospholipid environment can affect
the affinity of Arf for particular targets.

4.2 Acid phospholipid binding to Arf GEFs

The ARNO family of Arf GEFs (comprised of ARNO1, cytohesin

1/ARNO2, GRP1 and cytohesin 4 (Moss and Vaughan, 2002; Ogasawara et
al., 2000; Klarlund et al., 1997; Chardin et al., 1996)) and the EFA6 family
(Franco et al., 1999) have PH domains and are affected by phospholipids
(see Chapters 2 and 4-6; Jackson and Casanova, 2000; Moss and Vaughan,
2002) (Figure 1A). The lipid requirements for the ARNO family proteins
have been most extensively characterized. The function of the PH domain is
to recruit the GEF to a lipid surface (Paris et al., 1997b; Beraud-Dufour et
al., 1999; Chardin et al., 1996; Pacheco-Rodriguez et al., 1998). When using
a truncated Arf, Arf1-17, that is soluble in the GTP bound form, PI(4,5)P2
has no effect on activity. Furthermore, truncated ARNO lacking the PH
domain is active when using Arf1-17. As described above, acid
phospholipid binding to Arf also contributes to catalysis of exchange by
ARNO.
The independent regulation of ARNO family members may be in part
achieved through differential activation by specific phosphoinositide
isomers. Grp1 is specifically activated by PI(3,4,5)P3 (Klarlund et al., 1998;
Klarlund et al., 1997). ARNO1 and cytohesin 1 have been reported to
interact with either PI(4,5)P2 or PI(3,4,5)P3 (Bourgoin et al., 2002;
Venkateswarlu et al., 1998; Chardin et al., 1996; Moss and Vaughan, 2002;
Venkateswarlu et al., 1999). The basis for the differences in lipid specificity
was examined (Klarlund et al., 2000; Lietzke et al., 2000) and found to be
due to the replacement of a triglycine motif, found in ARNO and cytohesin,
with a diglycine motif in Grp1 (Figure 1B). Splice variation within the
mRNAs encoding each of these proteins produce a mixture of products
containing diglycine (PIP3 binding) and triglycine (promiscuous or PI(4,5)P2
binding) motifs, and therefore heterogeneity in phosphoinositide binding
(Klarlund et al., 2000; Moss and Vaughan, 2002; Ogasawara et al., 2000).
56
8 RANDAZZO, NIE, AND HIRSCH

Figure 1. Phospholipid-regulated Arf GEFs and GAPs. (A) Schematic showing the domain
organization of ARNO and EFA6 family Arf GEFs. (B) Amino acid sequences of regions
conferring lipid specificity of ARNO1 and GRP1. (C) Schematic of the Arf GAPs. Ankyrin
repeat, ANK; Arf GAP catalytic domain, Arf GAP; coiled-coil, CC; pleckstrin homology
domain, PH; proline-rich domain, PR; Rho GAP domain, Rho GAP.

4.3 Arf GAPs and lipids

To date, fourteen mammalian Arf GAPs have been described (see Chapters
7-9; Cukierman et al., 1995; Premont et al., 1998; Vitale et al., 2000;
Bagrodia et al., 1999; Brown et al., 1998; Premont et al., 2000; Miura et al.,
2002; Krugmann et al., 2002; Nie et al., 2002). The proteins can be divided
into three groups based on structure (Figure 1C). The first subfamily
contains Arf GAP1 and 3. The protein has a catalytic domain, found in all
proteins with GAP activity, comprised of a zinc finger motif. A second
domain targets Arf GAP1 to the Golgi apparatus by binding to ERD2 (Aoe
et al., 1999). A second subfamily contains the GITs (called APAP in the
nomenclature introduced in this volume). Similar to Arf GAP1 in containing
the Arf GAP catalytic domain at the amino terminus, the GITs also have
 ARF AND PHOSPHOLIPIDS 57
9

ankyrin (ANK) repeat domains and unique domains involved in targeting the
protein to the plasma membrane. The largest subgroup of Arf GAPs is the
ASAPs, defined by a core of PH, Arf GAP and ANK repeat domains. All
members of this subgroup contain additional domains N-terminal to this
core. Some also have additional domains C-terminal of the core. The lipid
dependence of Arf GAP1, GITs and ASAPs has been examined and are
discussed below.

4.3.1 Arf GAP1

Arf GAP1 has unique lipid dependence among the Arf GAPs. In the first
studies of Arf GAP1, PI(4,5)P2 was found to have a small but reproducible
effect on activity. Those studies were performed with nonmyristoylated
Arf1 (Randazzo 1997b; Makler et al., 1995). When myrArf1 was used, no
effect of PI(4,5)P2 was seen (Antonny et al., 1997b). The most likely
explanation for these results was that PI(4,5)P2 was recruiting the
nonmyristoylated Arf1 to the hydrophobic surface with which the GAP was
also associated. Myristoylated Arf1, bound to GTP, has a high affinity for
the vesicles and hydrophobic surfaces, and there was no further effect of
adding PI(4,5)P2.
When Antonny and colleagues subsequently re-examined the lipid
requirements of Arf GAP1 (Antonny et al., 1997b), they found that
diacylglyerols (DAG) with monounsaturated acyl groups both increased the
amount of GAP associated with vesicles and stimulated GAP activity. On
examination of the effects of a number of synthetic lipids, the extent of
stimulation was found to correlate with the relative volumes occupied by the
polar head group and the acyl groups. The smaller the head group relative to
the acyl group, the greater the activity. The monounsaturated acyl groups
have a kink that results in a relatively large occupied volume.
Dioleoylglycerol, with no head group and two kinked acyl groups, was the
most effective of the series they examined. Saturated phosphatidylcholine,
with a large head group and tightly packed acyl groups, was inhibitory.
Gcs1p, a yeast homolog of Arf GAP1, was similar in lipid dependency.
Based on this analysis, Cassel and colleagues proposed that Arf GAP1
binding to membranes is driven by hydrophobic contact of the protein with
the acyl groups of the lipids. Large head groups and tight packing of acyl
groups would tend to exclude the protein whereas small head groups and
loosely packed acyl groups would favor interaction with the hydrophobic
part of the protein.
The importance of the activation of Arf GAP1 by DAG was thought to be
related to the function of PLD. The product of PLD, PA, is rapidly
hydrolyzed to ortho-phosphate and DAG. This set of reactions could
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10 RANDAZZO, NIE, AND HIRSCH

constitute a negative feedback loop regulating Arf1 (see Figure 2, adapted

from Antonny et al., 1997b).

Figure 2. Postulated feedback loop regulating the activation status of Arf1 as a result of
changes in membrane lipids. The direct activation of PLD by Arf•GTP is shown, along with
the product of PLD, PA, being converted to DAG, an activator of Arf GAP1.

The idea that Arf GAPs are a target of lipid signaling is consistent with in
vivo studies in S. cerevisiae (Yanagisawa et al., 2002). Screens for secretory
mutants in yeast had led to the identification of Sec14p, a PI transfer protein
thought to regulate Golgi function by altering the lipid composition of Golgi
membranes (Bankaitis et al., 1989). Deletion of Gcs1, an Arf GAP, was
synthetically lethal with a temperature sensitive mutant of Sec14, sec14-1ts
(Yanagisawa et al., 2002). Similarly, replacement of Gcs1 with Gcs1-K54, a
mutant lacking GAP activity, was synthetically lethal with sec14-1ts. The
defect imposed by loss of Sec14 function can be partially overcome by
mutations (“bypass Sec14p” mutations) in genes that control
phosphatidylcholine or phosphoinositide metabolism. Consistent with Gcs1
functioning downstream of lipid changes mediated by Sec14, deletion of
Gcs1 re-imposed defects in growth and Golgi function in sec14-1ts strains
with “bypass Sec14p” mutations.

4.3.2 GITs

Less is known about the potential regulatory role of lipids for the function of
GITs. In one report, PIP3 stimulated the GAP activity of GIT, using Arf6 as
a substrate (Vitale et al., 2000). However, at least 100 M PIP3 was
required for a relatively modest (2 – 3 fold) stimulation (by comparison, 360
M PA with 45 M PI(4,5)P2 activate ASAP1 10,000 fold (Kam et al.,
2000).
 ARF AND PHOSPHOLIPIDS 59
11

4.3.3 ASAPs

Efforts to purify a phosphoinositide-dependent Arf GAP activity from

bovine brain led to the first identification of ASAP family proteins (Brown
et al., 1998). A core of PH, Arf GAP and ANK repeat domains defines these
Arf GAPs. Domains outside the core define subfamilies within the ASAP
family. ASAP1 and PAP (called AMAP1 and 2 in the nomenclature
introduced in this volume) contain a unique amino terminus, proline rich
domain and SH3 domain (Andreev et al., 1999; Brown et al., 1998). ACAPs
have unique amino termini containing one or two coiled coil domains
(Jackson et al., 2000). The AGAPs have a G-protein like domain (Nie et al.,
2002). The ARAPs have five PH domains and a Rho GAP domain (Miura et
al., 2002; Krugmann et al., 2002; personal observation). Two variants have
sterile  motif (SAM) domains.
ASAP1 has been the most extensively characterized of the ASAPs in
terms of lipid dependence (Randazzo and Kahn, 1994). The GAP activity of
ASAP1 purified from bovine brain or recombinant ASAP1 was
synergistically and specifically activated by PA and PI(4,5)P2 (Kam et al.,
2000; Brown et al., 1998; Randazzo, 1997a; Randazzo and Kahn, 1994). PA
was more potent than phosphatidylserine or phosphatidylinositol. PI(4,5)P2
could not be replaced with PI(3,4,5)P3 or other D-3-phosphorylated
phosphoinositides. A deletion mutant consisting of just the PH, Arf GAP
and ANK repeat domains was the minimal structure in which GAP activity
was detectable. The PI(4,5)P2 specificity was preserved in this protein.
PI(4,5)P2 binding to both ASAP1 and Arf is required for GAP activity.
As described above, Arf binding to PI(4,5)P2 facilitates its interaction with
ASAP1. Stimulation of GAP activity also correlated with GAP binding to
vesicles or micelles containing PI(4,5)P2 and PA. The introduction of
mutations into the PH domain of ASAP1 abrogated both PI(4,5)P2 binding to
ASAP1 and PI(4,5)P2 stimulation of GAP activity.
The PH domain of ASAP1 functions as an allosteric binding site for
PI(4,5)P2-dependent regulation of the GAP activity (Kam et al., 2000). This
possibility was examined after noting that recruitment to membranes and
stimulation of GAP activity could be dissociated. Probing the structure of
ASAP1 by limited proteolysis led to the identification of a trypsin cleavage
site within the Arf GAP domain that was exposed upon ASAP1 binding
PI(4,5)P2, consistent with a PI(4,5)P2-dependent conformational change in
the GAP. The possibility of a conformational change in Arf has not yet been
tested.
The phosphoinositide specificity within the ASAP family varies. The
ACAPs are activated by either PI(4,5)P2 or PI(3,5)P2 in vitro, with equal
efficacy (Z. Nie and P.A. Randazzo, unpublished observations). ARAPs are
60
12 RANDAZZO, NIE, AND HIRSCH

activated by PI(3,4)P2 and PI(3,4,5)P3 to a much greater extent than they are
activated by other phosphoinositide isomers (Miura et al., 2002; Krugmann
et al., 2002; personal observation). The contributions of each of the five PH
domains to this activation have not been extensively examined. PH domain
1, counting from the amino terminus, likely confers at least some of the
PI(3,4,5)P3 dependence. PH domain 2 is of particular interest, given the role
of the PH domain of ASAP1 as an allosteric binding site for the PI(4,5)P2-
dependent regulation of GAP activity. The in vitro studies of the AGAPs
have been the most complex to interpret (Nie et al., 2002). The PH domain
is split, with an 80 amino acid insert between the predicted 4 and 5
strands of the structure. Within the first 4  strands, AGAP1 has 9 identities
of the consensus sequence for PI(3,4,5)P3 binding (Lietzke et al., 2000). The
one difference is conservative, lysine in place of an arginine. Consistent
with this level of identity, PI(3,4,5)P3 stimulated this GAP. However, the
required concentrations were relatively high (10 – 50 M) compared to that
seen with ARAP1 (100 nM) and the addition of a second acid phospholipid,
PA, inhibited the PI(3,4,5)P3-dependent activity. PI(4,5)P2, in the presence
of PA, stimulated activity. This effect was specific for PA. Other acid
phospholipids, including phosphatidylserine and PI, were much less
effective in inhibiting PI(3,4,5)P3-dependent GAP activity and synergizing
with PI(4,5)P2.
The distinct phosphoinositide specificities of each Arf GAP and GEF
could allow for the independent regulation of each. For instance, ASAP1,
ACAP1 and ACAP2 have been found in the same cellular sites (Jackson et
al., 2000). Changes in PI(3,5)P2 could contribute to the regulation of
ACAP1 and ACAP2 whereas ASAP1 could be regulated by changes in
PI(4,5)P2. Other mechanisms could provide additional regulation; e.g.,
ASAP1 is dependent on PA and is also a target of other signaling proteins,
such as Src.

5. SYSTEMS OF PHOSPHOINOSITIDE
DEPENDENT REACTIONS

PI(4,5)P2 and PA could be elements of a system of regulatory feedforward

and feedback loops. In addition to PI(4,5)P2 and PA affecting Arf, Arf
affects the production of these lipids. PLD1 is activated synergistically by
Arf, Rho family GTP-binding proteins, protein kinase C (PKC) and
PI(4,5)P2 (see Chapter 11; Thomas et al., 1994; Liscovitch et al., 2000;
Exton, 1999; Frohman et al., 1999). PIP kinase I is directly activated by
Arf and PA (Anderson et al., 1999; Honda et al., 1999; Jones et al., 2000).
 ARF AND PHOSPHOLIPIDS 61
13

The effect of Arf GAPs on Rho family proteins adds further complexity
to this network of interactions. ARAP1 induces the activation of Cdc42 and
the inactivation of RhoA in NIH 3T3 fibroblasts (Miura et al., 2002).
ARAP1 has a Rho GAP domain and RhoA GAP activity in vitro and in vivo.
Based on the formation of membrane ruffles and filopodia observed when
the proteins are ectopically expressed, other ASAP family members also
appear to be able to activate Cdc42 and/or Rac (P.A. Randazzo, unpublished
observations). Furthermore, GIT family Arf GAPs bind the Rho family
exchange factor called Pix/Cool (Zhao et al., 2000; Turner et al., 1999). Rho
family proteins stimulate PLD and PIP kinase (Sternweis et al., 1997; Chong
et al., 1994). Thus, the effects on the Rho family proteins could generate
additional feedforward and feedback loops.

Figure 3. Proposed system of feedforward and feedback loops impinging on Arf and
regulating phospholipid concentrations. Substrates and products of specific enzymes are
indicated with a solid line and allosteric or regulatory effects with dotted lines. PI3-K, PI 3-
kinase, PI(4)P-5K, PI(4)P 5-kinase.

With the products of the PLD and PIP kinase I reactions synergizing with
Arf and Rho to activate other enzymes, a complex network of feedforward
and feedback loops can be assembled. The distinct phosphoinositide
specificities of GAPs and GEFs could provide further coordination to these
reactions. For instance, a PI(3,4,5)P3-dependent GEF could function with a
62
14 RANDAZZO, NIE, AND HIRSCH

PI(4,5)P2-dependent Arf GAP to drive the cycle of GTP binding and

hydrolysis on Arf. A summary of these reactions is shown in Figure 3.

6. FUNCTION OF ARF/LIPID REACTION

NETWORKS IN CELL PHYSIOLOGY

Systems of reactions driving cyclical changes in Arf and phospholipids

could function in several ways that need not be mutually exclusive. Here,
three possible roles for the dynamic relationships between Arf and
phospholipids are considered.

6.1 Rapid and precipitous switching

The simplest model for Arf action is that any particular effect (e.g.,
membrane traffic) is mediated by a single effector (e.g., a coat protein). Arf
function in membrane traffic is dependent on the regulated cycle of GTP
binding and hydrolysis. Not only is this cycle integral to Arf function, the
cycle must occur on a time scale that matches the events being regulated.
Therefore, one possible role of the feedforward and feedback loops is to
mediate rapid and precipitous switching of Arf between on and off states.
Lipid metabolites would be particularly suited for these functions as they
could be generated and diffuse rapidly within the membrane at which Arf
works.

6.2 PI(4,5)P2 and PA are Arf effectors

PI(4,5)P2 and PA could be Arf effectors, in which case the lipid loops could
be a means of regulating the levels of either or both PI(4,5)P2 and PA.
PI(4,5)P2 and PA regulate a number of events that impact membrane traffic
and actin polymerization and waves of PI(4,5)P2 coincide with phagocytosis,
an Arf6-dependent event (Takenawa and Itoh, 2001; Osborne et al., 2001;
Gillooly et al., 2001; Simonsen et al., 2001; Payrastre et al., 2001; Cremona
and De Camilli, 2001). Evidence supporting the function of PA and
PI(4,5)P2 as Arf effectors has been reviewed elsewhere (Chapter 11 of this
volume and Randazzo et al., 2000). Briefly, either blocking the production
or sequestering the lipids has been found to block Arf-dependent cellular
functions (Honda et al., 1999; Randazzo et al., 2000). Additionally,
overexpressing PIP kinase mimics part of the phenotype seen with the
constitutive activation of Arf6 (Honda et al., 1999; Brown et al., 2001).
Mechanistic details of PA and PI(4,5)P2 function as Arf effectors are
lacking. Two possibilities have been considered (Randazzo et al., 2000).
 ARF AND PHOSPHOLIPIDS 63
15

First, the lipids may recruit specific proteins to membranes. For example,
PA might recruit a coat protein or may activate a PIP kinase. PI(4,5)P2 may
activate PLD, directly bind coat proteins or affect coat protein binding by
influencing the association of actin cytoskeletal components with the
membranes. In this “mesh hypothesis”, a membrane microdomain with a
high content of PI(4,5)P2 recruits spectrin, an actin binding protein, which
loosens the actin mesh, allowing coat binding and subsequent vesicle
budding (Godi et al., 1999; Lorra and Huttner, 1999). A second possibility
is that the lipid changes affect the physical properties of the membranes.
The synthesis of PA may, by inducing curvature in the membrane, drive
budding and release of a transport vesicle (Weigert et al., 2000; Weigert et
al., 1999b; Weigert et al., 1999a; Schmidt et al., 1999).

6.3 Temporal regulation

The changes in lipids that would be driven by systems of reactions such as

those schematized in Figure 3 could have a third effect. Arf, by both
affecting lipid levels and having lipid-dependent protein targets, could order
a series of reactions. As discussed above, PA and PI(4,5)P2 inhibit Arf
binding to Arfaptin 2. PA promotes Arf binding to GGA; PA with PI(4,5)P2
promotes Arf interaction with Arf GAP. These effects create the potential
for temporal regulation. For instance, with low PA and PI(4,5)P2, Arf could
act through Arfaptin, perhaps mediating the recruitment of a Rho family
protein to a membrane. As these lipids increase, in part driven by a
feedforward loop activating Arf, which in turn activates both PIP kinase and
PLD, Arf would dissociate from Arfaptin and recruit a coat protein. This
event could result in the sorting of transmembrane “cargo” proteins (see
Chapters 12-13). As PI(4,5)P2 and/or PA concentrations increase further,
Arf GAPs could be activated, Arf•GTP hydrolyzed to Arf•GDP, and the
sequence of events terminated. The sequence depicted in Figure 4 is just one
of many possibilities by which Arfs may integrate lipid and protein
signaling.

7. CONCLUSION

The role of lipids in Arf function has been examined for over a decade and,
rather than being simplified, more relationships have been discovered among
the proteins and lipids in the Arf pathway. At the same time, the functions
of phosphoinositides and phosphatidic acid have been further defined. This
progress has led to an appreciation of the integral relationship between Arfs
and phospholipids and a number of testable models of the mechanisms by
64
16 RANDAZZO, NIE, AND HIRSCH

which membrane traffic and the actin cytoskeleton are temporally and
spatially regulated.

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Chapter 4
THE SEC7 FAMILY OF ARF GUANINE
NUCLEOTIDE EXCHANGE FACTORS

CATHERINE L. JACKSON
Cell Biology and Metabolism Branch, NICHD, NIH, Bethesda, USA

Abstract: Arf GEFs are all multidomain proteins that likely integrate upstream signals
with the downstream effects of GTPase activation. The presence of a highly
conserved domain, the Sec7 domain, allows prediction of the number of Arf
GEFs in multiple organisms and phylogenetic analyses. The profound effects
of brefeldin A on membrane systems in eukaryotic cells demonstrate that its
targets, the Arf GEFs, are central regulators of organelle structure and
function. Ultrastructural analyses, particularly those coupled to genetic studies
in S. cerevisiae, offer new insights into the roles of Arf GEFs in membrane
dynamics and protein transport.

1. INTRODUCTION

Conversion of Arf•GDP to Arf•GTP is catalyzed by guanine nucleotide

exchange factors (GEFs). Arf•GDP is largely soluble, with only a weak
affinity for membranes, whereas Arf•GTP is more tightly membrane bound
(Antonny et al., 1997). Activation of Arf results not only in a
conformational change in the protein that allows it to interact with numerous
effectors, but also in a change in localization from the cytoplasm to a
membrane. It is the localization of the Arf GEFs that determine where in the
cell Arf will be activated. Hence, the GEFs control not only the timing of
activation of Arf GTPases, but also the spatial regulation of activation.

71
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2 JACKSON

2. THE SEC7 DOMAIN CATALYZES GUANINE

NUCLEOTIDE EXCHANGE ON ARFS

The Arf GEFs began to be identified at the molecular level in 1996 and the
presence of a conserved domain, the Sec7 domain, as that responsible for
GEF activity was soon evident. A genetic approach in budding yeast,
Saccharomyces cerevisiae, identified the first Arf GEF (Peyroche et al.,
1996). In yeast, ARF1 and ARF2 encode functionally indistinguishable Arf
proteins, which in sequence and function are most similar to mammalian
Class I Arfs (see Chapter 1). ARF1 produces 90% of the total Arf protein in
cells, and ARF2 only 10% (Stearns et al., 1990). In a strain deleted for
ARF1 and expressing wild type Arf2p from the ARF2 gene alone, ARF2-N31
was expressed from a low-copy plasmid. This dominant negative phenotype
was conditional: at 37oC, cells expressing the ARF2-N31 allele were viable
but at 23oC, cells expressing the mutant protein failed to grow. A multi-
copy suppressor of the cold-sensitive growth defect was found and named
GEA1 (for Guanine nucleotide Exchange on Arf). There is a second gene
encoding a protein 50% identical to Gea1p, named GEA2. Gea1p and Gea2p
are functionally redundant. Strains carrying a deletion of one or the other
individually have no growth or secretion phenotype, but the double deletion
strain is inviable. To date, no clear differences in function have been
reported. At the time they were identified, the only sequence feature evident
in the Gea1p and Gea2p proteins was the presence of a so-called “Sec7
domain”. This domain was homologous to a region found in the Sec7p
protein. SEC7 was first identified in the screen for secretion mutants carried
out in S. cerevisiae (Novick et al., 1980). Other proteins such as
Arabidopsis thaliana GNOM/Emb30 and mammalian B-2/cytohesin had
also been found to have a Sec7 domain (Sec7d), although its function was
not clear from analysis of these proteins (Liu and Pohajdak, 1992; Shevell et
al., 1994; Zagulski et al., 1995). In collaboration with P. Chardin, we
searched for human proteins containing any sequence similarities to the
Gea1p and Gea2p proteins, and found an EST clone with a Sec7d. The
protein was expressed in E. coli and found to have very potent Arf GEF
activity (Chardin et al., 1996). The protein was named ARNO for ADP-
Ribosylation factor Nucleotide binding site Opener. The only sequence
similarity between ARNO and the Gea proteins was in the Sec7d. This
observation led to the prediction that the Sec7d was responsible for Arf GEF
activity, and indeed the ARNO Sec7d alone (purified from E. coli) had Arf
GEF activity (Chardin et al., 1996). Hence, the Sec7d is the catalytic
domain of the Arf GEFs.
Two other groups were taking different approaches to identify an Arf
GEF at about the same time. A classic biochemical approach aimed at
 THE SEC7 FAMILY OF ARF GEFS 73
3

identifying a protein responsible for in vitro Arf GEF activity culminated in

the cloning of the bovine BIG1 cDNA, encoding a mammalian ortholog of
yeast Sec7p (Morinaga et al., 1997; Morinaga et al., 1996). Shortly
afterwards, the human BIG1 and BIG2 proteins were cloned and all three of
these proteins were shown to have Arf GEF activity in vitro (Togawa et al.,
1999) (see Chapter 6). Also, a genetic approach was used to identify the
target of the drug brefeldin A (BFA; see below) in Chinese hamster ovary
cells. This led to the identification of GBF1, another Sec7d-containing
protein with demonstrated Arf GEF activity in vitro (Claude et al., 1999)
(see Chapter 5).

3. EVOLUTIONARY CONSERVATION OF THE ARF

GEFS

The complete genome sequences for humans, D. melanogaster, C. elegans,

S. pombe, S. cerevisiae and A. thaliana are now available. With the
identification of the Sec7d as that responsible for Arf GEF activity, it is now
possible to scan the predicted proteins from these organisms to determine all
predicted Arf GEF proteins. S. cerevisiae, S. pombe, C. elegans and D.
melanogaster each have 5 Sec7d proteins, A. thaliana has 7-8, and humans
have ~15. Each of the Arf GEFs in C. elegans and D. melanogaster belong
to a different subfamily (Figure 1). Humans have 3-4 members of four of
these five subfamilies, with GBF1 being the sole human member of the fifth
subfamily (Figure 2). Mice and humans have one additional putative Arf
GEF, the Fbx8 protein, which is not found even in Drosophila. In addition
to a Sec7d, Fbx8 contains an N-terminal F-box, normally found in E3
ubiquitin ligase complexes that target proteins for degradation via the
ubiquitination pathway. The function of this protein is not known.
Two subfamilies of Arf GEFs, Gea/GBF/GNOM and Sec7/BIG, are
common to all five organisms. Arabidopsis has no other subfamilies (Figure
1); it has three members of the Gea/GBF/GNOM family, and five members
of the Sec7/BIG family. One of the latter (BIG5-At) is quite divergent in
sequence in the Sec7d, and documentation of Arf GEF activity is required to
ensure its appropriate placement in the family. A member of the Sec7/BIG
family was identified in Paramecium tetraurelia in a screen for ciliary
proteins, and it may play a role in transport of components to cilia in this
organism (Nair et al., 1999). The two yeasts S. cerevisiae and S. pombe
have, in addition to the Gea/GBF/GNOM and Sec7/BIG subfamilies, two
more divergent Arf GEFs. S. cerevisiae Syt1p and S. pombe ArfGEF4 share
a significant level of sequence homology, as do S. cerevisiae Ybl060p and S.
pombe ArfGEF5 (Figure 1). The S. cerevisiae Syt1p Sec7d, although quite
74
4 JACKSON

Figure 1. Phylogenetic tree of Arf GEFs from different species. Included are Sec7 domain
proteins from five fully sequenced eukaryotic genomes, and published Sec7 domain
sequences from other organisms. Names of subfamilies are given on the right. Arf GEFs from
closely related organisms to those shown here are not included. See Table 1 for descriptions
of each Arf GEF sequence. Full-length Sec7 domain sequences were used for the analysis,
and the tree was generated using the EMBL European Bioinformatics Institute ClustalW
program at http://www.ebi.ac.uk/clustalw/.
 THE SEC7 FAMILY OF ARF GEFS 75
5

divergent in sequence from those of the Gea/GBF/GNOM and Sec7/BIG

subfamilies, has Arf GEF activity in vitro (Jones et al., 1999). The
ARNO/GRP/cytohesin subfamily is a branch of the Sec7/BIG family
restricted to metazoans. In humans, there are four members of this
subfamily: cytohesin-1/PSCD1, ARNO/PSCD2, GRP1/ARNO3/PSCD3 and
cytohesin-4/PSCD4 and close homologues are found in mice and rats.
Humans, D. melanogaster and C. elegans have two additional subfamilies of
Arf GEFs. These are the EFA6 subfamily, which includes EFA6A,
EFA6B/TIC, EFA6C and EFA6D (Derrien et al., 2002) (see Chapter 14),
and the ArfGEP100/BRAG subfamily, including BRAG1,
ArfGEP100/BRAG2 (Someya et al., 2001), and BRAG3 (see Chapter 5). S.
pombe ArfGEF4 and ArfGEF5 each have a PH domain downstream of their
Sec7d which shows weak homology to those of human and D. melanogaster
EFA6 proteins. Hence, these S. pombe Arf GEFs (and possibly their S.
cerevisiae counterparts Syt1p and Ybl060p) may be distantly related to the
EFA6 proteins of higher eukaryotes (Figure 1).
Included in the tree shown in Figure 1 are two Arf GEFs from human
pathogens that have been demonstrated to have in vitro exchange activity
towards class I Arfs. The RalF protein of Legionella pneumophila is
necessary for the localization of Arf on phagosomes containing the
bacterium, and plays an important role in the infection process (Nagai et al.,
2002). The malarial parasite Plasmodium falciparum has a single Sec7d
protein, Arf GEF-Pf, that has in vitro Arf GEF activity sensitive to BFA
(Baumgartner et al., 2001).

4. SUBSTRATE SPECIFICITY OF ARF GEFS

A major issue in the Arf GEF field is to determine which Arf substrate(s) are
used by a given GEF in the cell. Arf proteins are divided into three classes: I
(Arf1, 2, 3), II (Arf4, 5), and III (Arf6), based on sequence homology and
function (see Chapter 1). The primary means of assessing substrate
specificity for GEFs is in vitro nucleotide exchange assays. The
Gea/GBF/GNOM and Sec7/BIG Arf GEFs act on class I Arf proteins, as
determined by in vitro exchange assays (Jackson and Casanova, 2000; Moss
and Vaughan, 1998). The full length Gea1p and GNOM proteins use
mammalian class I Arfs as substrates very efficiently (Peyroche et al., 1996;
Steinmann et al., 1999). The in vitro activity of yeast Gea1p on human Arf1
is equivalent to its activity on yeast Arf1p or Arf2p (A. Peyroche and C. L.
Jackson, unpublished observations). However, GBF1 has not yet been
reported to have GEF activity on class I Arfs in vitro, although class I Arfs
76
6 JACKSON

Table 1. Sequence information for the Arf GEFs shown in Figures 1 and 2.
Name Organism Accession number Length
GBF1-Hs Homo sapiens NM_004193 1859
GBF1-Dm Drosophila melanogaster AE003822 2040
GBF1-Ce Caenorhabditis elegans AL032627 1820
Gea1p-Sc Saccharomyces cerevisiae P47102 1408
Gea2p-Sc Saccharomyces cerevisiae P39993 1459
Gea1p-Sp Schizosaccharomyces pombe NP_596613 1462
GNOM-At Arabidopsis thaliana S65571 1451
GNL1-At Arabidopsis thaliana NP_198766 1443
GNL2-At Arabidopsis thaliana NP_197462 1375
BIG1-Hs Homo sapiens Q9Y6D6 1849
BIG2-Hs Homo sapiens Q9Y6D5 1785
BIG1-Dm Drosophila melanogaster AAF53331 1643
BIG1-Ce Caenorhabditis elegans NP_493386 1628
Sec7p-Sc Saccharomyces cerevisiae AAB04031 2009
Sec7p-Pp Pischia pastoris AAK40234 1772
BIG1-Sp Schizosaccharomyces pombe NP_594555 1822
BIG2-Sp Schizosaccharomyces pombe NP_594954 1811
BIG1-At Arabidopsis thaliana NP_195533 1698
BIG2-At Arabidopsis thaliana NP_191645 1793
BIG3-At Arabidopsis thaliana NP_171698 1750
BIG4-At Arabidopsis thaliana NP_195264 1711
BIG5-At Arabidopsis thaliana NP_189916 1669
Sec7-Pt Paramecium tetraurelia AAF36486 1135
Cytohesin1-Hs Homo sapiens Q15438 398
ARNO-Hs Homo sapiens CAA68084 399
GRP1/ARNO3-Hs Homo sapiens NP_004218 399
Cytohesin4-Hs Homo sapiens NP_037517 394
ARNO-Dm Drosophila melanogaster AAF57230 348
ARNO-Ce Caenorhabditis elegans NP_498764 393
EFA6A/PSD-Hs Homo sapiens NP_002770 645
EFA6B/TIC-Hs Homo sapiens NP_036587 1056
EFA6C-Hs Homo sapiens CAB66494 771
EFA6D/KIAA0942-Hs Homo sapiens NP_056125 534
EFA6-Dm Drosophila melanogaster AAF56027 900
EFA6-Ce Caenorhabditis elegans NP_502416 818
BRAG1/KIAA0522-Hs Homo sapiens T00080 1560
ArfGEP100/BRAG2-Hs Homo sapiens NP_055684 841
BRAG3/KIAA 1110-Hs Homo sapiens BAA83062 740
BRAG-Dm Drosophila melanogaster AAF51675 1210
BRAG-Ce Caenorhabditis elegans NP_500420 614
Syt1-Sc Saccharomyces cerevisiae NP_015420 1226
ArfGEF4-Sp Schizosaccharomyces pombe NP_594894 657
ArfGEF5/Yb1060p-Sc Saccharomyces cerevisiae NP_009493 687
ArfGEF5-Sp Schizosaccharomyces pombe NP_594936 942
Ra1F-Lp Legionella pneumophila AAL23711 374
Arf GEF-Pf Plasmodium falciparum Not available 3384
 THE SEC7 FAMILY OF ARF GEFS 77
7

are likely to be a substrate for this human GEF in cells (Claude et al., 1999;
Kawamoto et al., 2002). Activity of GBF1 on the class II Arf, Arf5p has
been shown in vitro (Claude et al., 1999). The ARNO/GRP/cytohesin
subfamily of Arf GEFs can act on both class I and class III Arfs in vitro
(Franco et al., 1998; Frank et al., 1998; Macia et al., 2001). An apparent
exception to this rule is cytohesin-4, which has been reported to have no
activity towards Arf6, and which acts preferentially on class I Arfs in vitro
(Ogasawara et al., 2000).
A promising newer approach to the question of substrate specificity uses
pull-downs of Arf•GTP from cell extracts by the Arf effector GGA1, which
binds exclusively to the GTP-bound form of all Arfs (classes I, II and III)
(Boman et al., 2000; Dell'Angelica et al., 2000). This approach was used to
demonstrate that over-expressed ARNO specifically activates Arf6 to
modulate migratory potential and morphology in Madin-Darby canine
kidney (MDCK) cells (Santy and Casanova, 2001). Nakayama and
colleagues used this pull-down approach to demonstrate that BIG2 activates
class I Arfs in cells (Shinotsuka et al., 2002). In the case of the EFA6
family, biochemical and cell biological approaches converge, showing in
both cases that the preferred substrate is Arf6 (Franco et al., 1999; Macia et
al., 2001). ArfGEP100/BRAG2 has highest in vitro activity towards Arf6,
accelerating binding of GTPγS by 12-fold (Someya et al., 2001).
Immunofluorescence analyses indicate that ArfGEP100/BRAG2 and Arf6
co-localize in cells in peripheral areas, strongly supporting the conclusion
that Arf6 is the physiological substrate of this GEF (Someya et al., 2001).

5. BFA TARGETS A SUBFAMILY OF ARF GEFS IN

COMPLEX WITH ARF•GDP

5.1 Effects of BFA on cells

BFA has a profound effect on organelles of the secretory and endocytic

pathways in a wide range of cell types (Jackson, 2000; Klausner et al.,
1992). In the mid-1980’s, BFA was shown to specifically inhibit secretion
of proteins in mammalian cells, with little or no immediate effect on other
cellular processes (Misumi et al., 1986; Takatsuki and Tamura, 1985).
Subsequent studies revealed that BFA caused the disassembly of the Golgi
apparatus, and the redistribution of certain Golgi enzymes to the ER (Doms
et al., 1989; Fujiwara et al., 1988; Lippincott-Schwartz et al., 1989). This
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Figure 2 . Phylogenetic trees of the Arf GEFs from A) Homo sapiens B) Drosophila
melanogaster and C) Saccharomyces cerevisiae. See Table 1 for sequence information.

effect was rapid, with complete breakdown of this organelle occurring

within 10 minutes of treatment with high concentrations of BFA (Lippincott-
Schwartz et al., 1989). These experiments were the first to show the
incredibly dynamic nature of the Golgi apparatus, whose elaborate structure
 THE SEC7 FAMILY OF ARF GEFS 79
9

would seem to suggest a more stable state. BFA rapidly blocks transport of
secreted proteins out of the ER-Golgi (Klausner et al., 1992) and trans-Golgi
network (TGN)-endosomal systems, causing fusion of the TGN and
endosome (Hunziker et al., 1992; Lippincott-Schwartz et al., 1991; Reaves
and Banting, 1992; Wood et al., 1991). BFA treatment of particular cell
types also causes tubulation of lysosomes (Lippincott-Schwartz et al., 1991;
Wood and Brown, 1992). Two cell lines, MDCK and PtK1, are resistant to
the effects of BFA at the Golgi, but sensitive to the drug at the
TGN/endosome (Hunziker et al., 1992). This result suggests the existence of
two distinct BFA targets acting at the level of each organelle.
The most rapid effect of BFA is the release of specific peripherally
associated proteins from organelles, notably the Golgi and endosomes
(Boehm et al., 2001; De Matteis and Morrow, 2000; Jackson, 2000). The
first such protein identified was β-COP, a subunit of the COPI coat complex,
which is released from Golgi membranes within 1 minute of BFA treatment
(Donaldson et al., 1990; Presley et al., 2002). Class I Arfs are also rapidly
released from membranes upon BFA treatment (Donaldson et al., 1991;
Presley et al., 2002). GTPγS was found to antagonize the effects of BFA on
the binding of COPI to membranes, suggesting that Arfs might regulate this
rapidly reversible process. This prediction was elegantly verified by the
demonstration that binding of Arf1 to Golgi membranes was a prerequisite
for binding of β-COP, and that only the former step was inhibited by BFA
(Donaldson et al., 1992). Moreover, several groups demonstrated that Golgi
membranes were capable of stimulating guanine nucleotide exchange on
Arf, and that BFA inhibited this GEF activity (Donaldson et al., 1992;
Helms and Rothman, 1992; Randazzo et al., 1993). These results
demonstrated that at least one target of BFA was an Arf GEF or an
associated Golgi protein. Cells expressing the dominant negative mutant
Arf1-N31 had a phenotype indistinguishable from that of BFA-treated cells
(Dascher and Balch, 1994), thus providing further support for the role of Arf
and Arf GEFs in BFA action and COPI binding to Golgi membranes. Arf
controls binding of other coat complexes at the TGN/endosome; including
AP-1/clathrin, AP-3, AP-4 and the GGAs (see Chapters 12 and 13; Boehm
and Bonifacino, 2001; Drake et al., 2000; Liang and Kornfeld, 1997; Ooi et
al., 1998; Robinson and Bonifacino, 2001; Zhu et al., 1998).

5.2 Target of BFA: an Arf•GDP – Arf GEF complex

5.2.1 BFA targets in live cells

The cloning of the Arf GEFs provided an opportunity to test the hypothesis
that these proteins are direct targets of BFA. Surprisingly, although some
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Arf GEFs have in vitro exchange activity sensitive to BFA (Morinaga et al.,
1997; Peyroche et al., 1996), others such as ARNO, have in vitro GEF
activity that is completely resistant to the drug (Chardin et al., 1996). To
determine whether the Sec7d itself is responsible for specifying BFA
resistance/sensitivity, chimeric proteins were constructed. The Sec7ds of the
BFA-sensitive Gea1p and Sec7p GEFs were replaced with that of BFA-
resistant ARNO, and their sensitivity to BFA was determined in vivo. The
chimeras (Gea-ARNO-Gea1p and Sec7-ARNO-Sec7p) were expressed in
cells with chromosomal deletions of the wild type genes, and each
functioned like the endogenous proteins in terms of both growth and
secretion (Peyroche et al., 1999). Even a gea1-∆ gea2-∆ sec7-∆ strain
expressing Gea-ARNO-Gea1p and Sec7-ARNO-Sec7p as the sole copies of
Gea1/2p and Sec7p, respectively, grew normally. However, this strain was
now almost completely resistant to the effects of BFA. Both growth and
secretion in the presence of BFA were comparable to those of the untreated
strain (Peyroche et al., 1999). This result demonstrates that Sec7, Gea1p and
Gea2p are the major targets of BFA in the secretory pathway of yeast, that
the Sec7d itself is responsible for BFA sensitivity or resistance, and that the
cellular toxicity of BFA is directly attributable to its actions on these
proteins in yeast.

5.2.2 Residues in the Sec7 domain determine sensitivity to BFA

To determine residues important for BFA sensitivity, a portion of the GEA1

gene was mutagenized and BFA-resistant mutants were selected. All
mutants obtained had at least one amino acid substitution in a 35-amino-acid
region of the Gea1p Sec7d. Remarkably, this region also contained a high
frequency of residues that differed between BFA-resistant and BFA-
sensitive subfamilies, but were conserved between members within each
subfamily. Introducing one of these mutations, M699L, into the Sec7d of
Gea1p, expressed in E. coli, led to BFA-resistant exchange activity in vitro
(Peyroche et al., 1999). K. Lingelbach and colleagues isolated a BFA-
resistant malarial parasite line, and found that the mutation responsible for
conferring BFA resistance was present in a novel Plasmodium Arf GEF
(Baumgartner et al., 2001). Strikingly, the mutation was a Met→Ile
mutation in the residue corresponding to M699 of Gea1p.
Adjacent to the M699 residue are a pair of residues that differ between
BFA-resistant and BFA-sensitive classes of Arf GEFs. The double mutant
ARNO-YS190-191 was sufficient to convert ARNO into a protein with
BFA-sensitive Arf GEF activity in vitro (Peyroche et al., 1999). The Gea-
ARNO-YS190-191-Gea1p chimera, expressed in yeast, was made BFA-
sensitive, like wild type Gea1p (Peyroche et al., 1999). Hence, these
 THE SEC7 FAMILY OF ARF GEFS 81
11

residues are determinants of BFA resistance/sensitivity both in vitro and in

vivo. When the F→Y or A→S mutations were introduced separately into the
Gea-ARNO-Gea1p chimera, only the F→Y mutant was BFA-sensitive (A.
Peyroche and C. L. Jackson, unpublished observations), indicating that the
critical residue of this pair for BFA resistance/sensitivity is the tyrosine.
In a parallel study (Sata et al., 1999), chimeras were constructed between
the Sec7 domains of the BFA-sensitive Sec7p and BFA-resistant cytohesin-1
Sec7 domain. The different chimeras were purified and tested for in vitro
Arf GEF activity and their sensitivity to BFA. These authors narrowed
down a region (within the 35-aa region described above) that was
responsible for BFA resistance/sensitivity of these GEFs. In particular, two
residues that differed between BFA-resistant and sensitive GEFs were
identified as key for determining sensitivity to BFA. These were D965 and
M975 in Sec7p, which correspond to S199 and P209 in cytohesin-1 (Sata et
al., 1999).
Looking at an alignment of the Sec7ds of all of the human Arf GEFs, one
can ask whether it is possible to predict which ones will be BFA sensitive or
resistant based on these mutagenesis studies. Figure 3 shows the four
residues identified as being critical for determining BFA
resistance/sensitivity. Seven of the human Arf GEFs, those in the
ArfGEP100/BRAG, EFA6 and Fbx8 subfamilies, have a leucine at the
position equivalent to M699 of Gea1p (Figure 3), so would be predicted to
be BFA resistant. Although these seven human Arf GEFs all have aspartates
and methionines at the positions equivalent to cytohesin S199 and P209,
respectively, corresponding to those found in BFA-sensitive GEFs, none
except Fbx8 have a tyrosine at the position corresponding to Y695 in Gea1p
(F190 in ARNO/F191 in cytohesin-1) (Figure 3). Indeed, ArfGEP100 and
EFA6 have activity resistant to BFA in vitro (Someya et al., 2001; Franco et
al., 1999). Of the remaining GEFs, the cytohesin/ARNO/GRP subfamily has
been extensively studied and shown to be BFA-resistant (Jackson and
Casanova, 2000). Hence, only BIG1, BIG2 and GBF1 would be predicted to
be BFA-sensitive human Arf GEFs (Figure 3). Although GBF1 has been
described as a BFA-resistant GEF using Arf5 as a substrate, there is
evidence, at least in vivo, that GBF1 can act on class I Arfs as well. In this
case, it may well be that such activity towards a class I Arf can be inhibited
by BFA (Claude et al., 1999; Kawamoto et al., 2002).

5.2.3 Structure of the Sec7 domain-BFA-Arf complex

The crystal structure of a Sec7d bound to nucleotide-free Arf reveals that

the residues important for BFA resistance/sensitivity lie in the heart of the
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Arf binding region. This result might imply that BFA acts as a competitive
inhibitor, binding to the Sec7d and preventing Arf from binding. However,

Figure 3. Alignment of a portion of the Sec7 domains of the human Arf GEFs. Identical
residues are boxed and homologous residues shaded blue. White letters on black background
indicates residues important for determining BFA resistance/sensitivity. The asterisk denotes
the catalytic glutamic acid residue (Béraud-Dufour et al., 1998).

the mechanism of action of this drug turns out to be much more unusual.
BFA does not bind to either Arf or the Sec7d alone, but to a normally very
short-lived reaction intermediate, an Arf•GDP-Sec7d complex (Mansour et
al., 1999; Peyroche et al., 1999; Robineau et al., 2000) (Figure 4). This
Arf•GDP-BFA-Sec7d quaternary complex is relatively stable, with a
dissociation rate (τoff) for BFA of 30 min (Robineau et al., 2000). If BFA
binds to an Arf•GDP-Sec7d protein complex, residues of Arf would be
expected to influence BFA binding. Indeed, B. Antonny and colleagues
identified a mutation in the Switch II region, Arf1-A80, which results in a
decrease in the dissociation rate of BFA from the complex (Robineau et al.,
2000). Another striking feature of the mechanism of action of BFA is the
type of complex stabilized by the drug. The only complex between a small
G protein and its GEF that normally can be isolated is one in which
nucleotide has been lost from the complex. Robineau et al. demonstrate that
BFA does not bind to such a complex (Robineau et al., 2000). The unusual
mechanism of action of BFA provides a paradigm for development of novel
drugs. Instead of the usual search for drugs competing for a given substrate,
 THE SEC7 FAMILY OF ARF GEFS 83
13

one could design screens for drugs that block reactions through stabilization
of reaction intermediates (Chardin and McCormick, 1999).

Figure 4. Mechanism of action of brefeldin A (BFA).

6. MOLECULAR ANALYSIS OF THE ARF

EXCHANGE FACTORS

6.1 Protein Partners of Arf GEFs

An important step in determining the roles of Arf GEFs in cells is to identify

interacting partners. Several partners of the ARNO/cytohesin/GRP
subfamily have been identified. This work is less advanced for other
subfamilies of Arf GEFs, but one partner for the GNOM protein has been
reported (Grebe et al., 2000). An N-terminal region conserved in all
members of this subfamily, the DCB domain, is involved both in
dimerization of the GNOM protein, and in binding to cyclophilin (Grebe et
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14 JACKSON

al., 2000). The physiological relevance of this interaction is not known, but
it may represent an important regulatory mechanism. Indeed, interactions
between cyclophilins and regions of proteins involved in their
oligomerization have been previously reported (see Grebe et al., 2000) for
references).
Two-hybrid screens with the Sec7 domains of different Arf GEFs have
identified a surprisingly large number of potential partners. These results
support the idea that the catalytic domain itself is involved in regulatory
interactions. We identified the yeast TGN-localized protein Drs2p as a
direct binding partner of the Sec7d of Gea2p (S. Chantalat and C. L.
Jackson, manuscript in preparation). Drs2p is a 12-transmembrane domain
P-type ATPase that likely functions as an aminophospholipid translocase
(flippase) (Chen et al., 1999). Drs2p co-localizes with Kex2p in a late Golgi
compartment in yeast, and is required for the formation of clathrin-coated
vesicles. There are four other proteins in yeast that are grouped into the
same subfamily of P-type ATPases along with Drs2p, which have distinct
localizations and which function in different transport steps (Hua et al.,
2002).
The region of the Gea2p Sec7d that interacts with Drs2p was mapped by
a reverse two-hybrid approach. Interestingly, the residues identified as
important for the interaction are clustered in the C-terminal portion of the
Sec7d, adjacent to the Arf binding site (C. Jackson, unpublished
observations). This region is quite divergent among different Arf GEFs, and
may represent a region generally used for regulation of Arf GEF activity.
Screens with different Sec7 domains identified a number of partners (E.
Smirnova, S. Chantalat, and C. L. Jackson, unpublished observations) and in
at least two other cases, this same C-terminal portion of the Sec7d was found
to be important for interaction. This C-terminal region is also the region in
which residues affecting BFA resistance/sensitivity were found (Peyroche et
al., 1999). Thus, the C-terminal portion of the Sec7d is important for the
binding of other proteins and likely regulation of Arf GEF activity.

6.2 Interaction with Membranes

Members of the ARNO/cytohesin/GRP and EFA6 subfamilies have PH

domains that mediate membrane binding through specific interaction with
phosphoinositides (Jackson and Casanova, 2000). It is not known if the PH
domains are the only localization determinants, or whether other
mechanisms exist for specific membrane targeting of these GEFs. Members
of the ArfGEP100/BRAG subfamily also have PH-like domains, but their
sequences are quite different than those for the subfamilies mentioned
above, and it is not known if they mediate membrane binding (Someya et al.,
 THE SEC7 FAMILY OF ARF GEFS 85
15

2001). There are no easily identifiable PH domains present in the other

subfamilies of Arf GEFs, so the mechanism of membrane localization of the
Sec7/BIG and Gea/GBF/GNOM proteins remains a major question.
For the Gea/GBF/GNOM subfamily of Arf GEFs, is Drs2p likely to be a
major membrane localization determinant? Our data for the yeast proteins
indicates that there must be additional mechanisms for localization, since the
Gea2p protein is still localized in cells with a complete deletion of the DRS2
gene. Subcellular fractionation of wild type and drs2∆ cells shows the same
profile for Gea2p, again suggesting that another mechanism for localization
exists (S. Park and C. L. Jackson, unpublished observations). A novel
transmembrane domain partner of the Gea1p and Gea2p proteins, Gmh1p,
has been identified, which may be involved in localization of the Gea1p and
Gea2p proteins to the Golgi apparatus (Chantalat et al., 2002). Gmh1p has
orthologs in all organisms examined to date, and the human homolog,
hGmh1, localizes to the Golgi complex in mammalian cells (Chantalat et al.,
2002).

7. ROLE OF THE ARF GEFS IN PROTEIN AND

MEMBRANE TRAFFIC

The molecular mechanism of action of BFA indicates that the effects of this
drug on cells is a result not only of failure to activate Arf, but also of
trapping sensitive Arf GEFs in an inactive complex. As described above,
BFA exerts profound effects on both the structure and function of organelles
along the secretory and endocytic pathways. Understanding precisely how
BFA affects traffic has never been easy in the context of the classic models
of intracellular traffic (Griffiths, 2000; Klausner et al., 1992; Sciaky et al.,
1997), and suggests that new ways of understanding membrane dynamics
are needed.

7.1 Membrane Transformation Events that Accompany

Traffic through the Golgi Apparatus

The current unifying principle for all traffic events in eukaryotic cells is the
idea that vesicles carrying cargo bud from a donor compartment membrane,
then target to and fuse with an acceptor compartment. This paradigm is
based on the idea that transport pathways in the cell are divided into distinct
compartments defined by a set of characteristic resident proteins.
Morphological studies based on electron microscopy (EM) of ultra-thin
sections do not provide evidence to contradict this view of transport.
However, thin section EM has significant limitations, which prompted two
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researchers to develop alternative methods to probe the structures of the

endomembrane systems of cells (Rambourg and Clermont, 1990).
Beginning in 1974, Alain Rambourg, Yves Clermont and colleagues
developed novel EM techniques that allowed accurate visualization of larger
segments of eukaryotic cells than had traditionally been viewed at the
ultrastructural level. They used selective impregnation methods that
provided high levels of contrast between the membrane and the surrounding
cytoplasm. Thick sections (from 200 nm to over 1µm) were observed with
the aid of stereoscopic techniques. These techniques permitted the
visualization of the three-dimensional structure of cellular organelles
(Rambourg and Clermont, 1990). Rambourg and Clermont demonstrated
that the Golgi apparatus of mammalian cells does not consist of independent,
separate compartments (stacks of saccules) but rather is one continuous
entity with the appearance of a continuous ribbon (Rambourg and Clermont,
1997). The cis compartment (or cis-tubular network) consists of a system of
closely anastomosed tubules, which form a network of regular polygonal
mesh. The medial compartment consists of stacks of saccules interconnected
by intersaccular tubular zones. Each stack of saccules is formed by closely
superposed flattened elements (saccules), poorly fenestrated, and strictly
parallel with each other. The trans compartment comprises several
superposed sacculotubular elements which appear as independent units along
the main axis of the Golgi ribbon and curve away from the latter into the
cytoplasm as if they were peeling off (Figure 5; Rambourg and Clermont,
1997).
In secretory cells, the secretory material is first uniformly distributed in
the lumen of the Golgi elements. This material then begins to accumulate in
dilations located at apparently random locations of a saccular element of the
compact zone situated at any level along the cis-trans axis of the Golgi
ribbon (although most commonly in a saccule of the trans compartment)
(Figure 5; Rambourg et al., 1987). The drainage of the secretory material
towards these dilations is accompanied by a progressive perforation and
tubulation of the large saccular portions of the trans element (Rambourg et
al., 1987). Hence, segregation of the secretory material (which results in
formation of a progranule from the original dilation) is tightly coupled to
membrane transformation (from saccular to fenestrated to tubular). When the
tubular elements connecting the dilations/progranules become devoid of
secretory content, they fragment to liberate the pro-secretory granules at the
trans face of the Golgi apparatus (Figure 5). This tubulation of the saccular
portions which accompanies the segregation and maturation of the secretory
material, and the subsequent rupture of the tubular systems at the trans face
of the Golgi apparatus are the manifestations of an intense level of protein
 THE SEC7 FAMILY OF ARF GEFS 87
17

and lipid sorting activity at this level of the organelle (Rambourg et al.,
1987; Robinson and Bonifacino, 2001).
The membranes of the Golgi apparatus are the object of permanent
reorganization, as demonstrated by studies of Golgi structure under
conditions of physiological stimulation and inhibition of secretion. In
prolactin cells of the pituitary gland of lactating rats or acinar cells of the
mammary gland of such animals, the Golgi rapidly breaks down when the
secretion stimulus is removed, and then reforms within minutes of the pups
being returned to their mother for lactation (Clermont et al., 1993;
Rambourg et al., 1993). The results demonstrate that the Golgi apparatus
arises as a result of continual renewal at the cis side, and consumption at the
trans side.
Contrary to what is observed in mammalian cells, the Golgi apparatus of
S. cerevisiae comprises approximately thirty independent units, distributed
seemingly randomly throughout the cytoplasm (Rambourg et al., 2001).
Each unit possesses a structure that is exclusively tubular and in the form of
small tubular networks of polygonal meshes (Rambourg et al., 2001). There
are at least two reasons for the dispersed nature of Golgi elements in S.
cerevisiae, as opposed to mammalian cells (or even other yeasts such as
Pichia pastoris). First, S. cerevisiae lacks the microtubule-dependent system
that carries ER-Golgi transport intermediates to the pericentriolar region
(Huffaker et al., 1988). Second, there are more ER exit sites present in S.
cerevisiae than in other organisms because the former lacks a system that
assembles exit sites together at the level of the ER (Rossanese et al., 1999).
However, despite the differences, the fundamental process of secretion is
highly conserved between S. cerevisiae and higher eukaryotic cells (Duden
and Schekman, 1997; Ferro-Novick and Jahn, 1994; Kaiser and Huffaker,
1992).
Morphological analysis of wild type S. cerevisiae strains reveals that
formation of secretory vesicles/granules occurs by a mechanism remarkably
similar to that in mammalian cells. Nodular swellings resembling secretion
vesicles/granules form at the intersections of polygonal meshes made up of
interconnected tubules (Rambourg et al., 2001). In structures representing
the next stage of development, nodules that have the appearance of fully
formed secretory vesicles are connected by very fine tubes, and are adjacent
to free secretory vesicles (Rambourg et al., 2001). Thus, it is likely that in
yeast, as in mammalian cells (Rambourg and Clermont, 1997), the liberation
of secretion vesicles/granules occurs by rupture of tubular areas and not by
budding from the edges of saccular elements.
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Figure 5. Diagram showing a small section of the Golgi ribbon in cross section (top).
Selected elements are illustrated in the three dimensional depiction shown in the lower panel.
ER, endoplasmic reticulum; CE, cis element; TTN, trans-tubular network; Pg, progranule; g,
granule. Taken with permission from Rambourg et al., 1987.

7.2 Temperature-sensitive mutants in yeast: Evidence

For Vectorial Forward Membrane Flow

In the original selection for secretory pathway mutants (sec mutants) in S.

cerevisiae, a mutation in the gene encoding what would turn out to be the
founding member of the Arf GEF family was isolated (Novick et al., 1981;
Novick et al., 1980). At the restrictive temperature (37oC), this mutant,
sec7-1, has a block in the secretory pathway at the level of exit from the
Golgi apparatus (Franzusoff and Schekman, 1989; Julius et al., 1984;
Nishikawa et al., 1990; Novick et al., 1981; Stevens et al., 1982). At steady
 THE SEC7 FAMILY OF ARF GEFS 89
19

state, Sec7p localizes primarily to late Golgi compartments, partially co-

localizing with Kex2p, a marker protein for the yeast TGN (Franzusoff et al.,
1991). In the sec7-1 mutant over a time course of incubation at 37oC, the
Golgi tubular networks were progressively transformed into stacks of
saccules whose superimposed elements were interconnected along a cis-
trans axis to form a membrane structure that is anatomically continuous
(Figure 6; Rambourg et al., 1993). This process can be divided into several
steps. First, after only seven minutes at 37oC, one or a few of the numerous
Golgi tubular networks became more extensive, but with the same type of
organization as in a wild type strain. At this time, secretory
vesicles/granules were rarely seen (Figure 6B). After 15 minutes at the
restrictive temperature, the tubular networks within a single Golgi element
had become aligned in a parallel fashion. Some cells had accumulated
highly fenestrated cisternae, suggesting that the tubular elements, once
aligned in parallel, had begun to undergo filling in of the tubular mesh to
form fenestrated saccules (tubulation- fenestration transition) (Figure 6C).
After 30-60 minutes, only a few Golgi elements per cell were seen, and all
were composed of five to eight stacked, poorly fenestrated saccules. Hence
the next membrane transformation step, fenestration to saccularization, had
been completed (Figure 6D). Morphologically, these Golgi elements closely
resemble Golgi stacks in mammalian cells (Rambourg et al., 1993). That
these structures which accumulate in the sec7-1 mutant are bone fide Golgi
elements is supported by the fact that Golgi-localized proteins such as Ypt1p
(Segev et al., 1988), Rer1p (Sato et al., 1995), Vps1p (Rothman et al., 1990)
and the TRAPP ER-Golgi vesicle-targeting complex (Sacher et al., 1998), as
well as proteins in transit through the secretory pathway such as DPAP B
(Roberts et al., 1989), are associated with them.
The Arf GEFs, through activation of Arf, act in the process of budding of
a transport vesicle from a donor membrane. However, as illustrated by
Rothman and Warren, vesicle budding is topologically equivalent to fission
of tubules and fenestration of cisternal membranes (see Figures 5 and 6 in
(Rothman and Warren, 1994)). The sec7-1 mutant has a phenotype
consistent with defects in breaking of tubules and in fenestration of
cisternae. Interestingly, BFA has a very different phenotype. In both S.
cerevisiae and mammalian cells, BFA causes the Golgi to disappear rather
than accumulate. These morphological results are consistent with the
molecular differences in the two lesions. BFA binds to the Sec7d of
sensitive Arf GEFs such as yeast Sec7p, whereas mutations in the sec7-1
mutant protein lie outside of the Sec7d (Deitz et al., 2000). It would be
interesting to know if the residues mutated in sec7-1 are involved
specifically in interaction with proteins that mediate membrane tubule
breakage and saccule fenestration.
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Figure 6. Diagrammatic representation of the modifications of the Golgi apparatus of an S.

cerevisiae sec7-1 mutant following shift to the non-permissive temperature of 37oC. A) A
Golgi saccule (S) from a wild type S. cerevisiae strain or a sec7-1 mutant at the permissive
temperature of 24oC. B) A Golgi element (S) in a sec7-1 mutant at 37oC for 7.5 to 15 minutes.
Thin plates (P) of electron-dense material appear at this stage. C) A transverse section through
a stack of saccules in the sec7-1 mutant maintained at 37oC for 15-30 minutes. Four flattened
saccules (S), interconnected with each other, are stacked and separated by a space showing
thin plates (P) of electron-dense material. D) A transverse section through a Golgi stack from
the sec7-1 mutant incubated at 37oC for 30 min to 1 hour. Fenestrated spheres (FS) often
appear at this stage. Taken with permission from Rambourg et al., 1993.

In vivo morphological analysis of ts mutants in the Gea1p-Gea2p Arf

GEF pair also support a role for Arf GEFs both in Golgi structure and in
 THE SEC7 FAMILY OF ARF GEFS 91
21

regulation of vectorial membrane flow. Gea1p and Gea2p have redundant

functions that are largely interchangeable (Peyroche and Jackson, 2001;
Peyroche et al., 1996; Spang et al., 2001). Single mutant strains have no
growth or secretion phenotypes, whereas a gea1∆ gea2∆ strain is inviable
(Peyroche et al., 1996). In three ts mutants, gea1-4, gea1-6 and gea1-19,
proteins exit the ER, but are blocked or slowed at an earlier stage in
transport than for sec7-ts mutants. All of the gea-ts mutants have severe
defects in glycosylation, much more severe than in sec7-ts mutants
(Peyroche et al., 2001).
Interestingly, gea1-4 accumulated larger-than normal Golgi tubular
networks, but for the most part their structure was similar to wild type Golgi
elements (Peyroche et al., 2001). The slow-down in transport in the gea1-4
mutant occurred at a point prior to that in the sec7-1 mutant, after exit from
the ER but before Golgi exit. The structures that accumulated were also
upstream in the membrane transformation pathway compared to those
accumulated in the sec7-1 mutant. In contrast, gea1-6 and gea1-19 had
essentially complete blocks to transport at the level of the cis-Golgi. In these
mutants, no Golgi tubular network structures accumulated, but rather
unfenestrated membrane structures (Peyroche et al., 2001).
Like mutations in the Arf GEFs, mutations in Arf itself have phenotypes
consistent with an important role for this protein in regulation of vectorial
membrane flow, and in membrane fenestration and tubulation in yeast (Deitz
et al., 2000; Rambourg et al., 1995). The arf1∆ mutant is viable at all
temperatures, but grows more slowly, especially at low temperatures. This
mutant has defects in glycosylation and the rate of secretion of all proteins
traversing the secretory pathway is slowed (Gaynor et al., 1998). Even more
pronounced than the transport defects are marked alterations in Golgi and
endosome structure (Deitz et al., 2000; Gaynor et al., 1998; Rambourg et al.,
1995). Again, slowing of the rate of secretion results in an accumulation of
larger-than-normal Golgi structures. Isolation of a number of randomly
generated ts mutants in ARF1 showed that yeast Arf acts at multiple steps of
intracellular transport, including ER-Golgi, Golgi-endosome, endosome-
vacuole and Golgi-PM (Yahara et al., 2001). Analysis of these mutants is
consistent with Arf regulating both organelle structure and protein transport
at each of these steps (Yahara et al., 2001).
The studies described above show that understanding the roles of Arf
GEFs even at the single cell level is currently a challenge. An even bigger
challenge is understanding the roles of these proteins at the level of the
organism, for example in development. The most information to date on this
subject has come from studies of Arabidopsis thaliana. The gnom mutant
was identified as a pattern-formation mutant, and has defects in polarized
growth and asymmetric cell division (Mayer et al., 1993). GNOM is
92
22 JACKSON

involved in polarized secretion of auxin, a plant growth hormone (Geldner et

al., 1999). Polar auxin transport is important for a number of developmental
processes in plants, and in Arabidopsis, is dependent on the polarized
localization of PIN1, an auxin efflux carrier. PIN1 localization is severely
perturbed in gnom mutant embryos, and is also disrupted by BFA treatment
(Geldner et al., 2001; Steinmann et al., 1999). Since GNOM is a BFA-
sensitive Arf GEF, both of these lines of evidence indicate that GNOM-
dependent membrane traffic is important for polarized localization of PIN1
and thus polarized auxin secretion. Interestingly, in contrast to Gea2p and
GBF1, GNOM does not appear to be localized primarily to the Golgi
apparatus at steady state. Instead, GNOM is associated with multiple
membrane compartments, including the plasma membrane (Steinmann et al.,
1999).

8. FUTURE DIRECTIONS

This is an exciting time for research in the Arf GEF field. The profound
effects of BFA on membrane systems in eukaryotic cells demonstrate that its
targets, the Arf GEFs, are central regulators of organelle structure and
function. The Arf GEFs are all multidomain proteins that, as is the case for
the Rho family of GEFs, likely integrate upstream signals with the
downstream effects of GTPase activation. Molecular analysis of the Arf
GEFs is just now getting underway, and should soon lead to the
identification of membrane localization determinants, and both positive and
negative regulators of exchange activity.
Analysis of membrane systems both by electron and fluorescence
microscopy leads to the concept that regulated membrane flow is an
essential aspect of membrane dynamics in eukaryotic cells. In vivo analysis
of yeast Arf GEF mutants shows that a specific type of lesion in different
Arf GEFs leads to accumulation of larger-than-normal Golgi structures. In
these cases, the type of structure that accumulates depends on the point in
the secretory pathway that is inhibited by the mutation. It will be interesting
to determine whether these lesions abolish interaction with a particular
protein or membrane partner. A given structure is a manifestation, at a given
time and place, of an underlying dynamic process. The challenge for the
future is to understand this process at the molecular level, and the role of the
Arf GEFs in integrating upstream signals with the generation of dynamic
membrane structures.
 THE SEC7 FAMILY OF ARF GEFS 93
23

9. ACKNOWLEDGEMENTS

I thank Alain Rambourg and Yves Clermont for many stimulating

discussions, and for allowing me to present their views. I apologize to
authors whose work could not be cited due to space limitations.

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C. J. (1995). The sequence of 24.3 kb from chromosome X reveals five complete open
reading frames, all of which correspond to new genes, and a tandem insertion of a Ty1
transposon. Yeast, 11, 1179-1186.
Zhu, Y., Traub, L. M., and Kornfeld, S. (1998). ADP-ribosylation factor 1 transiently
activates high-affinity adaptor protein complex AP-1 binding sites on Golgi membranes.
Mol. Biol. Cell, 9, 1323-1337.
Chapter 5
LARGE ARF GEFS OF THE GOLGI COMPLEX
In search of mechanisms for the cellular effects of BFA

PAUL MELANÇON, XINHUA ZHAO, AND TROY K. R. LASELL

Department of Cell Biology, University of Alberta, Edmonton, Canada

Abstract: Class I and class II Arfs localize to the Golgi complex where they activate
lipid-modifying enzymes and also promote the recruitment of several coat
proteins implicated in the sorting of cargo into budding transport vesicles.
Three families of large GEFs spatially and temporally regulate Arf activation
in this organelle. GBF1, a BFA-resistant GEF with specificity for Class II
Arfs in vitro, localizes to cis-compartments of the Golgi and appears to
regulate assembly of the COPI coat. In contrast, BIG1 and BIG2 are BFA-
sensitive GEFs that concentrate on trans-compartments where they control the
recruitment of clathrin adaptor proteins. BRAG1, a representative of the third
family, is confined to early Golgi compartments, but unlike GBF1 displays
broader Arf substrate specificity. These large GEFs are likely multidomain
proteins with complex interactions responsible for regulating activity,
membrane association and possibly the selection of downstream effectors.
Ongoing efforts in several laboratories focus on the identification of partners
for these GEFs.

1. INTRODUCTION

Dynamic and bi-directional transport pathways link organelles of the central

vacuolar system of eukaryotic cells. The efficient and selective transport of
proteins between these organelles is made possible by a complex molecular
machinery that drives the budding, targeting and fusion of transport vesicles
into which cargo and specific address molecules are selectively inserted
(Antonny and Schekman, 2001; Spang, 2002). The initial sorting and
budding processes are driven by the recruitment of specific coat proteins that
are spatially and temporally regulated by small GTPases of the Arf and
Sar1p families. The coatomer complex, or COPI, directs retrograde
movement of escaped ER proteins from the Golgi and the vesiculo-tubular
101
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2 MELANÇON, ZHAO, AND LASELL

clusters (VTCs) of the ER-Golgi Intermediate Compartment (ERGIC) that

ferry cargo to the Golgi stack; it has also been implicated in transport of
cargo across the Golgi stack (Marsh and Howell, 2002; Pelham and
Rothman, 2000). In agreement with such diverse functions, COPI localizes
to several compartments, with greatest abundance in VTCs and cis-elements
of the Golgi stack (Griffiths et al., 1995; Oprins et al., 1993). In contrast, the
clathrin coat directs transport of specific cargo to endosomes. Several types
of adaptor complexes, or APs (see Chapters 12 and 13), regulate association
of clathrin with membranes of the trans-Golgi network and endosomes (Hirst
and Robinson, 1998; Robinson and Bonifacino, 2001).
An approach to identify and characterize components of this transport
machinery has involved elucidating the mechanism of action of the drug
Brefeldin A (BFA). BFA is a fungal metabolite that blocks protein secretion
and dramatically alters the morphology of several organelles of the secretory
pathway (Jackson, 2000). Current thinking is that these effects result from
blocking the activation of Arfs by BFA-sensitive GEFs, which in turn
prevents assembly of several cytoplasmic coats (Donaldson et al., 1992;
Helms and Rothman, 1992; Randazzo et al., 1993). Efforts to identify those
cellular targets and describe how BFA disrupts so many aspects of protein
traffic led to the characterization of 3 families of large Golgi-associated Arf
GEFs termed Golgi-specific BFA resistance Factor 1 (GBF1), BFA-
Inhibited GEFs (BIGs), and BFA-Resistant Arf GEFs (BRAGs). As
summarized below, those studies paint a surprisingly more complex picture
than was suggested from in vitro studies. Furthermore, they demonstrate
that large Arf GEFs all localize to the Golgi complex but have distinct
properties that may explain how Arfs can simultaneously regulate the
function of so many distinct processes in the Golgi complex. As the
properties of yeast Arf GEFs were reviewed in Chapter 4, we will focus on
results obtained with mammalian systems.

2. BFA-RESISTANT CELL LINES: A STARTING

POINT

2.1 Resistance to BFA cytotoxicity correlates with

stabilization of the COPI coat

Several years ago, we and others set out to develop a genetic approach to
identify the cellular target(s) of BFA (Yan et al., 1992; Yan et al., 1994).
We generated, by chemical mutagenesis, several mutant lines of Chinese
hamster ovary (CHO) cells resistant to BFA in order to better understand the
effects of this drug on various organelles. These studies led to the isolation
 LARGE ARF GEFS OF THE GOLGI COMPLEX 1033

of 24 CHO mutant lines (termed BFY1-24) that can grow in the presence of
BFA concentrations (1-2 µg/ml) 5-10 fold greater than the IC50. These BFY
lines displayed organelle-specific resistance to BFA: whereas the Golgi
complex maintained its appearance and function at high BFA concentrations,
the endosomal system retained sensitivity (Yan et al., 1994). A similar
selection procedure by Nakayama and colleagues led to isolation of several
BFA resistant CHO lines termed BAR1-30 (Torii et al., 1995). In these
mutant cells, COPI showed reduced sensitivity to BFA whereas γ-adaptin
was released at BFA concentrations similar to those effective in the parental
line. These two studies provided direct evidence for the presence of
multiple, organelle-specific targets for BFA. In addition, they demonstrated
that the cytotoxicity of BFA correlated with effects on the COPI coat and the
Golgi complex and not on events associated with the TGN or endo-
lysosomal systems. We concluded that characterization of factors that alter
BFA cytotoxicity would lead to the discovery of proteins involved in
regulation of the COPI coat, and the possible identification of those Arf
GEFs or effectors impacting cell viability.

2.2 BFY cells express a BFA resistance factor

As a first step towards understanding the mechanism of resistance, we chose

some of the most resistant lines for detailed biochemical analysis. BFY1
and BFY2 cells display normal Golgi morphology at a BFA concentration as
high as 20 µg/ml and have been the focus of this analysis (Yan et al., 1994;
Yan and Melançon, 1994). This work led to the unexpected discovery of a
dominant activity that confers BFA resistance to several in vitro assays and
is present in both cytosolic and membrane fractions. We then established
that this “resistance factor” was clearly distinct from Arfs and coat proteins
and acted either at or upstream of the nucleotide exchange reaction that
activates Arfs and initiates coat recruitment. The observation that the BFA
resistance factor acted in a dominant manner was particularly important
since it demonstrated the feasibility of expression cloning as a method for its
identification.

3. GBF1: A GOLGI-SPECIFIC BFA RESISTANCE

FACTOR

3.1 Identification of GBF1

To identify the BFA resistance factor, we constructed cDNA libraries from

mutant BFY1 and BFY2 cells using an episomal vector that allowed
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4 MELANÇON, ZHAO, AND LASELL

recovery of stable HEK-293 transformants at high frequency (Claude et al.,

1999). Plasmids recovered from populations of BFA-resistant transformants
were screened individually for their ability to confer BFA-resistance. This
procedure yielded a single plasmid encoding the putative BFA “resistance
factor”. This Golgi-specific BFA resistance factor, or GBF1, appears to be
the only one that can be recovered by this approach since several subsequent
selections with either the BFY1 or BFY2 libraries yielded the same cDNA.
Sequence analysis revealed a novel 210 kDa protein with a central sec7
domain (Sec7d) which is now known to catalyze nucleotide exchange on
Arfs and is present in all characterized Arf GEFs (see Chapter 2). As a first
test of GBF1 function, we prepared Golgi-enriched membrane fractions from
cells overexpressing the cDNA and confirmed that those membranes now
displayed BFA-resistant Arf-specific GEF activity.

3.2 BFY and parental cells express similar levels of

multiple splice forms of GBF1

Two approaches established that BFY cells express wild-type GBF1. First,
we constructed a full-length cDNA for human GBF1 and characterized it
(Mansour et al., 1998). To our surprise, moderate overexpression of the
wild-type protein led to BFA resistance similar to that observed with the
GBF1 recovered from mutant CHO cells (S. Mansour and P. Melançon,
unpublished observation). In parallel, we cloned and sequenced several full-
length cDNAs from a library prepared from the parental CHO line (Zhao et
al., 2002a). Analysis of these GBF1 transcripts revealed no point mutations
but did identify three nucleotide positions (at bp 1010, 1864 and 4479 in the
open reading frame) displaying frequent in-frame deletions or insertions. A
combination of genomic DNA sequencing, RT-PCR and biological assays
demonstrated that (1) the small variations were consistent with the use of
alternative splicing sites, (2) the splice variants were made at similar
frequencies in wild-type and BFA-resistant cells, and, (3) those variants
displayed activity identical to the original GBF1 cDNA (Zhao et al., 2002a).
These results led us to conclude that contrary to expectations, GBF1 was not
mutated in BFY cells and that its isolation in our screen resulted from its
intrinsic ability to induce BFA resistance when overexpressed. Interestingly,
overexpression of the BFA-sensitive Geas similarly induces BFA resistance
(Peyroche et al., 1999). This property appears unique to the GBF1/Gea sub-
family of Arf GEFs: overexpression of BIGs does not reduce BFA
sensitivity (see section 6.2 below) while that of ARNOs actually mimics
BFA and causes dispersal of the COP1 coat (Franco et al., 1998; Monier et
al., 1998).
 LARGE ARF GEFS OF THE GOLGI COMPLEX 1055

3.3 GBF1 is a BFA resistant GEF with specificity for

Class II Arfs in vitro

The guanine nucleotide exchange activity of GBF1 was first confirmed by

using a His6-tagged form of GBF1 expressed in HEK-293 cells (Claude et
al., 1999). As expected, GBF1 displayed a robust and BFA-resistant GEF
activity. However, whereas GBF1 activated both Arf1 and Arf5 at µM
concentrations of MgCl2, under more physiological (> 1 mM) MgCl2
concentrations GEF activity was only observed with Arf5. The Nakayama
group also cloned GBF1 and recently confirmed this result using crude,
recombinant GBF1 and Arf (Kawamoto et al., 2002). Interestingly,
overexpression of GBF1 was found to promote recruitment of both Class I
and Class II Arfs in intact cells, suggesting that the substrate specificity of
GBF1 may be different in vivo.

Figure 1. Alignment of Sec7d of several GEFs showing residues of motif 2 implicated in

sensitivity to BFA. Residues found to strongly correlate with BFA resistance and sensitivity
are shown on first and second line, respectively. Positions marked with asterisk correspond to
mutations shown to affect BFA sensitivity.

The BFA resistance observed in vitro is actually surprising considering

the sequence of GBF1. Mutational analysis and comparison of several
Sec7d proteins identified several residues involved in BFA binding
(Peyroche et al., 1999; Sata et al., 1999). These residues are located in or
near a conserved hydrophobic helix and participate in the flexibility of the
C-terminal region of the Sec7d that appears critical for formation of the
complex with BFA (Renault et al., 2002). The consensus motif for BFA
resistant proteins is FAVIMLNTSL-X7-KP and that for BFA sensitive
proteins is YSIIMLTTDL-X7-KM (see Figure 1). The hydroxyl moiety of
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6 MELANÇON, ZHAO, AND LASELL

the tyrosine residue may be particularly crucial as it makes a key contact in

the BFA-Arf•GDP-GEF complex. Not surprisingly, the FY mutation is
sufficient to convert the Arf GEF activity of the Sec7d of ARNO from BFA-
resistant to BFA-sensitive (see Chapter 4 for details). The fact that motif 2
of GBF1 (YAVIMLNTDQ-X10-PM) contains the critical tyrosine would
suggest a BFA-sensitive activity. The presence of three additional amino
acid residues within motif 2, relative to the consensus sequence (see Figure
1), may account for the BFA resistance and unique Arf specificity of GBF1.

3.4 GBF1 associates with Golgi membranes

As observed for most Arf GEFs, GBF1 is a peripheral membrane protein

(Claude et al., 1999). GBF1 is readily detected on membranes (Kawamoto
et al., 2002), however, careful analysis of proportional amounts of cytosol
and membrane fractions established that the microsome fraction corresponds
to <10% of the total GBF1 in the post-nuclear supernatant (Claude et al.,
1999). A proteomics-based analysis of Golgi-associated proteins identified
GBF1 in the detergent phase of a TX-114 extraction of a highly stacked rat
liver Golgi fraction (Bell et al., 2001). These results suggest that a fraction
of membrane-associated GBF1 may be tightly bound to an integral
membrane protein of the Golgi complex. This conclusion is supported by
analysis of the membrane-association of GNOM, one of several orthologs of
GBF1 encoded by the A. thaliana genome. GNOM is a BFA-sensitive GEF
that can use Arf1 as a substrate and can complement loss of Gea function in
a ¨gea2/gea1-19 temperature-sensitive strain (Steinmann et al., 1999). This
study revealed that GNOM was released from microsomes by TX-100 and
urea, but not by incubation with buffers of high salt concentration or alkaline
pH. It may be important to note that treatment with BFA causes a
significant increase in the extent of GNOM membrane association
(Steinmann et al., 1999).
GBF1 co-localized with well-characterized Golgi markers to perinuclear
structures, as determined by indirect immunofluorescence using antiserum
raised against a C-terminal peptide (Claude et al., 1999). This result was
confirmed using in situ immunogold electron microscopy on rat hepatocyte
cryosections. Quantitative analysis of these images established that even
though GBF1 was present at the highest density on Golgi membranes, the
majority of it was associated with a smooth tubular compartment adjacent to
Golgi stacks that correspond to the ERGIC in this tissue (Claude et al.,
1999). Subsequent analysis of rat parotid cryosections by Nakayama and
colleagues confirmed this clear enrichment in vesicular and tubular
structures in the vicinity of the Golgi stack (Kawamoto et al., 2002). In both
studies, gold particles were also observed on ER-like elements suggesting
 LARGE ARF GEFS OF THE GOLGI COMPLEX 1077

that GBF1 may cycle between the Golgi complex and the ER. Recent
observations with a new antibody (9D2), raised against a fragment of GBF1,
support the possibility that GBF1 functions on membranes in transit to the
Golgi complex. Contrary to what is observed with most of our sera (e.g., see
Figure 2A), this new antiserum reveals not only the expected juxtanuclear
pattern but also shows a number of peripheral puncta (arrows in Figure 2B).

Figure 2. GBF1 localizes to the Golgi complex (A; 9D5) and peripheral puncta (B; 9D2).

3.5 GBF1: a homodimer of unknown domain structure

As is the case for large GEFs that regulate other small GTPases, such as
SOS (Hall et al., 2002) and Dbl (Hoffman and Cerione, 2002), GBF1 is
likely to be a multidomain protein with complex interactions responsible for
regulating its membrane association and activity. Unfortunately, with the
exception of the central Sec7d and some proline-rich regions near the C-
terminus, the GBF1 sequence does not contain recognizable protein motifs.
The only other characterized domain regulates dimerization and was
identified through characterization of GNOM. The possibility that GNOM
is a homodimer was first suggested by unusual allelic complementation
between three classes of GNOM alleles (Busch et al., 1996). Subsequent
characterization using a yeast two-hybrid assay confirmed that GNOM is a
homodimer and established that the region responsible for dimerization is
present at the N-terminus (residues 1 to 246), in the region encoded by the
first exon (Grebe et al., 2000). This region is also able to bind cyclophilin-5,
a protein with folding activity that may be important for regulation of
GNOM dimerization and function. This dimerization and cyclophilin-
binding, or DCB domain, is also found in Geas and GBF1 but its function in
these proteins has not been confirmed. However, GBF1 and Geas likely
function as dimers because other well-characterized Arf GEFs such as BIGs
(Yamaji et al., 2000) and ARNOs (Chardin et al., 1996) clearly form dimers.
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To identify regions implicated in membrane recruitment, a large number

of tagged forms of various N- and C-terminal truncations were constructed.
Contrary to what was observed with BIG1 and BIG2 (see section 4 below),
this approach proved unsuccessful as no chimeras localized to the Golgi
complex (S. Mansour, unpublished observations).

4. BIGS: GOLGI-LOCALIZED TARGETS OF BFA

To further investigate the mechanism of action of BFA, we cloned and

characterized the cDNA of human BFA Inhibited GEF1, or BIG1 (Mansour
et al., 1998). This 205 kDa GEF was first isolated from bovine brain
extracts by Joel Moss and colleagues on the basis of its ability to promote
guanine nucleotide exchange on purified Arfs (Morinaga et al., 1997;
Morinaga et al., 1996). It was later shown to co-purify as a heterodimer with
another BFA-sensitive Arf GEF with 60% sequence identity termed BIG2
(Togawa et al., 1999; Yamaji et al., 2000).

4.1 BFA is an non-competitive inhibitor of BIGs

Using in vitro Arf GEF assays, we first characterized the activity of a 39

kDa recombinant fragment that spans the central Sec7d of BIG1 (Mansour et
al., 1999). Previous work with ARNO (Chardin, 1996) had established that
this domain was sufficient to catalyze nucleotide exchange (see Chapter 2).
The N- and C-terminal boundaries of the BIG1 fragment were selected on
the basis of our experience with numerous fragments of GBF1 and the
presence of a sequence motif lying outside the Sec7d, termed the HUS box
(homology upstream of Sec7d), that is shared by several large Arf GEFs.
This fragment was particularly active and promoted nucleotide exchange on
Arfs at enzyme:substrate ratios as low as 1:100 (Mansour et al., 1999). In
contrast, full-length forms of BIGs or truncated forms of the yeast Sec7p
show much lower specific activity and necessitate near equimolar amounts
of enzymes and substrate (Sata et al., 1998; Togawa et al., 1999; Yamaji et
al., 2000). More importantly, this high specific activity allowed
confirmation that the Sec7d was the direct target of BFA. Kinetic analyses
established that BFA did not compete with Arf for interaction with BIG1
but, rather, acted as a non-competitive inhibitor that bound the BIG1-Arf
complex with a KI of 7 µM (Mansour et al., 1999). Peyroche et al. (1999)
further confirmed that BFA promoted formation of a stable complex
between Arf and a BFA-sensitive GEF that could be recovered by size-
exclusion chromatography. Interestingly, GDP remained associated with
Arf1 in this complex.
 LARGE ARF GEFS OF THE GOLGI COMPLEX 1099

4.2 BIGs: Golgi-localized BFA targets

To investigate the intracellular location of BIG1, we constructed tagged

forms of both full-length and various fragments of the protein. HA-tagged
full length BIG1 localized to tight ribbon-like juxtanuclear structures that
overlapped with the Golgi marker mannosidase II (Mansour et al., 1999).
Similar results were subsequently reported for BIG1 and tagged BIG2
(Shinotsuka et al., 2002b; Yamaji et al., 2000). Analysis of several truncated
forms established that fragments containing the N-terminal third of BIG1
contained sufficient information to direct localization to the Golgi complex
(Mansour et al., 1999). A similar analysis established that a tagged N-
terminal fragment containing the first 550 residues of BIG2 likewise
localized to the Golgi complex (Zhao et al., 2002b). It is not clear at this
point whether this domain contains targeting information or is responsible
for dimerization with a properly targeted endogenous BIG.

5. BRAGS: A NOVEL FAMILY OF BFA-RESISTANT

ARF GEFS

BLAST searches of the human genome with the GBF1 sequence identified
13 human proteins with a Sec7d, including three previously uncharacterized,
large, putative Arf GEFS encoded by partial human cDNAs (KIAA0522,
KIAA0763 and KIAA1110). Phylogenetic analysis of metazoan Sec7d
proteins encoded by the genomes of H. sapiens (n=13), D. melanogaster
(n=5) and C. elegans (n=5) revealed that these three large proteins define a
fifth subclass of Arf GEFs (see Figure 3). Interestingly, whereas humans
express several members of each subfamily, the D. melanogaster and C.
elegans genomes encode only one member of each of the 5 groups.
Surprisingly, as is also true for ARNOs and EFA6s, the genomes of S.
cerevisiae, S. pombe or A. thaliana do not encode a member of this family.
Furthermore, these Arf GEFs are unique among the large Arf GEFs in
containing a pleckstrin homology (PH) domain, located approximately 55
residues downstream of the Sec7d.
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10 MELANÇON, ZHAO, AND LASELL

Figure 3. Phylogenetic analysis of metazoan Sec7d proteins identifies 5 sub-families.

5.1 BRAGs are BFA-resistant Arf GEFs

The Sec7d of KIAA0522 and KIAA0763 were expressed in bacteria as GST

fusion proteins. These proteins demonstrated robust Arf GEF activity
(Lasell and Melançon, 2001). Of three fragments tested, the most active
contained the Sec7d of KIAA0522 and also included the downstream PH
domain. This fragment displayed Arf GEF activity towards myristoylated
forms of all three classes of Arfs but appeared most active on Class I Arfs.
The sequences of the Sec7d of all three members of this family contain 2 key
residues (shaded in Figure 1) that strongly correlate with BFA resistance.
We confirmed that the recombinant fusion protein was not affected by BFA,
under conditions in which the activity of BIG1 measured in parallel
experiments was inhibited by more than 80%. These proteins were renamed
BRAGs for their BFA-resistant Arf GEF activities (personal
communication).
J. Moss and colleagues recently reported the characterization of the ORF
encoded by KIAA0763/BRAG2 (Someya et al., 2001). A recombinant form
of the His6-tagged-100 kDa protein, termed GEP-100, was expressed in
insect cells and found to promote nucleotide exchange on recombinant forms
 LARGE ARF GEFS OF THE GOLGI COMPLEX 111
11

of all three classes of Arfs, but with significantly greater activity towards
Arf6. Furthermore, as expected, this activity was BFA resistant. Previous
work established that the PH domain present in members of the ARNO and
EFA6 families regulates GEF activity by facilitating recruitment to
membranes containing phosphoinositides (Chardin et al., 1996; Macia et al.,
2001; Paris et al., 1997). Surprisingly, varying levels of PS, PIP2 or PIP3
between 0 to 50 µM had no impact on GTP loading by GEP-100/BRAG2 on
either Arf1 or Arf6 (Someya et al., 2001). The authors concluded that the
PH domain does not mediate regulation of GEF activity by
phosphoinositides. However, one should note that all nucleotide exchange
assays were performed with Arfs lacking the essential myristate and used PS
or phosphoinositides bound to BSA rather than as liposomes. Arf activation
is coupled to membrane interaction and assays with full-length Arf are
usually performed in the presence of vesicles (Franco et al., 1993; Paris et
al., 1997). Reactions carried out with non-myristoylated ARFs in absence of
liposomes may therefore not reflect the true specificity of the GEF.
Furthermore, since the authors did not use liposomes doped with varying
levels of phosphoinositides, their results do not exclude regulation of
membrane recruitment by the PH domain.

5.2 BRAGs may localize to distinct compartments

Antibodies raised against a recombinant form of the BRAG1 Sec7d localize

the endogenous protein to juxtanuclear structures (see Figure 4) that co-stain
with the Golgi marker mannosidase II in NRK cells (Lasell and Melançon,
2001). Interestingly, BRAG1 is rapidly displaced from Golgi membranes
upon treatment with BFA. This observation is an important distinction
because GBF1 and BIGs remain membrane-associated after BFA treatment.
As discussed in 7.1 below, this displacement could depend on the PH
domain and result from changes in lipid composition. The displacement of
BRAG1 from the Golgi could explain some of the effects of BFA on the
Golgi complex of animal cells. In contrast, endogenous GEP-100/BRAG2
localizes to coarse puncta near the nucleus that coincide with the early
endosomal marker EEA1 in human glioblastoma T98G cells (Someya et al.,
2001). Contrary to GBF1/Geas and BIGs that are restricted to the Golgi
complex, or the EFA6s/ARNOs that function in the endosomal system,
BRAGs may localize to both of these compartments.
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12 MELANÇON, ZHAO, AND LASELL

Figure 4. Endogenous BRAG1 localizes in NRK cells to cis-elements of the Golgi complex
(A), well resolved from trans-elements positive for the clathrin adaptor protein GGA1 (B).

6. ARF GEFS CONTROL DISTINCT REACTIONS

IN THE GOLGI COMPLEX

6.1 GBF1 and BIGs localize to cis- and trans-

compartments of the Golgi complex, respectively

The results summarized in the previous sections established that three

families of Arf GEFs localize to the Golgi complex. To test the possibility
that these GEFs have specialized function that may be reflected in distinct
localization, we examined their relative distribution using quantitative
confocal laser scanning microscopy (Zhao et al., 2002b). Co-staining for
endogenous GBF1 and either HA-tagged or endogenous BIG1 revealed clear
differences in the staining patterns of these two Arf GEFs. Such separation
between GBF1 and BIGs was observed in at least two cell types and with
various combinations of endogenous or HA-tagged-forms of BIGs. Further
work showed that GBF1 co-localizes with the cis-marker p115 (86%) while
BIGs overlap extensively with the trans-Golgi marker TGN38 (83%). This
localization of GBF1 is consistent with its relative abundance on vesiculo-
tubular structures detected by immunogold labeling (Claude et al., 1999;
Kawamoto et al., 2002). These observations suggest that GBF1 and BIGs
activate Arfs in specific locations to regulate different coating reactions. In
agreement with this possibility, the COPI coat overlaps to a greater extent
with GBF1 (64%) than BIG1 (31%), while clathrin shows limited overlap
with BIG1, and virtually none with GBF1. A similar analysis of BRAG1
(see Figure 4) suggests that it localizes preferentially to cis-compartments
where it may participate with GBF1 in regulation of the COPI coat.
The localization of GBF1 and BIGs to early and late compartments of the
secretory pathway, respectively, is supported by their response to various
treatments. As expected of a cis-Golgi protein, GBF1 partially redistributes
 LARGE ARF GEFS OF THE GOLGI COMPLEX 113
13

from the Golgi complex to peripheral sites following incubation at 15˚C,

while it remains membrane-associated and appears in a diffuse, reticular,
pattern (consistent with an ER location) in the presence of BFA (Kawamoto
et al., 2002; Zhao et al., 2002b). In contrast, the perinuclear localization of
BIGs is not altered at 15ºC (Zhao et al., 2002b). Furthermore, BIGs are not
released from membranes upon BFA treatment but, instead, redistribute (like
TGN38) to a dense collection of fine punctate structures in the perinuclear
region. The apparent discrepancy with the conclusion of Vaughan and
colleagues, that BFA causes little change in BIGs’ distribution (Yamaji et
al., 2000), most likely reflects the slow kinetics of BFA effects on the TGN
and the short (10 min) treatment used by those authors (X. Zhao,
unpublished data).

6.2 GBF1 and BIGs regulate the recruitment of distinct

coats

GBF1 was originally identified by its ability to allow moderately

overexpressing cells (2-6 fold) to grow at BFA concentrations that blunt the
growth of control cells (Claude et al., 1999). This effect is unique to GBF1
since dramatic overexpression of BIG1 (10-20-fold) has minimal impact on
the BFA-sensitivity of transformants (Claude et al., 2003). GBF1 mitigates
the effects of BFA on cell growth by stabilizing the COPI coat (Claude et al.,
2003; Kawamoto et al., 2002). As expected from its intra-cellular
localization, GBF1 overexpression does not protect BFA-induced
redistribution of the trans-Golgi marker furin (Claude et al., 2003).
In contrast, overexpression of BIG1 does not protect either Arf or COPI
from BFA-induced dissociation. The Golgi disperses with normal kinetics
in cells overexpressing BIG1 (Claude et al., 2003). Similarly,
overexpression of BIG2 has no protective effects on COPI or Golgi
organization (Shinotsuka et al., 2002b). However, overexpression of BIG2
does antagonize some of the effects of BFA, but only those related to the
trans-Golgi. For example, overexpression of BIG2 prevents the BFA-
induced release of the clathrin adaptor complex, AP-1, to cytosol.
Furthermore, a dominant-negative mutant of BIG2 lacking a critical Glu
residue in its Sec7d does not interfere with COPI recruitment but specifically
affects membrane traffic from the TGN through inhibition of clathrin
adaptors such as AP-1 and GGA (Shinotsuka et al., 2002a).
Interestingly, overexpression of BIG2 does not prevent tubulation of the
TGN; the resulting tubules appear as queues of spherical swellings labeled
with BIG2, AP-1 and low levels of Arf1 (Shinotsuka et al., 2002a).
Preliminary results show that endogenous BIG1 does not appear in BFA-
induced TGN tubules (Figure 5). The presence of HA-BIG2 in TGN-
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14 MELANÇON, ZHAO, AND LASELL

derived tubules could result from its overexpression. Alternatively, BIG2

homodimers may localize differently and have functions distinct from
BIG1homo and heterodimers within the TGN.

Figure 5. BFA-induced tubules from the TGN contain TGN38 (A) but lack BIG1 (B).

6.3 GBF1 and BIG1 have distinct but interrelated

functions in the Golgi complex

Analysis of Arf GEF expression levels in single and double GBF1/BIG1

transformants revealed unexpected relationships between these two GEF
families (Claude et al., 2003). Expression of any of several GBF1 splice
variants substantially decreased endogenous BIG1 protein levels. This
reduction in BIG1 levels was even observed when BIG1 synthesis was
driven from a plasmid’s viral promoter suggesting effects on translation or
stability rather than transcription. In contrast, overexpression of BIG1 had
no impact on GBF1 levels. However, BIG1 overexpression clearly
interfered with GBF1 function: in double transformants, GBF1 no longer
protected COPI from BFA-induced dissociation, and no longer caused an
increase of the LD50 in cytotoxicity assays. These results underscore the
poorly understood and probably complex functional relations between Arf
GEF families at the Golgi complex.

7. EXPLANATION FOR BFA EFFECTS ON THE

GOLGI COMPLEX REMAINS ELUSIVE

7.1 BARS-50 and BFA effects on early compartments of

the Golgi complex

It is generally assumed that the effects of BFA result from inhibition of an

Arf GEF. However, as summarized above, BIGs are the only mammalian
 LARGE ARF GEFS OF THE GOLGI COMPLEX 115
15

Arf GEF clearly inhibited by BFA and they localize and function within
trans-elements of the Golgi complex. How then can one explain the effects
of BFA on early steps in protein traffic? Although the precise mechanism
remains unclear, several possibilities are discussed here. For example,
BARS-50 is a lysophosphatidate acyl transferase (thus, like phospholipase D
produces phosphatidic acid) that becomes ADP-ribosylated in response to
BFA treatment of cells (Weigert et al., 1999). BARS-50 also promotes
scission of COPI vesicles in in vitro assays (Spano et al., 1999; Weigert et
al., 1999). Thus, BFA-induced ADP-ribosylation of BARS-50 may alter its
enzymatic activity and result in alterations in the lipid composition of the
Golgi stacks and thereby interfere, directly or indirectly, with COPI
recruitment or prevent the release of VTCs from ER exit sites. Changes in
lipid composition could also be responsible for the rapid loss of BRAG1
from Golgi membranes observed after BFA treatment. Some of the effects
of BFA on the Golgi could therefore result not from blocking a cis-acting
GEF but from interfering with its recruitment. We also cannot exclude the
possibility that GBF1 is BFA sensitive, under physiological conditions.

7.2 The mechanism of BFA resistance in BFY cells

remains unknown

Several lines of evidence established that GBF1 was not the “resistance
factor” originally detected in extracts of BFY1 and BFY2 cells. Comparison
of GBF1 levels in wild-type and mutant cells first confirmed that BFA
resistance did not result from GBF1 overexpression (Claude et al., 1999).
As described in section 3.2 above, subsequent analysis eliminated the
possibility that mutant lines expressed unusual splice forms of GBF1 with
differential effects on BFA sensitivity (Zhao et al., 2002a). These
observations prompted us to examine if a mutation in the Sec7d of BIGs
might be responsible for the reduced BFA-sensitivity of mutant lines.
Sequencing of several RT-PCR products encoding the Sec7d of BIG1 and
BIG2 established that mutant cells expressed wild-type forms of both of
these BFA-sensitive Arf GEFs (personal communication). The nature of the
mutation in our BFY lines therefore remains unknown and open to
investigation. The mechanism responsible for the “natural” BFA resistance
of cell lines such as Ptk1 (Ktistakis et al., 1991) and MDCK (Hunziker et al.,
1991) remains similarly unknown. Clearly the cellular effects of BFA are
more complex than originally anticipated, and further investigation of the
BFY lines may shed new light on regulation of Arf activation in early Golgi
compartments.
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16 MELANÇON, ZHAO, AND LASELL

8. CONCLUDING REMARKS: ARF GEFS AS

MOLECULAR SCAFFOLDS

The work summarized above provides convincing evidence that the Arf
GEFs that function at the Golgi complex display both substrate preference
and distinct localization. These properties may help define which Arf is
activated where. What remains largely unexplored, however, is the
mechanism(s) that controls the choice of effector targeted by the activated
Arf to elicit a specific cellular response. As discussed in several other
chapters, Arf•GTP promotes not only membrane recruitment of COPI and
the several classes of clathrin adaptor proteins; it also regulates several non-
coat effectors, including the membrane remodeling enzymes phospholipase
D and PI(4)P 5-kinase (Brown et al., 1993; Godi et al., 1999; Roth, 1999), as
well as proteins of poorly defined function such as Arfaptins and Arfophilin
(Kanoh et al., 1997; Shin et al., 1999; Van Valkenburgh et al., 2001;
Williger et al., 1999). By acting as transient molecular scaffolds, the large
Arf GEFs could provide some of the required specificity to direct specific
interactions between GEFs and downstream effectors in a spatially and
temporally regulated manner. Ongoing efforts in several laboratories to
identify partners for Arfs and their regulators will in time test this
hypothesis.

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Chapter 6
BIG1 AND BIG2: BREFELDIN A-INHIBITED
EXCHANGE FACTORS FOR ARFS
Actions of brefeldin A

G. PACHECO-RODRIGUEZ, J. MOSS, AND M. VAUGHAN

Pulmonary-Critical Care Medicine Branch, NHLBI, NIH, Bethesda, USA

Abstract: Brefeldin A-inhibited guanine nucleotide exchange factor 1 (BIG1) and BIG2
were purified as components of a ~670 kDa protein complex. BIG1 and BIG2
partially colocalize with the Golgi 58 kDa protein and Ȗ-adaptin, a component
of the AP-1 complex. The distribution of BIG1 in the TGN is distinct from
that of GBF-1, a brefeldin A (BFA)-resistant GEF that was confined to the cis-
Golgi. Recently reported studies implicate BIG2 in the transport of lysosomal
enzymes between the TGN and late endosomes. Recognition that BFA
inhibition of BIG1 and Gea1 is non-competitive was the key to understanding
its mechanism of action. [3H]BFA binding was used to define structural
requirements for the formation of a BFA-Arf-Sec7 domain complex. A
recently published synthesis of structure-function information includes a
valuable model of the Arf-Sec7 domain interaction and the mechanism of BFA
inhibition.

1. INTRODUCTION

Brefeldin A (BFA) is a fungal, fatty acid metabolite described over 30 years

ago as an anti-viral agent (Tamura et al., 1968). Later, investigation of its
effect on protein synthesis in cultured rat hepatocytes revealed that protein
secretion was completely inhibited by BFA at concentrations that were
without effect on protein synthesis (Misumi et al., 1986). This and
subsequent work demonstrated that BFA reversibly blocked transport of
protein from the ER to Golgi and caused apparent disintegration of Golgi
structure (Fujiwara et al., 1988). Only three years later, studies of BFA
action by several groups had resulted in significant clarification of the
processes of ER-Golgi transport, anterograde and retrograde (Orci et al.,
1991).

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2 PACHECO-RODRIGUEZ, MOSS, AND VAUGHAN

Since Palade (1975) described a vesicular transport process for protein

secretion, the use of cell-free systems to characterize biochemically and
microscopically individual steps in membrane traffic pathways has
accelerated the recognition that analogous molecular mechanisms and
machinery are employed at multiple sites in all eukaryotic cells. Present
understanding of these processes is based on work of many investigators
including major contributions, experimental and conceptual, from the
Rothman laboratory. In the Rothman model (Rothman, 1994), inactive,
cytosolic Arf•GDP interacts with a guanine nucleotide-exchange protein
(GEF), which catalyzes the replacement of bound GDP with GTP generating
Arf•GTP that moves to a Golgi membrane, where it recruits coatomer (a
cytosolic complex of seven coat proteins) to initiate vesicle formation.
Sequential accumulation of Arf•GTP and coatomer results in deformation of
the membrane to form a bud. By fusion of the donor membrane at the base
of the bud (fission), a transport vesicle is released. Demonstration of bud
and vesicle formation from lipid model membranes (liposomes) later
confirmed that the only additions required were Arf•GTP and coatomer
(Spang et al., 1998).
After the arrival of a coated vesicle at its target membrane and before
membrane fusion can complete the transport process, release of coatomer
must occur. This was believed to be triggered by the hydrolysis of Arf-
bound GTP, followed sequentially by dissociation of Arf•GDP and
coatomer. Earlier this year, a publication by Lippincott-Schwartz and co-
workers provided valuable new insight into the dynamics of Arf and
coatomer interactions in vesicle formation (Presley et al., 2002). Based on
detailed quantification and comparison of movements of fluorescently
labeled Arf1 and ß-COP in living cells, they established clearly that
inactivation of Arf and release of Arf•GDP is required for release of
coatomer. In the cell, however, dissociation of Arf from Golgi membranes
was much more rapid than that of coatomer, i.e., release of Arf did not
“trigger” release of coatomer. As was pointed out, the continuing Arf-
dependent recruitment and independently regulated release of coatomer
during vesicle formation is consistent with the critical functions of coatomer
in the modification of membrane morphology (composition) and the
assembly/accumulation of appropriate cargo through direct and indirect
interactions with phospholipids and other proteins (Presley et al., 2002).
BFA was, in fact, employed to advantage in these studies to inhibit vesicle
formation from Golgi membranes.
In 1992, two groups had reported simultaneously that Golgi membranes
accelerated in vitro binding of GTP to Arf, and this GEF activity was
inhibited by BFA (Donaldson et al., 1992; Helms and Rothman, 1992).
Purification of a BFA-inhibited GEF was finally reported in 1996 (Morinaga
 BIGS: BFA-SENSITIVE ARF GEFS 1233

et al., 1996). That protein, p200 (later named BIG1), and a second BFA-
inhibited GEF (p190 or BIG2) were present together in a ~670 kDa complex
(Morinaga et al., 1996; Togawa et al., 1999).
Although many effects of BFA on cells are due to its interference with
Arf activation via GEF inhibition, BFA also alters Golgi structures by
stimulating the ADP-ribosylation of a 50 kDa protein termed BARS, for
BFA-ADP-ribosylated substrate (Spanfó et al., 1999). A 38 kDa
glyceraldehyde-3-phosphate dehydrogenase is also modified by apparently
the same BFA-stimulated ADP-ribosyltransferase. Comparisons of effects
of a number of inhibitors of bacterial ADP-ribosyltransferases revealed
parallel effects of coumarin and quinone class compounds on the ability of
BFA to influence Golgi morphology and to inhibit ADP-ribosylation in
vitro. This result is consistent with the involvement of an ADP-ribosylated
protein in the disruption of Golgi architecture (Weigert et al., 1997). BFA-
induced dissociation of coatomer from Golgi membranes, which results from
its inhibition of Arf activation, was unaffected by prevention of the protein
ADP-ribosylation and the perturbation of Golgi structure (Mironov et al.,
1997). These observations implicated the ADP-ribosylated BARS in Golgi
disintegration, and were consonant with the notion that this effect of BFA
does not result from its inhibition of Arf activation.
With the demonstration that BARS is a lysophosphatidic acid:acyl-CoA
transferase, it was suggested that this activity (or the phosphatidic acid that it
produces) is required for fission of the Golgi tubular
compartments/elements, which in its absence become massively
enlarged/elongated as vesicle formation ceases (Weigert et al., 1999). It is
notable that endophilin, a protein that interacts with dynamin and is essential
for endocytosis, is also a lysophosphatidic acid:acyl-CoA transferase
(Schmidt et al., 1999). Both groups suggested similar mechanisms for the
effects of phosphatidic acid on membrane structure at the site of vesicle
fission. It is of interest that BARS is very similar in structure to CtBP1 and
CtBP2, proteins involved in transcription, which interact with the C-
terminus of the adenovirus transforming protein E1A. As those proteins
were also substrates for BFA-stimulated ADP-ribosylation, it was suggested
that BARS be designated CtBP3, and that dual roles for these proteins in the
nucleus and in Golgi be considered (Spanfó et al., 1999).

2. STRUCTURE OF BIG1 AND BIG2

The human BIG1 gene was mapped to chromosome 8q13 (Mansour et al.,
1999) and BIG2 to 20q13.13. cDNA clones for bovine BIG1 (Morinaga et
al., 1997), human BIG1 (Togawa et al., 1999; Mansour et al., 1999), and
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4 PACHECO-RODRIGUEZ, MOSS, AND VAUGHAN

human BIG2 (Togawa et al., 1999; Shinotsuka et al., 2002a) have been
reported, BIG1 and BIG2 proteins and mRNAs are present in many bovine
and human tissues (Morinaga et al., 1997; Togawa et al., 1999; Mansour et
al., 1999).
BIG1 and BIG2 were initially purified together in a ~670 kDa protein
complex (Morinaga et al., 1996). Amino acid sequences of peptides from
BIG1 resembled portions of the yeast Sec7 protein, an Arf GEF involved in
protein secretion (Franzusoff and Schekman, 1989). BIG1 and BIG2 were
later cloned (Morinaga et al., 1997; Togawa et al., 1999; Mansour et al.,
1999; Shinotsuka et al., 2002a), and found to contain Sec7 domains of ~200
amino acids that are 50% identical to the yeast Sec7 domain and 90%
identical to each other. The Sec7 domain of yeast Sec7 synthesized as a
recombinant protein exhibited GEF activity that was inhibited by BFA (Sata
et al., 1998), as did the Sec7 domains of BIG1 (Morinaga et al., 1999), BIG2
(Togawa et al., 1999), and Gea1 (Peyroche et al., 1999). BFA analogues
that had not inhibited the GEF activity of Golgi membranes (Donaldson et
al., 1992) also failed to inhibit the Sec7 domain of yeast Sec7 (Sata et al.,
1998).
Until very recently, the only functional region identified in these
proteins, was the Sec7 domain, initially recognized in the Saccharomyces
cerevisiae Sec7 protein (Achstetter et al., 1988). Sec7 domain proteins, like
Arfs, are ubiquitous in eukaryotes. Structures of the BIG1 and BIG2 Sec7
domains have not been reported, but those for other Sec7 domains are
known, including those for ARNO or cytohesin-2 (Mossesova et al., 1998;
Cherfils et al., 1998), cytohesin-1 (Betz et al., 1998), and Gea2, a BFA-
inhibited GEF from yeast (Goldberg, 1998; Renault et al., 2002). The Sec7
domain is formed by two elements, each composed of five Į-helices, which
contain motifs 1 and 2. These short amino acid sequences are critical for Arf
binding and catalysis of nucleotide exchange. A diagram of the domain
organization of BIG1 is shown in Figure 1. Specific amino acids in a site
formed by apposition of the two motifs appear to interact with
complementary side chains in switch 1 (residues 41-55) and switch 2
(residues 70-80) of Arf (Mossesova et al., 1998; Goldberg, 1998; Betz et al.,
1998; Cherfils et al., 1998; Renault et al., 2002).
 BIGS: BFA-SENSITIVE ARF GEFS 1255

Figure 1. Functional domains of BIG1. Mansour et al. (1999) reported that the N-terminal
fragment (residues 1-560) was required for Golgi localization and identified the HUS
(homology upstream of the Sec7 domain) box as important for protein stability. The central
Sec7 domain contains Motifs 1 and 2. Asterisk indicates region that increases GEF activity of
the Sec7 domain 100-fold (Morinaga et al., 1999).

Functions of regions outside the Sec7 domains of BIG1 and BIG2 are, for
the most part, obscure. Arf GEF activity of BIG1(1-885), which lacks the
964 residues C-terminal to the Sec7 domain, was equivalent to that of the
full-length protein, when recombinant proteins synthesized in Sf9 cells were
compared. Additional studies of BIG1(1-885), truncated at the N-terminus,
indicated that removal of amino acids 1-594 (BIG1 (595-885) had no effect,
but an additional truncation of 35 residues (BIG1 (630-885) resulted in the
loss of 99% of the Arf GEF activity (Morinaga et al., 1999).This sequence,
with a predicted pI of 6.30, appears to form a coil, integrated by three helices
that are connected by segments without defined structure, which probably
represent loops. The effect of this segment is consistent with earlier
observations that regions outside the Sec7 domain altered cytohesin Arf GEF
activity (Chardin et al., 1996; Pacheco-Rodriguez et al., 1998). Mansour et
al. (1999) identified a sequence in BIG1 (amino acids 562-
VVDIYVNYDCDLNAANIFERLVNDLSKI-590), which they referred to as
the region of “homology upstream of the Sec7 domain (HUS)” that is also
present in other large GEFs, such as Sec7, GBF1, and EMB30. They
suggested that this sequence, which is identical in BIG1 and BIG2 (Togawa
et al., 1999), could be important for protein folding and stability. The region
N-terminal to it appears to contain a Golgi localization signal. Among five
epitope-tagged BIG1 mutants that were overexpressed in BHK cells, only
the one containing residues 1-560 coincided with the Golgi marker
mannosidase II, as did the full-length BIG1 (Mansour et al., 1999).
Subcellular localization signals have not been recognized in other Arf GEFs
leaving in question the determinants of their subcellular distributions, which
126
6 PACHECO-RODRIGUEZ, MOSS, AND VAUGHAN

undoubtedly depends on specific molecular interactions and very possibly

reversible covalent modification.
FBS (F-box/Sec7), a human protein of 319 amino acids, was identified in
a data base search for new E3 ubiquitin-protein ligases that contain F-box
motifs. Its deduced sequence contains also a Sec7-like domain (residues
139-203), which includes the glutamate that is critical for GEF activity in
other Sec7 domains (Ilyin et al., 2000). The F-box was recognized as a
structural element required for the binding of Skp1p, a cyclin F-binding
protein functionally implicated in cell cycle regulation (Bai et al., 1996).
Regulated proteolytic degradation of these proteins is critical to the control
of their function. Many proteins in the F-box family contain additional
conserved structural motifs (e.g., leucine-rich repeats, WD40 domains) that
are involved in specific protein-protein interactions (Ilyin et al., 2000).
More information regarding FBS and its potential GEF activity is clearly
needed.
Sec7 domains contain at least some of the determinants for specificity of
Arf GEF activity and/or molecular interaction (see also section 3,
Mechanism of BFA inhibition). The cytohesin-1 Sec7 domain, however,
had lower substrate specificity than the intact molecule (Pacheco-Rodriguez
et al., 1998). Cytohesin-1 accelerated GTPȖS binding to recombinant human
Arf1, yeast Arf3, and ARD1, a 64 kDa protein that contains a C-terminal Arf
domain (Mishima et al., 1993). The cytohesin-1 Sec7 domain, on the other
hand, was active with human Arfs 1, 5, and 6, yeast Arfs 1, 2, and 3, ARD1,
two ARD1 mutants that contain the Arf domains, and ǻ13Arf1, which lacks
the N-terminal Į-helix and terminal glycine. Neither cytohesin-1 nor its
Sec7 domain increased GTPȖS binding to human ARL 1, 2, or 3. It was
concluded that the N-terminus of Arf is important for functional interaction
with cytohesin-1 and that critical determinants of substrate specificity reside
outside the Sec7 domain of cytohesin-1 (Pacheco-Rodriguez et al., 1998).
This seems likely to be true also for BIG1 and BIG2, but remains to be
investigated.
Knowledge of the functional specificity of Arf-Arf GEF interactions in
cells is, at best, limited, albeit improving in the last few years. The GEF
activity of a protein assayed in vitro by its acceleration of GTPȖS binding to
a particular Arf substrate has no necessary or predictable relationship to the
physiological actions of the molecules studied. Inside the cell, Arfs and Arf
GEFs operate in an environment of numerous proteins and lipids, which
dynamically influence multiple simultaneous and sequential interactions.
An in vitro system with GTPȖS includes none of these and is lacking also the
cyclic interconversion of Arf•GDP and Arf•GTP that is required for its
biological function. Application of the procedure described by Santy and
Casanova (Santy and Casanova, 2001) to “pull down” Arf•GTP from cell
 BIGS: BFA-SENSITIVE ARF GEFS 1277

lysates using an immobilized fragment of GGA3 could prove very useful for
identifying and quantifying specific intracellular Arf-Arf GEF interactions.
A recent publication compared the crystal structure of the free (without
ligand) Sec7 domain of Gea2 (Renault et al., 2002) with that of Gea2 bound
to nucleotide-free Arf1 that lacks the N-terminal myristate and amino acids
1-17 (Goldberg, 1998). This information dramatically increases our
understanding of the molecular mechanisms of Arf GEF action and its
inhibition by BFA (reviewed in the next section). Renault et al. (Renault et
al., 2002) suggested also that differences among Arf GEFs in the width of
the empty catalytic groove that accommodates the Arf molecule could serve
to discriminate among different structures of the individual Arfs (Amor et
al., 1994; Ménétrey et al., 2000). Additional detailed data of this kind will
surely facilitate identification of the specific roles and interactions of these
molecules.

3. INTRACELLULAR LOCALIZATION OF BIG1

AND BIG2

BIG1 and BIG2 had been purified from bovine brain cytosol as components
of a ~670 kDa complex (Morinaga et al., 1996), and were, to a large extent,
immunoprecipitated together from HepG2 cell cytosol by antibodies specific
for BIG1 or BIG2 (Yamaji et al., 1999). BIG1 and BIG2 were also partially
co-immunoprecipitated from CHAPS-solubilized, microsomal fractions.
After centrifugation of microsomal preparations on a sucrose density
gradient, BIG, BIG2, and ß-COP (one of seven proteins that compose the
COPI coatomer) were recovered only in the 1.15 M/0.85 M sucrose (heavy
Golgi) fraction (Yamaji et al., 1999). By fluorescence microscopy, both
endogenous BIG1 and BIG2 in HepG2 cells were partially colocalized with
the Golgi-specific 58 kDa protein and partially with Ȗ-adaptin, a component
of the AP-1 complex. The distribution of over-expressed BIG2 and
endogenous BIG1, BIG2, and also appeared quite similar in HepG2 and
HeLa cells. After exposure of HepG2 cells to BFA for 10 min with
complete release of ȕ-COP from Golgi structures, both BIG1 and BIG2 were
still Golgi-associated (Yamaji et al., 1999). Mansour, et al. (Mansour et al.,
1999), however, found complete dispersal of over-expressed BIG1 from
Golgi in BHK2 cells incubated for 10 min with BFA, which likely reflects
simply a difference in the time required for BFA inhibition in different cells.
Zhao et al. (2002) reported that endogenous BIG1 in NRK cells was
localized in the TGN. Its concentration in the Golgi compartment was
contrasted with that of GBF1, a 208 kDa, BFA-resistant Arf GEF that
preferentially activates Arf5 (Claude et al., 1999), which was confined to a
128
8 PACHECO-RODRIGUEZ, MOSS, AND VAUGHAN

cis-Golgi compartment. Using quantitative confocal microscopy, Zhao et al.

(Zhao et al., 2002) demonstrated less than 25% overlap of the two proteins.
This methodology was also employed to quantify the extent of overlap of the
two Arf GEFs with Golgi and coat protein markers. The distribution of
over-expressed BIG2(2-552) carrying an N-terminal hemagglutinin (HA) tag
was very similar to that of endogenous BIG1 (Zhao et al., 2002). Whereas
BIG1 and BIG2 most likely function with class I Arfs in BFA-sensitive
anterograde traffic from the TGN, GBF1 may operate with Arf5 in
retrograde transport from Golgi to ER. It seems possible that the BFA-
insensitive binding of Arf5 to Golgi membranes from rat brain reported by
Tsai et al. (Tsai et al., 1993) was a reflection of GBF1 activity. An
association of BIG1 and BIG2 with the Ȗ-adaptin component of AP-1 is
consistent with the reported BFA inhibition of formation of clathrin-coated
vesicles (Robinson and Bonifacino, 2001).
Earlier this year, Shinotsuka et al. (Shinotsuka et al., 2002a) reported that
HA- or Myc-tagged BIG2 over-expressed in human HeLa cells or Clone 9
rat hepatocytes was localized in the perinuclear region. Staining overlapped
with that for ȕ-COP, mannosidase II, Ȗ-adaptin, and mannose-6-phosphate
receptor, but not with LAMP1 or EEA1. They used a glutathione S-
transferase (GST)-tag attached to the domain of GGA1 that specifically
binds Arf•GTP to show that the amount of activated Arf in cell lysates was
increased by overexpression of BIG2. BFA caused dispersal of over-
expressed Arf1-HA from the perinuclear area to the cytosol. When BIG2
was also overexpressed it appeared localized with Arf1-HA. On exposure of
cells to BFA, both Arf1-HA and Myc-BIG2 were seen on tubulovesicular
structures radiating from the perinuclear region. Comparison of the effects
of BFA on the behavior of ȕ-COP and Ȗ-adaptin revealed that whereas
overexpression of BIG2 did not interfere with BFA-induced release of ȕ-
COP, it did cause retention of Ȗ-adaptin which was seen along the tubular
structures with which BIG2 was associated. The authors concluded that
BIG2 was acting primarily at the TGN in the process of AP-1 recruitment to
form clathrin-coated vesicles. Over-expression of BIG2 did not prevent the
rapid BFA-induced release of ȕ-COP or the slower disruption of Golgi
structure evidenced by dispersal of mannosidase, consistent with the notion
that its function differs from that of BIG1, which presumably activates class
I Arfs for anterograde transport from the cis-Golgi (Shinotsuka et al.,
2002a).
Following their demonstration that BIG2 is responsible for the initiation
of clathrin-coated vesicles by activating Arf to recruit Ȗ-adaptin, Shinotsuka
et al. (Shinotsuka et al., 2002b), reported that over-expression in HEK 293
cells of dominant negative BIG2-K738, which lacks Arf GEF activity,
caused redistribution of Ȗ-adaptin from the perinuclear region, without
 BIGS: BFA-SENSITIVE ARF GEFS 1299

affecting ȕ-COP. They showed that the adaptor protein GGA1, like Ȗ-
adaptin and BIG2-K738, was dispersed along tubular structures resembling
those seen in BFA-treated cells. These and other observations implicated
BIG2 in transport of lysosomal enzymes between the TGN and late
endosomes (Shinotsuka et al., 2002b).
Earlier conclusions regarding the apparently similar subcellular
distributions of BIG1 and BIG2, both of which appeared to colocalize
partially with both Ȗ-adaptin and coatomer, were undoubtedly due to
insufficiently precise techniques for immunofluorescence microscopy
(Yamaji et al., 1999). They were perhaps accepted with less question than
they might otherwise have been because of the knowledge that BIG1 and
BIG2 had remained associated throughout six steps of purification and were
immunoprecipitated together, both phenomena that demand, now more than
ever, detailed investigation and explanation.

4. MECHANISMS OF BFA INHIBITION

The recognition that BFA inhibition of Gea1 (Peyroche et al., 1999) and
BIG1 (Mansour et al., 1999) is non-competitive was the key to
understanding its mechanism of action. Peyroche et al. (Peyroche et al.,
1999) demonstrated directly that BFA stabilized and thus increased the
amount of a stoichiometric complex containing the Gea1 Sec7 domain and
Arf1-∆17•GDP (lacking the N-terminal myristate and 17 amino acids).
They identified two amino acids in the Sec7 domain that were critical for
BFA inhibition of secretion in the mutant yeast used to test activity of the
recombinant proteins. A chimeric Gea1 containing an ARNO Sec7 domain
was used to demonstrate that replacement of F190 and A191, respectively,
with the residues found in the corresponding position in Gea1 (tyrosine and
serine), resulted in BFA sensitivity and the capacity to form a stable BFA-
Arf•GDP-Sec7 domain complex, each of which was lacking in the parent
molecule (Peyroche et al., 1999). Sata et al. (Sata et al., 1999) showed that
replacement of S198 and P208 in the Sec7 domain of cytohesin-1 (an Arf
GEF very similar to ARNO) with aspartate and methionine, respectively, as
found in the BFA-inhibited BIG1 (p200), made it sensitive to BFA.
Sequences in these regions of BFA-inhibited and BFA-insensitive GEFs are
aligned in Figure 2. In the crystal structure of the ARNO Sec7 domain, these
four amino acid side chains are exposed on the surface, in or near the
hydrophobic groove in which the Arf molecule binds through its switch 1
and switch 2 regions (Mossesova et al., 1998). Specific interactions of
individual amino acids in the two switch regions of Arf with complementary
residues in motifs 1 and 2 of the Sec7 domain were identified in crystal
130
10 PACHECO-RODRIGUEZ, MOSS, AND VAUGHAN

structures of the Gea2 Sec7 domain complex with a mutant Arf1, either
nucleotide-free or bound to Gpp(NH)p, a poorly hydrolysable GTP analogue
(Goldberg, 1998).

Figure 2. Amino acids in the Sec7 domain that influence sensitivity to BFA. Sequences
shown contain Motif 2 (Mossesova et al., 1998), which forms a side of the Arf-binding
groove and includes one pair of residues (**) that modify BFA sensitivity (Peyroche et al.,
1999). The adjacent sequence contains another pair of amino acids that affects BFA
sensitivity, (*) and the boxed position, in which interchanging Q (as in BIG1) with N, (as in
cytohesins), did not affect BFA sensitivity (Sata et al., 1999). Five BFA-sensitive GEFs,
from human (h), yeast (y), and Arabadopsis (a), are shown, along with four BFA-insensitive
cytohesins.

Robineau et al. (Robineau et al., 2000) used [3H]BFA-binding to define

structural requirements for the formation of a BFA-Arf•GDP-Sec7 domain
complex. BFA binding was similar whether the Sec7 domain contained
Y190 and S191 (Peyroche et al., 1999) or D198 and M208 (Sata et al.,
1999), but was essentially doubled when both pairs of amino acids were
present. Differences in the kinetics of [3H]BFA binding and release from
complexes with Sec7 domains containing either only two or all four of the
 BIGS: BFA-SENSITIVE ARF GEFS 131
11

critical amino acids provided further insight into molecular interactions

(Robineau et al., 2000). All Sec7 domains contain a specific glutamic acid
that is responsible for accelerating the release of bound guanine nucleotide
from Arf, i.e., for GEF activity. Replacement of this E156 with lysine in the
BFA-sensitive quadruple mutant Sec7 domain markedly accelerated its
[3H]BFA binding, indicative of complex formation. Without Arf GEF
activity the concentration of Arf•GDP remains high, whereas on interaction
with an active Sec7 domain, Arf•GDP is rapidly converted to nucleotide-
free Arf, which forms a much more stable interaction, so that the
concentration of the short-lived Arf•GDP-Sec7 domain that can bind BFA is
always very low.
Effects of mutations in switch 2 of Arf1 that had little effect on Arf GEF
activity were also evaluated using the BFA-binding assay. When H80 in Arf
1 was replaced with alanine, release of BFA from the complex formed with
the quadruple mutant Sec7 domain was slowed. Comparison of Sec7
domains containing only one of the two pairs of mutant amino acids,
demonstrated it was D198 and M208 (found in BIG1) that were necessary
for this effect of the A80 mutation, one of the examples of contributions of
elements in both of the molecules to interaction specificity (Robineau et al.,
2000). These observations, together with the three-dimensional structures,
provide important insights into mechanisms of Arf GEF action, as well as
BFA inhibition. Wittinghofer and colleagues have made major contributions
to the understanding of mechanisms by which guanine nucleotide-binding
proteins function as molecular switches in the regulation of cell signaling
and metabolism. A recent review presents a valuable analysis of the
differences, as well as similarities, among the diverse families of GTPases,
including the Arfs (Vetter and Wittinghofer, 2001).
In considering the mechanism of Sec7 domain actions in Arf activation
and BFA sensitivity, Renault et al. (Renault et al., 2002) began with
evidence of the structural flexibility of Arf, relative to that of the Sec7
domain. To evaluate flexibility, they compared the crystal structure of the
Gea2 Sec7 domain alone with that of the domain bound to nucleotide-free
Arf (Goldberg, 1998). They found that the N- and C-terminal halves of the
Sec7 domain, which form sides of the Arf-binding groove, were significantly
closer together when Arf was bound. This was termed “Sec7 domain
closure” and was accompanied by changes in the hinge region that links the
two halves, involving the conformation of helix Į7 (Renault et al., 2002). A
similar effect of Arf binding on the structure of cytohesin-1 had been
reported (Betz et al., 1998). Renault et al. (Renault et al., 2002) considered
the behavior of Gea2 in light of the published data on the structure of the
ARNO Sec7 domain. They noted that three of the four positions that had
been implicated in determination of BFA sensitivity, form more bonds in the
132
12 PACHECO-RODRIGUEZ, MOSS, AND VAUGHAN

empty Sec7 domain when they are occupied by tyrosine, aspartate, and
methionine (as in BFA-sensitive Arf GEFs) than when occupied by
phenylalanine, serine, and proline (BFA-insensitive). They concluded that
these three positions are important in the flexibility of the Sec7 domain that
is necessary for alternation between the open (empty) and closed (Arf-
bound) conformations of the Arf-binding groove. Both flexibility and the
capacity to form hydrogen bonds may contribute to BFA sensitivity.
Renault et al. (Renault et al., 2002) observed that the specificity of an Arf-
Arf GEF interaction should probably be established by the initial reaction of
the two molecules. They suggested that differences among GEFs in the
width of the empty catalytic groove, which is significantly wider in ARNO
than Gea2, could serve to discriminate among structures of the GDP-bound
forms of individual Arfs (Amor et al., 1994; Ménétrey et al., 2000) in the
switch 1 and switch 2 regions with which they interact.

5. CONCLUSIONS

BFA continues to be a valuable tool in the dissection of molecular processes

responsible for vesicular transport, very recently in the direct demonstration
that during vesicle formation in the Golgi, release of coatomer does not
immediately follow the inactivation and dissociation of Arf. Active Arf is
required for the recruitment of coatomer, but not for its continued function in
the assembly of cargo and modification of membrane morphology (Presley
et al., 2002).
BIG1 and BIG2 were isolated together from bovine brain cytosol through
six steps of purification (Morinaga et al., 1996), and were to a large extent
co-immunoprecipitated from HepG2 cell cytosol by specific antibodies
against one or the other (Yamaji et al., 1999). Growing evidence that they
can regulate different intracellular transport pathways (Shinotsuka et al.,
2002a,, 2002b), however, indicates the necessity of further investigation and
explanation of those phenomena.
Extensive analysis of the interactions of individual amino acids in the
Sec7 domain of Gea2, which is identical to BIG1 and BIG2 in the residues
that determine BFA inhibition, with different nucleotide-bound forms of
Arf1, which is also a preferred substrate for BIG1 and BIG2, has provided
detailed new insight into mechanisms of GEF action and BFA inhibition
(Renault et al., 2002).
 BIGS: BFA-SENSITIVE ARF GEFS 133
13

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Chapter 7
ARF GTPASE-ACTIVATING PROTEIN 1

DAN CASSEL
Department of Biology, Technion-Israeli Institute of Technology, Haifa, Israel

Abstract: Regulators of Arf activity include a family of proteins with a shared domain,
the cysteine-rich Arf GAP domain, that is responsible for activating the latent
GTPase activity of Arfs. The first of these to be discovered, Arf GAP1 is the
focus of this chapter. It’s role in the cellular actions of Arfs, particularly
vesicular traffic, and the regulation of Arf GAP1 by other factors, e.g., lipids,
is discussed.

1. PERSPECTIVES AND SCOPE

Like all regulatory GTPases, Arfs function as molecular switches, oscillating

between the active, GTP-bound form and the inactive, GDP-bound form.
These oscillations are tightly regulated by guanine nucleotide exchange
factors (GEFs) that “switch on” the Arf proteins, and GTPase-activating
proteins (GAPs) that act as off switches by facilitating the GTP hydrolysis
(GTPase) activity of Arfs. Accompanying the on/off switching of Arfs is a
cycle of membrane association/dissociation. In the GTP-bound state Arfs
associate with cellular membranes to which they recruit their various
effector molecules, thereby facilitating membrane traffic. GTP hydrolysis,
brought about by an Arf GAP, results in the rapid dissociation of Arfs from
membranes. Although Arf proteins were discovered in the 1980s (Kahn and
Gilman, 1986), their regulators began to be identified only in the next
decade. To date, multiple regulators of each part of the GTPase cycle of
Arfs have been identified. These regulators possess domains that function in
the binding to Arf and subsequent effects on GTP binding or hydrolysis.
Thus, all Arf GEFs contain a Sec7 domain, and all Arf GAPs contain an
“Arf GAP domain”.
This chapter begins with the introduction of the family of mammalian
Arf GAPs. A detailed discussion of Arf GAP1, the first Arf GAP to be

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purified and cloned, is then presented. This is the smallest of the many Arf
GAPs found in mammals, is localized to the cytosolic surface of Golgi
membranes, and acts to regulate traffic through this organelle. Where
appropriate, discussions will be extended to Arf GAPs from the yeast S.
cerevisiae that are structurally and/or functionally related to Arf GAP1.

2. ARF GAP PROTEINS

2.1 Conserved features and the catalytic domain

All Arf GAPs known to date contain a highly conserved “signature”

sequence of approximately 50 amino acids that comprises the Arf GAP
domain. The most notable feature of this sequence is the presence of a zinc
finger motif, consisting of 4 cysteine residues arranged in the sequence
CX2CX16CX2C, where X is any amino acid (see Figure 1). Residues
between pairs of cysteines frequently include a charged amino acid, whereas

Figure 1. Expression of Arf GAP1 in rat tissues. Tissues were homogenized in isotonic
sucrose in the presence of a cocktail of protease inhibitors, separated by centrifugation
(100,000xg, 1 h) into cytosolic fraction and total membrane fractions, and analyzed by
Western blotting with polyclonal, rabbit anti-Arf GAP1 antibodies.

the X16 “loop” is enriched in neutral and hydrophobic amino acids, reflecting
the fact that this loop is localized in the core of the protein and does not face
the solvent (Goldberg, 1999; Mandiyan et al., 1999). Particularly high
sequence conservation is observed in a 20-25 amino acid stretch that follows
the last cysteine residue of the zinc coordination site. This sequence is
thought to be part of the GAP “catalytic domain” that mediates the
stimulation of GTP hydrolysis on ARFs. In all, the GAP domain includes
approximately 90 residues starting from the last cysteine of the zinc
coordination site (see section 3.3, below). While some short sequence
 ARF GTPASE-ACTIVATING PROTEIN 1 1393

elements are conserved between certain subgroups of Arf GAPs in the part
of the catalytic domain that extends beyond the above-mentioned conserved
sequence, sequences in this region are in general quite variable, and do not
show any consensus when all Arf GAPs are compared.

2.2 Arf GAP subfamilies

The completed sequence of the human genome has revealed more than 20
genes encoding proteins with an Arf GAP signature. Many of these proteins
have been shown to possess Arf GAP activity. A notable exception is
centaurin-α (Hammonds-Odie et al., 1996), for which no GAP activity could
be demonstrated. Arf GAPs can be divided into five subtypes or
subfamilies, according to similarities in sequence and domain structure (see
Table 1). One type is Arf GAP1, which does not contain any domains found

Table 1: Mammalian Arf GAPs. Notes: 1Size of the longest splice variant (when multiple
variants exist). 2Paxillin does not interact with Git2. 3Absent in ACAPs. 4Defective in
ARAP2. 5Absent in ARAP1. 6Present only in ARAP2. Abbreviations: PH, plekstrin
homology domain; ANK, ankyrin repeats; PrR, proline-rich, SH3-binding domain; SH3, Src-
homology-3 domain; RA, Ras associating/Ral GDS domain; SAM, sterile α motif; RHD,
Rab13 homology domain; GRK, G protein receptor kinase; FA, focal adhesions; ND, not
determined.
Family Size Other Substrate Location Binding Pathways
kD1 Domains Preference Proteins Effected
Arf GAP1 45 - Arf1, 3, 5>6 Golgi KDEL ER-Golgi
receptor traffic
Arf GAP3 57 - ND Peri- ND ND
nuclear
Git1/Cat1 85 ANK Arf1, 3, 5, 6 PM/ GRK, Surface
Git2/Cat2 85 Arf1, 3, 5, 6 Endo- paxilin2, receptor
somes PIX internal-
ization,
integrin
signaling
ASAP1 130 CC, PH, Arf1, 5>6 FA Src, Crk Focal adhe-
Papα/ ANK, Arf1, 5>6 Pyk2, sions,
PAG3 PrR3, SH33 Arf6>1, 5 paxillin Integrin
ACAP1, 2 signaling
ARAP1 PH (x5), Arf1,5>6 Golgi/PM ND Coordi-
ARAP2 RhoGAP, ND ND nation of
ARAP3 ANK, RA4, Arf6 PM Arf and
SAM5, Rho family
RHD6 GTPases

in other proteins besides the Arf GAP domain (Cukierman et al., 1995).
This is also the case for Arf GAP3 (Zhang et al., 2000), a more recently
discovered Arf GAP. Arf GAP1 and Arf GAP3 have orthologs in yeast,
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named Gcs1 and Glo3, respectively (Poon et al., 1999; Poon et al., 1996).
The second subfamily is the Git proteins, including three highly related
members that contain ankyrin repeats, also present in most other Arf GAPs
(Premont et al., 1998; Premont et al., 2000; Turner et al., 1999). Two
additional Arf GAP families, the ASAPs/ACAPs (Andreev et al., 1999;
Brown et al., 1998; Jackson et al., 2000; Kondo et al., 2000) and ARAPs
(Krugmann et al., 2002; Miura et al., 2002) contain PH domain(s) and show
phosphoinositide-dependent Arf GAP activity. Alternative splicing is
common in most Arf GAP genes, adding further to the diversity of the Arf
GAP family of proteins.
The domain structure, substrate specificity, cellular localization and the
general function of Arf GAPs are summarized in Table 1. For detail on
some of these proteins, see subsequent chapters (e.g., Chapters 8 and 9).

3. ARF GAP1

3.1 Arf GAP role in the COPI system

Because Arf GAP1 is thought to function in the COPI system, we will first
briefly introduce this system. For additional details of the role of Arfs in
vesicle coat protein recruitment at Golgi and other membranes, see Chapters
12 and 13. The COPI system functions primarily in the retrograde traffic of
proteins from the Golgi to the endoplasmic reticulum (ER), although there is
evidence for its function in anterograde transport from ER to Golgi as well
(reviewed in (Lippincott-Schwartz et al., 1998; Rothman and Wieland, 1996;
Schekman and Orci, 1996)). The COPI coat (termed coatomer) is a seven-
subunit complex that is recruited en block from cytosol onto Golgi
membrane. Coatomer recruitment is initiated when Arf1 is converted to the
GTP-bound form by the action of a GEF at the Golgi. Subsequently, Golgi-
bound Arf1•GTP acts to recruit coatomer, apparently by direct binding to
coatomer subunits (Eugster et al., 2000; Zhao et al., 1997; Zhao et al., 1999).
Coatomer also interacts with cytoplasmic domains of membrane proteins
bearing the KKXX retrograde transport (sorting) signal, thereby
incorporating this cargo into COPI vesicles (Cosson and Letourneur, 1994;
Letourneur et al., 1994). p23/p24 transmembrane proteins, that are abundant
in the ER-to Golgi shuttle, also interact with coatomer through signals on
their cytoplasmic domain (Dominguez et al., 1998; Fiedler et al., 1996;
Nickel et al., 1997; Sohn et al., 1996). The interaction with coatomer is
likely to function in the recruitment of p23/24 proteins into COPI vesicles,
but the precise function of these proteins remains uncertain. Following its
binding to the Golgi membrane, coatomer is thought to polymerize and
 ARF GTPASE-ACTIVATING PROTEIN 1 1415

facilitate the deformation of the membrane, that is required to initiate vesicle

budding.
Before fusion with the target membrane, the vesicle must shed its protein
coat. This process of vesicle uncoating depends on the hydrolysis of Arf1-
bound GTP (Tanigawa et al., 1993; Teal et al., 1994). When COPI vesicles
are formed in the presence of the hydrolysis-resistant GTP derivative,
GTPγS, both uncoating and fusion are prevented. Because Arfs are devoid
of any intrinsic GTPase activity, GTP hydrolysis is completely dependent on
the action of an Arf GAP. Whether Arf GAP acts as an “uncoating factor” at
a time after completion of vesicle biogenesis or whether GTP hydrolysis is
initiated prior to the completion of the budding process, is still a matter of
some debate.
It is noteworthy that Arf1 also regulates the interaction of coat proteins
with the trans-Golgi network. Because evidence so far has not indicated a
role for Arf GAP1 in this organelle, we will limit our discussion to the COPI
system, but one should bear in mind that a role of Arf GAP1 at the TGN
cannot be ruled out at present.

3.2 Discovery of Arf GAP1 and its basic features

Arf GAP1 was identified following its purification from rat liver cytosol
(Cukierman et al., 1995; Makler et al., 1995). The purification was
monitored with a GAP assay in which the substrate was myristoylated Arf1
that had been loaded with [γ-32P]GTP in the presence of dimyristoyl-
phosphatidylcholine and cholate. The absence of phosphoinositides in this
assay probably precluded the detection of the phosphoinositide-dependent
Arf GAPs. Purification was greatly facilitated by the finding that GAP
activity could be readily recovered following denaturing conditions,
allowing for a critical purification step in the presence of 8 M urea, and
reverse-phase HPLC in organic solvents. The ability to renature Arf GAP1
by simple dilution with buffer is shared by yeast Arf GAPs Gcs1 and Glo3
(Poon et al., 1999; Poon et al., 1996), and is probably attributable to the zinc
coordination domain, as this domain retains the bound zinc even after
treatment in the presence of 6 M guanidine and 1 mM EDTA (I. Huber and
D. Cassel, unpublished observation). However, this resistance to
denaturants is not a property of all Arf GAPs, possibly because additional
domains that are required for GAP activity (such as the PH domain in
phosphoinositide-dependent GAPs) may not refold under these conditions.
Molecular cloning of the Arf GAP1 cDNA from a rat liver cDNA library
(Cukierman et al., 1995) revealed a protein containing 415 amino acids
(accession NP_659558) (Figure 1). Database entries indicate that the mouse
ortholog of Arf GAP1 (accession XP_130740) contains 414 amino acids,
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whereas two human splice variants encode proteins of 406 (NP_060679) and
414 (AAH28233) amino acids. Sequencing of different rat liver cDNA
library-derived clones has revealed the presence of multiple splice variants
(Cukierman et al., 1995). One variant had an insert containing an early stop
codon, and thus encoded a truncated protein that nevertheless had full Arf
GAP activity when expressed in E. coli, while another variant lacked the
entire zinc finger domain. In this variant the initiating methionine became
out of frame, but translation could be initiated from the second methionine in
the sequence (Met64), resulting in an inactive protein. Additional splice
variants were identified but have not been characterized in detail. Western
blot analysis of different rat tissues and cell lines showed that the long form
(containing 415 amino acids) that we consider the “normal” protein is the
most abundant form expressed. In some cases the expression of smaller
proteins was observed (see Figure 2), but whether these forms are splice
variants or degradation products has not been definitively established.
Attempts to express the full-length Arf GAP1 protein in E. coli were
unsuccessful, whereas fragments containing up to approximately 80% of the
amino terminal sequence could be readily expressed in bacteria (Cukierman
et al., 1995). Amino terminal fragments with full catalytic activity were
employed in numerous studies of the catalytic mechanism (see below).
More recently, full-length Arf GAP1 was successfully expressed in insect
cells using the baculovirus system (Huber et al., 2001; Vitale et al., 2000).
An amino terminal fragment containing the first 257 amino acids of Arf
GAP1 was employed for immunization of rabbits, generating antibodies that
recognized the protein with high affinity and specificity. Western blot
analysis (Figure 2) showed that the protein is ubiquitously expressed in rat
tissues, with the highest levels observed in brain and less in liver and lung.
In all tissues examined, the protein was distributed between cytosol and total
cellular membranes. Comparison to purified, recombinant protein revealed
that Arf GAP1 comprises ~0.004% of liver cytosolic proteins, and is yet less
abundant in most cultured cells.
Use of the antibodies in indirect immunofluorescence showed that Arf
GAP1 distributes between cytosol and the Golgi complex, where it
colocalizes with coatomer. This distribution pattern was observed in normal
rat kidney (NRK) cells (Cukierman et al., 1995) as well as in many other cell
types, including baby hamster kidney (BHK), HeLa and COS-7 cells (E.
Cukierman and D. Cassel, unpublished observations).
When examined in GAP assays in vitro, Arf GAP1 was reported to prefer
Arf1, as compared with Arf6 ((Brown et al., 1998; Vitale et al., 2000) and B.
Antonny and D. Cassel, unpublished observation). The preference for Arf1
over Arf6 may be further increased in vivo, due to the localization of Arf
 ARF GTPASE-ACTIVATING PROTEIN 1 1437

GAP1 to Golgi, and the differential distribution of Arf1 and Arf6 to Golgi
and plasma membrane/endosomes, respectively.

Figure 2. (A) The sequence of rat Arf GAP1 is shown with the four cysteines that comprise
the zinc finger (ZF) motif and the essential arginine residue (R50) highlighted. (B) The
domain organization of Arf GAP1 is shown. The N-terminal GAP catalytic domain is
conserved in all known Arf GAPs, whereas the non-catalytic domain is unique to Arf GAP1
and does not contain any motif present in other proteins.

When overexpressed in mammalian cells, Arf GAP1 caused the

disappearance of the Golgi complex and its fusion with ER, whereas inactive
mutants bearing substitutions in the scaffold cysteines of the zinc finger
(Cukierman et al., 1995) or in an invariant arginine residue (Arg50, Pick and
Cassel, unpublished observation) had no effect. A simple model, whereby
an excess of Arf GAP activity causes the conversion of Arf1 to the GDP
form with subsequent dissociation of coat protein from the Golgi, may
explain these findings. This would result in the fusion of the Golgi with the
ER, an effect similar to that of the drug brefeldin A (BFA), which inactivates
Arf by inhibiting a subset of Arf GEFs (Donaldson, 1992b; Donaldson and
Jackson, 2000; Helms and Rothman, 1992; Peyroche et al., 1999).
Structure-function studies showed that the catalytic domain of Arf GAP1 by
itself was ineffective in causing Golgi dissociation, and that this effect
requires determinants positioned in the carboxyl-terminal, non-catalytic part
of Arf GAP1 (Huber et al., 1998). Apparently, these determinants increase
the efficiency of GAP-induced Golgi disassembly by mediating GAP
binding to the Golgi, thereby increasing its proximity to its substrate,
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membrane-bound Arf1-GTP. This prediction has recently received strong

support by a study employing Arf GAP1 fragments fused to the green
fluorescence protein (GFP) (Yu and Roth, 2002), which demonstrated that
carboxyl terminal residues are both necessary and sufficient for targeting Arf
GAP1 to the Golgi. Additionally, this study has suggested a role of
phosphorylation by casein kinase-I in Arf GAP1 binding to the Golgi.

3.3 Catalytic core

When various amino terminal fragments of Arf GAP1 were tested for in
vitro GAP activity, high activity was observed in proteins containing the first
146 amino acids or more. Substantial, although much lower, activity was
still observed in a mutant protein containing only the first 121 amino acids,
whereas shorter fragments were inactive (Cukierman et al., 1995). These
findings suggested that the minimal GAP catalytic core resides within the
first ~120 amino acids. An essential role of the zinc finger motif was
initially suggested by the finding that mutations in its scaffold cysteines
abolished GAP activity (Cukierman et al., 1995). However, more recent
crystallographic data (Goldberg, 1999) indicated that the 16 amino acid
“loop” of the zinc finger domain is buried within the protein core, and is thus
unlikely to directly participate in catalysis. The loss of activity upon
mutation of the scaffold cysteines is probably the result of unfolding of the
protein upon the destruction of the zinc finger structure that results from the
loss of the chelated zinc atom. That this is indeed the case is suggested by
the decreased solubility of the mutant proteins and their aberrant
chromatographic behavior (D. Cassel, unpublished observations).
Limited proteolysis of the catalytically active Arf GAP1(1-146) resulted
in a fragment consisting of the first 136 amino acids, presumably
representing the complete catalytic core. The crystal structure of the
catalytic core of the Arf GAP domain was solved by Goldberg (Goldberg,
1999) and demonstrated a unique fold, not found in GAPs for other families
of GTPases. In this structure the zinc finger, composed of β strands, is
nested within an array of one β strand and six α helices. Some mechanistic
implications of this structure are discussed below (see also Chapter 2).

3.4 Catalytic mechanism

Small GTPases catalyze the hydrolysis of GTP by a conserved mechanism,

whereby a glutamine residue presents the water molecule at the active site
(Scheffzek et al., 1998). This mechanism has a built-in low catalytic
efficiency due to poor positioning of the glutamine residue for catalysis.
Structural studies of Ras and Rho GAPs complexed with their respective
 ARF GTPASE-ACTIVATING PROTEIN 1 1459

GTPases (Nassar et al., 1998; Rittinger et al., 1997; Scheffzek et al., 1997)
showed that the GAPs stimulate GTP hydrolysis in two ways. First, the
GAPs bind to the switch 2 regions of the GTPases, acting to stabilize this
region and to orient the glutamine residue in a position that is optimal for
catalysis. Second, the GAPs possess an “arginine finger” mechanism,
whereby an arginine residue is directly introduced into the active site of the
GTPase. This arginine residue is thought to facilitate GTP hydrolysis by
interacting with the glutamine residue and further orienting it for catalysis,
and by interacting with the phosphate groups of GTP, leading to a decrease
in their effective charge and thereby assisting in the nucleophilic attack of
the water molecule. However, recent studies have indicated that the arginine
finger mechanism does not take place in all GTPases, and is absent in Ran
and Rap GAPs (Brinkmann et al., 2002; Seewald et al., 2002)
The mode of interaction between Arf GAP1 and its substrate has been
studied by Goldberg (Goldberg, 1999), who co-crystallized Arf1•GDP and
the catalytic core of Arf GAP1. The structure revealed multiple interactions
of helix α3 of Arf1 with the GAP catalytic core. A number of interactions
were also observed between the GAP catalytic core and switch 2 of Arf1, but
the significance of these interactions remained unclear as switch 2 undergoes
a significant conformational change upon the GDP-to GTP switch, and
presumably the transition state of the GTPase reaction resembles the GTP
state of Arf1 rather than the GDP state which was used in this study. The
mode of interaction between Arf1 and GAP as observed in the above study
has subsequently been challenged (Click et al., 2002; Mandiyan et al., 1999),
although an alternative model has yet to be presented.
The mechanism that emerged from the structural study by Goldberg
suggested that Arf GAP1 does not contribute an arginine residue to facilitate
catalysis. The single arginine residue in the GAP catalytic core that is
invariant in all known Arf GAPs (Arg50 in Arf GAP1) pointed away from
the protein-protein interface. Additionally, the same study demonstrated that
coatomer greatly stimulated the activity of the catalytic core of GAP. These
findings raised the possibility that coatomer is required to complement the
catalytic activity of GAP, possibly by supplying the arginine residue to the
active site. The effect of coatomer on GAP activity will be further discussed
below. However, site-directed mutagenesis studies by several groups have
shown that the invariant arginine residue in Arf GAPs (always positioned 5
residues from the carboxyl-terminal cysteine of the zinc finger (see Figure
1)) is essential for GAP activity. Even conservative replacements with a
lysine residue completely abolished catalytic activity of Arf GAP1 (Szafer et
al., 2000), and the invariant arginine was also found essential for activity in
other Arf GAPs (Jackson et al., 2000; Mandiyan et al., 1999; Miura et al.,
2002; Randazzo et al., 2000). These findings suggested that this residue
146
10 CASSEL

might in fact act in an arginine-finger mechanism. Once again, structural

studies of the complex between Arf GAPs and their substrate in the GTP-
bound state will be required in order to resolve this issue, as well as other
mechanistic detail of Arf GAP action.

3.5 Regulation of Arf GAP1 activity

3.5.1 Effect of membrane lipids

The substrate of GAP, Arf•GTP, is tightly associated with membrane lipids.

This is due to the myristoyl group attached to the amino terminus, as well as
the amphipathic amino-terminal helix, that interacts with the protein core in
the GDP-bound state and is thus shielded from solvent, but become exposed
in the GTP-bound state (Antonny et al., 1997a; Cherfils et al., 1998;
Goldberg, 1998). The loading of Arf1 with GTP (in preparation for a GAP
assay) can be achieved using a large variety of phospholipids and detergents.
By systematically varying the composition of liposomes used during Arf1
loading, Antonny (Antonny et al., 1997b) found that the activity of Arf
GAP1(1-257) (a fragment of Arf GAP1 that can be readily expressed in
bacteria) is increased in the presence of phospholipids with small head
groups such as phosphatidylethanolamine, and is dramatically stimulated
upon addition of a small percentage of diacylglycerols bearing at least one
unsaturated fatty acid chain. The effects of the different lipid compositions
on Arf GAP1 activity closely correlated with the binding of Arf GAP1 to the
liposomes. From these results, it was concluded that a hydrophobic domain
in GAP can bind to membrane lipids, that this binding is facilitated by the
presence of open spaces in the lipid bilayer created by cone-shaped lipids,
and is sterically inhibited by the presence of large phospholipid head groups.
The good correlation between GAP binding to liposomes and GAP activity
suggested a mechanism whereby binding of Arf GAP1 to membrane lipids
results in an increase in GAP activity simply due to an increase in the
effective GAP concentration in the proximity of its Arf•GTP substrate, the
latter being tightly bound to liposomes regardless of their composition.
Phosphoinositides, such as PI(4,5)P2, were reported to increase Arf
GAP1 activity in some studies (Makler et al., 1995; Vitale et al., 2000), but
not in others (Antonny et al., 1997b). How this effect is brought about is
uncertain because unlike ASAP- and ARAP-like Arf GAPs, Arf GAP1 does
not have a PH domain. It should be borne in mind that the interactions of
proteins with lipid vesicles may differ markedly from their interaction with
biological membranes. Whereas reported effects of lipids on Arf and Arf
GAP activities are suggestive of regulatory events in vivo, the latter are
difficult to demonstrate and thus remain uncertain.
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3.5.2 Role of coatomer

Goldberg has demonstrated that coatomer, which in itself has no effect on

GTP hydrolysis on Arf1, dramatically stimulates Arf GAP1-dependent GTP
hydrolysis (Goldberg, 1999). In this study GAP activity was assayed in
solution, by employing a truncated Arf1 mutant that lacks the first 17 amino
acids (∆17-Arf1). Unlike wild-type Arf1 that depends on a lipid
environment (Antonny et al., 1997a; Paris et al., 1997) or effectors (Zhu et
al., 2000) for GTP binding, ∆17-Arf1 can bind GTP to high stoichiometry in
solution (Paris et al., 1997). This is apparently due to an alleviation of a
restriction brought about by the amphipathic amino terminus of the wild type
Arf1 protein, which becomes exposed in the GTP state and must be shielded
by binding to a hydrophobic surface (Amor et al., 1994; Goldberg, 1998).
The stimulation of Arf GAP1 activity was demonstrated using the catalytic
core of Arf GAP1 (residues 1-136). One obvious conclusion from these
findings was that Arf GAP1 and coatomer do not compete for the same
binding site on Arf1. Interestingly, the opposite situation was recently
reported for the coat adaptors termed GGAs, which compete with GAP for
Arf1 binding and thus inhibit GAP activity, thereby stabilizing their binding
to membranes (Puertollano et al., 2001).
Our group has extended the studies of the role of coatomer to more
naturally-occurring conditions, using wild type, myristoylated Arf1 in place
of ∆17-Arf1, and wild type, full length Arf GAP1 in place of the catalytic
core. When using myr-Arf1 loaded with GTP in the presence of liposomes
as substrate, Arf GAP1 activity was higher by two orders of magnitude than
that observed with ∆17-Arf1, and this activity was not further increased in
the presence of coatomer (Szafer et al., 2000). This suggested that while
coatomer strongly stimulates Arf GAP1 activity in a soluble system, its
effect vanishes when Arf1 and Arf GAP1 are brought to proximity by their
mutual binding to phospholipids (see 3.5.1 above). Additionally, with a
different type of Arf GAP, the phosphoinositide-dependent ASAP1 (Brown
et al., 1998), coatomer did not increase GAP activity even when using ∆17-
Arf1 as substrate (Szafer et al., 2000). We concluded from these studies that
coatomer might not be an essential component of the mechanism of GTP
hydrolysis on Arf1, and that Arf GAPs may possess the entire catalytic
machinery of this mechanism.
In a subsequent study (Szafer et al., 2001) we analyzed the effect of
coatomer using Golgi membranes, the biological site of coatomer assembly.
We designed an assay in which myr-Arf1 is loaded with GTP by Golgi
membrane-bound GEFs and thus becomes bound to the membrane. In rat
liver Golgi membrane preparations endogenous GAP activity is relatively
low (particularly so after salt wash), and thus the “spontaneous” rate of
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decay of the GTP state of Arf1 is slow. The effect of external GAPs on GTP
hydrolysis can then be readily measured. Arf GAP1 exhibited high activity
in this assay, but unlike the liposome assay, addition of coatomer
approximately doubled this activity. A similar two-fold effect of coatomer
was observed when using the whole cellular repertoire of Arf GAPs,
represented by coatomer-depleted liver cytosol. Immunodepletion
experiments suggested that although Arf GAP1 contributes significantly to
cytosolic GAP activity, most of the activity in cytosol is mediated by
additional Arf GAPs under these conditions. The identity of these soluble
Arf GAPs is currently unknown.
A different pattern was observed with an Arf GAP from S. cerevisiae.
This organism contains at least 3 Arf GAPs, termed Gcs1, Glo3 and Age2
(Poon et al., 1999; Poon et al., 2001; Poon et al., 1996). When Glo3 was
tested in the Golgi assay, its GAP activity showed large dependency on
coatomer, while in the liposome assay Glo3 activity was low both in the
absence and presence of coatomer (Szafer et al., 2001). These quantitatively
different effects of coatomer on GAP1 and on Glo3 can be rationalized as
follows. The above mentioned studies in Golgi membranes suggest that
coatomer can increase the efficiency of Arf GAP1 stimulation of GTP
hydrolysis in a biological setting. Moreover, evidence for a similar role of a
coat in GTP hydrolysis was recently presented for the COPII system, whose
mechanisms appear to resemble those of the COPI system (Antonny and
Schekman, 2001; Springer et al., 1999). However, unlike in the soluble
system (Goldberg, 1999), a recruitment of GAP to its site of action, either by
binding to membrane lipids (Antonny et al., 1997b) or by interaction with
membrane proteins (Aoe et al., 1997), should provide significant GAP
activity also in the absence of a coat complex, as suggested by our
experiments with liposomes and Golgi membranes. The role of coatomer
would then be to fine-tune the rate of GTP hydrolysis at COPI assembly
sites, a process that could play an important role in cargo sorting (see
below). In the case of Glo3, the large effect of coatomer on GAP activity
might be related to the fact that this protein forms a complex with coatomer
(Eugster et al., 2000), which should bring this GAP into proximity with
Arf1•GTP. Glo3 is not an ortholog of Arf GAP1, and perhaps unlike Arf
GAP1, Glo3 does not interact with membrane lipids, thus explaining its low
activity in the presence of liposomes. The reason for the low activity of
Glo3 with Golgi membranes is less clear; one possible reason might be the
use of a heterologous system consisting of a yeast Arf GAP and a
mammalian Golgi. Future studies should reveal whether the mammalian
ortholog of Glo3 (Arf GAP3) shows a coatomer-dependent GAP activity on
Golgi membranes.
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13

3.5.3 Regulation by cytoplasmic domains of p23/24 proteins

p23/24 proteins are abundant membrane proteins that shuttle between the
ER, the ER-Golgi intermediate compartment (ERGIC) and cis-Golgi.
Multiple members of this family are found in hetero-oligomeric complexes
(Emery et al., 2000; Fullekrug et al., 1999; Gommel et al., 1999). They
present short cytoplasmic tails, many of which bind coatomer through a
diphenylalanine (FF) and/or a dibasic motif (Dominguez et al., 1998; Fiedler
et al., 1996; Nickel et al., 1997; Sohn et al., 1996). Recent studies suggest
that some of these tails may act as inhibitors of Arf GAP1 activity. The first
demonstration of this effect came from Goldberg’s study (Goldberg, 2000),
where he showed that a peptide representing the cytoplasmic tail of a p24
protein (with the sequence YLKRFFEVRRVV, termed hp24a) selectively
inhibited coatomer-stimulated activity of the catalytic core of Arf GAP1 in
solution. The peptide had no effect on GAP activity observed in the absence
of coatomer. Several peptides representing cytoplasmic tails of other p24
proteins, as well as p24a variant peptides containing replacements in
residues that are critical for coatomer binding, had no effect of coatomer-
dependent Arf GAP1 activity. Based on these findings, Goldberg suggested
a model for cargo sorting on the basis of GTP hydrolysis on Arf1. In this
model, the interaction of coatomer with cargo (such as the p24 tails)
prevents coatomer stimulation of GAP, resulting in stabilization of
Arf1•GTP. In this way the correct match of cargo, coat complex, and Arf is
“proofread” and specificity in vesicle assembly can be assured. In the
absence of the cargo component, coatomer-dependent Arf GAP activity
rapidly hydrolyzes GTP on Arf1, thereby preventing the formation of COPI
vesicles that lack cargo. Although the effect on GTP hydrolysis was
restricted to p24a, the existence of p24 proteins in hetero-oligomeric
complexes could explain how at least some other members of this family
may be sorted into COPI vesicles.
Recently, Nilsson’s group has reported the results of their studies of the
role of GTP hydrolysis in cargo sorting, using an in vitro assay for vesicle
budding from liver Golgi membranes (Lanoix et al., 1999; Lanoix et al.,
2001). In this system, Golgi enzymes as well as the p23/24 proteins are
sorted into vesicles by a COPI-dependent mechanism, which is in
accordance with the model of Golgi maturation (Bonfanti et al., 1998;
Mironov et al., 2001; Pelham, 2001). Significantly, sorting was observed in
the presence of GTP, but not in the presence of the hydrolysis-resistant GTP
derivative, GTPγS. Several lines of evidence suggested that p23/24 proteins
might be involved in a GTP hydrolysis-dependent sorting event. Addition of
low concentrations of certain p23/24 peptides prevented the sorting of all
Golgi markers examined. The most active peptide was similar to the one
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found by Goldberg to inhibit coatomer-dependent GAP activity

(CQIYYLKRFFEVRRVV, derived from the same p24 protein but named in
this study p24β1). When examined for their effect on Arf GAP1, some p24
peptides were found to bind the GAP and to inhibit its activity. Comparison
of different p24 peptides, as well as variant p24β1 peptides, revealed a good
correlation between Arf GAP1 binding capacity, effect on Arf GAP1
activity, and the ability of the peptides to inhibit cargo sorting. Unlike the
study by Goldberg (Goldberg, 2000), the peptides were found in this study to
inhibit GAP activity in the absence of coatomer. Possible reasons for these
different results are difference in the lengths of the p24 peptides employed,
as well as types of assays and conditions employed. The p24 tail-derived
peptides used in both studies contain a number of hydrophobic residues as
well as multiple positive charges, and could therefore exhibit non-specific
effects, particularly so when membranes are concerned. Thus, additional
approaches are required in order to establish a role for cytoplasmic tails of
p24 proteins in sorting cargo into Golgi-derived vesicles and the role of GAP
activity.

3.6 Role of Arf GAP1 in KDEL receptor signaling

The KDEL receptor (KDEL-R) serves as a specialized mechanism for the

retrieval of escaped soluble ER-resident proteins, many of which bear the
Lys-Asp-Glu-Lys (KDEL) signal (Andres et al., 1991). The receptor is a
seven-pass transmembrane protein with a short cytoplasmic tail. It shuttles
between the Golgi and the ER, based on a cycle of ligand-induced Golgi to
ER transport followed by the release of the ligand in the ER and the
recycling of the empty receptor back to the Golgi. The directionality of
these cycles is apparently determined by an increased affinity of the receptor
for KDEL ligands at the lower pH that prevails at the Golgi lumen as
compared with the ER (Wilson et al., 1993).
The KDEL-R is transported from the Golgi through COPI vesicles
(Majoul et al., 1998; Orci et al., 1997). Studies by Hsu and colleagues have
suggested a role of Arf GAP1 in this process. Co-immunoprecipitation
experiments demonstrated that the KDEL-R interacts with the GAP,
apparently following receptor oligomerization (Aoe et al., 1997). Moreover,
using cells harboring a plasmid that allows the regulated expression of a
KDEL ligand, Aoe (Aoe et al., 1998) demonstrated that interaction to the
KDEL-R with Arf GAP1 depends on ligand binding to the receptor. A non-
catalytic domain at the carboxyl-terminal part of Arf GAP1 that is required
for the ability of overexpressed GAP to disassemble the Golgi in transfected
cells (Aoe et al., 1999) was also required for GAP-KDEL-R interaction.
More recently, an interaction between Arf GAP1 and the KDEL-R, as well
 ARF GTPASE-ACTIVATING PROTEIN 1 151
15

as a KDEL ligand-induced recruitment of Arf GAP1 to the Golgi, were

demonstrated in living cells using fluorescence resonance energy transfer
(FRET) (Majoul et al., 2001).
What could be the function of Arf GAP1 recruitment to the Golgi
membrane following ligand binding to KDEL-R? One possibility is that
GAP interaction with the receptor inhibits its catalytic activity. Such
(putative) inhibition would promote coatomer assembly onto membrane
patches containing the KDEL-R, as suggested for p24 proteins (section
3.5.3). In this way the KDEL-R may act analogously to the p24 proteins to
help sort cargo into Golgi-derived, COPI coated vesicles.
Another way to envision the role of GAP in cargo sorting is based on
lessons from the COPII system (Antonny and Schekman, 2001; Springer et
al., 1999). Like the COPI system, coat binding in the COPII system is
triggered by the activation of the GTPase, Sar1, at the ER membrane.
However, the GAP in the COPII system (the Sec23/24 complex) plays a
direct and essential role in the assembly of the coat protein complex, by
binding directly to both Sar1 and the other coat components, Sec13/31.
Additionally, interactions of Sec23/24 with SNAREs are thought to ensure
the inclusion of the fusion machinery in COPII vesicles. There is growing
evidence for parallel mechanisms in the two systems. Thus, the yeast Arf
GAP Glo3 was shown to interact with coatomer (Eugster et al., 2000),
although this has not so far been demonstrated for either Arf GAP1 or its
yeast ortholog Gcs1. As in the COPI system, the Sec13/31 coat was found
to stimulate GTP hydrolysis mediated by Sec23/24 (Antonny et al., 2001).
Yeast Arf GAPs show genetic interactions with SNAREs (Poon et al., 1999),
and recent evidence suggests that a transient interaction of yeast Arf GAPs
with v-SNAREs may activate the v-SNAREs for Arf1 binding to membranes
(Rein et al., 2002). Together, these findings suggest that Arf GAPs could
function as an intimate component of the COPI complex. According to this
view, cargo such as the KDEL-R could be recruited to COPI vesicles
through its interaction with GAP, rather than vice versa.
However, there is one obvious distinction between the COPI and COPII
systems. Unlike the COPII system, coatomer can bind directly to Arf,
without any obvious requirement for GAP (Zhao et al., 1997), and can
induce vesicle budding from liposomes in a minimal system containing only
Arf1 and the guanine nucleotide (Donaldson et al., 1992a). Thus, further
studies will be required to find out how far the analogy between the two
systems can be taken.
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4. CONCLUSION

GTPase-activating proteins acting in the early secretory system appear to

function not merely as signal terminators but also as components in key
processes of the system such as cargo sorting. While accumulating evidence
suggests such a role for Arf GAP1, there are still many questions that need
to be answered more definitely. One should also bear in mind that Arf
GAP1 may not be the only player at the Golgi, and other Arf GAPs that
might fulfill this function have been described (Andreev et al., 1999; Liu et
al., 2001; Mazaki et al., 2001; Miura et al., 2002). Likewise, at least two Arf
GAPs were reported to function in the ER-Golgi shuttle in S. cerevisiae
(Poon et al., 1999; Poon et al., 1996). Thus the specific role of each of these
GAPs in COPI-mediated trafficking will have to be further analyzed. The
recent advances in the area of membrane traffic, as well as the introduction
of modern techniques that allow studies in living cells, seem to promise
rapid progress in the understanding of the role of GAPs in this system.

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Chapter 8
GIT PROTEINS: ARF GAPS AND SIGNALING
SCAFFOLDS

ROBERT SCHMALZIGAUG AND RICHARD PREMONT

Department of Medicine (Gastroenterology), Duke University Medical Center, Durham, USA

Abstract: GIT proteins are GTPase-activating proteins (GAPs) for the ADP-ribosylation
factor (Arf) family of GTPases. The two GIT family members, GIT1 and
GIT2, stimulate GTP hydrolysis on all the known Arf subtypes about equally
well, and this activity is stimulated by PI(3,4,5)P3. As such, GITs are negative
regulators of the cellular functions of Arfs, such as intracellular vesicle traffic
and cytoskeletal rearrangement. In addition, GITs form the core of a large
protein complex involved in integrating signals through multiple pathways,
involving G protein-coupled receptors, receptor tyrosine kinases, integrins,
and at least two classes of small GTP-binding proteins, the Arfs and
Rac/Cdc42, Rho families.

1. INTRODUCTION

GIT proteins are members of the fast-growing family of Arf GAP domain-
containing proteins, which presently has 22 known human members (Figure
1). Based on overall sequence similarity and domain organization, the
members of this family can be divided into two major subgroups that contain
subfamilies with multiple members. We classify these into the smaller
GAPs (Arf GAP1, Arf GAP3/Zfp289, RIP/Hrb, SMAP1/2, centaurin-
α1/α2), and the large, multidomain GAPs (GITs, ASAP1/PAP, ACAPs,
ARAPs, AGAPs). These proteins all share the ~100 amino acid
CX2CX16CX2C zinc finger-containing Arf GAP domain, although several
members have yet to be directly tested for GAP activity. In another
classification scheme, the centaurin subfamily of Arf GAPs has been
proposed for those Arf GAPs also containing ankyrin repeats and PH
domains, and includes two centaurin-α proteins, ASAP1/PAP (centaurin-
β’s), ACAPs (also centaurin-β’s), AGAPs (centaurin-γ’s) and ARAPs
159
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2 SCHMALZIGAUG AND PREMONT

(centaurin-δ’s) (Jackson et al., 2000a). In all these GAPs (except the

centaurin-α subgroup), the GAP domain is found centrally in the protein. In
contrast to this centaurin group, GITs do not have identifiable PH domains,
and share the amino terminal GAP domain localization with the small GAP
proteins and centaurin-α’s. GITs have a multidomain structure and
participate in multiple intracellular signaling events (as detailed below).
This suggests that other, less well-characterized multidomain GAP family
members will also be found to participate in multiple cellular signaling
pathways.

Figure 1. Mammalian Arf GAP Family Domain Structure. PBS, paxillin binding sequence;
PH, pleckstrin homology domain; Rho GAP, GTPase-activating protein domain for Rho
family small GTPases; SH3 Src homology type 3 domain; Spa2, region of homology with
yeast Spa2p and Sph1p

2. GITS: DISCOVERY AND NOMENCLATURE

Over the past few years, GITs have been identified in several distinct
contexts by many laboratories. This has led to a wealth of information about
GITs on the one hand, and to multiple names and a confusing literature on
the other hand.
In short, there are only two GIT genes in humans or mouse, and two
identified GIT sequences from chicken. These sequences have been called
GIT1/Cat-1/p95-APP1 and GIT2/KIAA0148/Cat-2/p95-PKL/p95-APP2.
These are summarized in Table 1.
 GITS: ARF GAPS AND SIGNALING SCAFFOLDS 1613

Table 1. GIT protein nomenclature. °Mouse Cat-1 was partially peptide sequenced, but not
cloned or fully sequenced. *PKL was originally reported as being a human cDNA sequence,
but has since been shown to be from chicken and identical to p95-APP2.
GIT1 GIT2 Species Reference
GIT1 Rat Premont et al., 1998
GIT1 GIT2 Human Premont et al., 2000
(Cat-1)º Cat-2 Mouse Bagrodia et al., 1999
PKL Chicken* Turner et al., 1999
p95APP1 Chicken Di Cesare et al., 2000
p95APP2 Chicken Paris et al., 2002
KIAA0148 Human Nagase et al., 1996

The initial discovery of the GIT proteins came from a search for G
protein-coupled receptor kinase 2 (GRK2) interacting proteins. In a yeast
two-hybrid screen, GRK2 was employed as the bait and a rat cDNA library
served as the prey. One positive clone was found to encode the C-terminus
of a previously unknown protein that was named GRK interactor 1, or GIT1
(Premont et al., 1998). The full-length rat GIT1 sequence was found to
contain an N-terminal zinc finger-like motif with similarity to the then
recently identified rat Arf GAP1 GAP domain (Cukierman et al., 1995),
suggesting that it too was a GAP for Arf proteins. Searching DNA databases
for rat GIT1-related sequences revealed two classes of GIT1-related
sequences: the human KIAA0148 sequence encoding a putative protein of
unknown function with high similarity to the first two-thirds of GIT1
(Nagase et al., 1995), and single human and mouse ESTs with similarity to
the extreme C-terminus of GIT1. Reverse transcriptase PCR revealed that
these two sequences were in fact derived from the same alternatively spliced
gene, and then named GIT2 (Premont et al., 2000).
GITs were next found in connection with two small GTPases, Rac1 and
Cdc42, and their effectors, the p21-activated protein kinases (PAKs).
Bagrodia et al. (1999) immunoprecipitated PAK3 from mouse fibroblasts
and co-purified a tyrosine phosphorylated protein of 95 kDa. Analysis of the
protein revealed an indirect binding to PAK3 via the PIX/Cool protein, a
guanine nucleotide exchange factor (GEF) for Rac and Cdc42. Because the
protein directly bound to Cool and was found to be tyrosine phosphorylated,
it was called Cool-associated tyrosine phosphorylated protein 1 or Cat-1.
From the limited peptide sequences obtained from this purified protein, it is
likely mouse GIT1. cDNA cloning based on Cat-1 peptide sequences
yielded the related mouse Cat-2 cDNA, the mouse ortholog of human GIT2
(Bagrodia et al., 1999). Zhao et al. (2000b) used a similar strategy to
identify GIT1 as a binding partner of PIX/Cool and PAK complexes. Both
GIT1 and GIT2 bind to PIX proteins, and indirectly to PAKs (Bagrodia et
al., 1999; Zhao et al., 2000b; Premont et al., 2000). Another study
investigated the link between membrane transport and Rac-mediated actin
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4 SCHMALZIGAUG AND PREMONT

reorganization by identifying proteins that specifically bound to Rac•GTP.

Two proteins, p95-APP1 and p95-APP2 (95 kDa, Arf GAP-putative, PIX-
interacting, Paxillin-interacting proteins), were isolated (Di Cesare et al.,
2000; Paris et al., 2002). These are the chicken orthologs of mammalian
GIT1 and GIT2.
Yet another group applied a GST-paxillin pull-down assay to isolate
paxillin-interacting proteins, and identified a protein that linked paxillin to
the PIX/PAK complex, which they named p95-PKL (paxillin-kinase linker)
(Turner et al., 1999). PKL binds to a very small region of paxillin, the LD4
motif, and this motif directs the localization of PKL (and the PIX/PAK
complex) to focal adhesions. The PKL clone was reported to come from a
human HeLa cell cDNA library, and originally was thought to encode a third
mammalian GIT family member. Despite much effort, no authentic PKL
sequences could be amplified from human cDNA, and no human gene for
PKL could be mapped or found in database searches (R. T. Premont,
unpublished observations). The PKL cDNA is identical to the chicken p95-
APP2 cDNA, and has since been acknowledged to be derived from a
chicken cDNA library (Turner et al., 2001). Thus, PKL/p95-APP2 is the
chicken ortholog of mammalian GIT2. Another study sought to find
proteins that interact with paxillin specifically in the perinuclear region
(Mazaki et al., 2001). From a GST-paxillin pulldown experiment, the
shortest splice variant of GIT2 (GIT2-short, KIAA0148) was isolated.
GIT2-short co-localized with paxillin in the perinuclear region but not in
focal adhesions (Mazaki et al., 2001).
An elegant screen for substrates of the receptor-type protein tyrosine
phosphatase ζ (PTPζ) used a yeast substrate-trapping screen, where proteins
were phosphorylated by over-expressed Src and trapped by an inactive
mutant of PTPζ (Kawachi et al., 2001). GIT1 is a known Src substrate
(Bagrodia et al., 1999), and was found to interact with the mutant PTPζ and
to be a substrate for dephosphorylation by active PTPζ. As GIT1
phosphorylation by protein kinases is associated with focal adhesions and
cell attachment, this finding potentially completes the regulatory cycle of
GIT1 phosphorylation by extracellular stimuli.
In summary, GIT proteins have been identified multiple times in very
different biological contexts. The current challenge is to organize these
diverse functional roles into a better understanding of the physiological
function of these proteins.
 GITS: ARF GAPS AND SIGNALING SCAFFOLDS 1635

3. GIT GENES, SPLICING AND TISSUE

DISTRIBUTION

3.1 Genes and Genomic location

Human GIT1 and GIT2 proteins are encoded by two genes, located at
chromosome bands 17p11.2 and 12q24.1, respectively (Premont et al.,
2000). The two mouse GIT genes are located on chromosomes 1 and 5 (R.
T. Premont, unpublished observation). No additional GIT-like sequences
were detected in the human or mouse genomes. Chickens also appear to
have two GIT genes (Di Cesare et al., 2000; Paris et al., 2002). To date,
neither GIT gene locus has been associated with any disease. The human
GIT1 gene consists of 20 coding exons distributed over 15 kb. The mouse
GIT1 gene has 19 exons, as there is no intron separating the sequences
equivalent to human exons 13 and 14. The human and mouse GIT2 genes
have an identical structure of 21 coding exons spread over 50 kb. The
location of introns is highly conserved between the two GIT genes, with 18
introns inserted in equivalent positions in the two human and mouse
sequences (R. T. Premont, unpublished observations). In GIT1, an extra
exon is present in a non-conserved part of the C-terminus, and in GIT2, an
additional intron is present within the region encoding the second ankyrin
repeat, and the 21st exon encodes the C-terminal nine amino acids of the
GIT2-short splice variant (see below).

3.2 Alternative splicing

GIT1 appears to be expressed only as the full-length protein, as splice

variants have not been found in human or rat tissues using RT-PCR
(Premont et al., 2000). However, the rat GIT1 sequence contains a nine
amino acid insertion after residue 253, as compared to the human, mouse
and chicken GIT1 sequences, due to a short extension at the junction of
exons 7 and 8. Thus, rat GIT1 is 770 residues long, while human and mouse
GIT1 have 761 residues.
In contrast, human GIT2 mRNA is expressed as multiple splice variants
(Premont et al., 2000), and chicken GIT2/PKL was suggested to have
multiple splice variants as well (Turner et al., 1999). Analysis of mRNA
from multiple human tissues suggests that the GIT2 transcript undergoes
tissue-specific, alternative splicing. The longest form of the protein, called
GIT2-long, is co-linear with GIT1 and uses 20 exons, but alternative splicing
leads to two classes of shorter GIT2 variants, in which either internal
sequences appear to be deleted or the C-terminus is truncated (see Figure 2).
First, direct sequencing of eight distinct human GIT2 cDNAs identified five
164
6 SCHMALZIGAUG AND PREMONT

contiguous and independently spliced blocks of sequence between residues

333 and 578 (originally termed sequences A through E) that are deleted in
individual clones. Sequence A corresponds to exons 12 and 13, B plus C
correspond to exon 14 (which appears to have an internal splice acceptor site
in the codon for residue 449), and sequences D and E match exons 15 and
16, respectively. Totally independent splicing of these five blocks would
potentially lead to 32 internally spliced variants. In the second class of
alternative splicing, exon 14 is spliced to a distinct exon (i.e., exon 21,
shown as D’ in Figure 2) that leads to early translation termination,
producing the shortest known variant of GIT2 (called GIT2-short or
KIAA0148) that lacks just over 300 amino acids at the C-terminus
(including the major paxillin binding site). However, it remains unknown
whether all GIT2 mRNA splice variants are translated into distinct variants
of the GIT2 protein. Other groups have identified GIT2-long from chicken
(i.e., p95-APP2/PKL) (Turner et al., 1999; Di Cesare et al., 2000), and one
shorter GIT2 version (i.e., mouse Cat-2) that lacks sequences B and C (exon
14) (Bagrodia et al., 1999). The GIT2-short/KIAA0148 variant has been
reported as well (Nagase et al., 1995; Mazaki et al., 2001).

Figure 2. Domains identified in GITs and alternative splicing of GIT2.

3.3 GIT tissue distribution

The mRNAs encoding GIT proteins appear to be ubiquitously expressed in

rat, human and chicken (Premont et al., 1998; Premont et al., 2000; Paris et
al., 2002). Low levels of GIT1 mRNA are found in liver and spleen,
medium levels in lung, skeletal muscle, heart and kidney and highest levels
in testes and brain (Premont et al., 1998; Premont et al., 2000). Direct RT-
PCR amplification of GIT2 splice variants from a variety of human tissues
suggests that this message undergoes extensive tissue-specific alternative
splicing, as detailed above. GIT2 mRNA is expressed at moderate to high
levels in many tissues including the heart, brain, kidney and spleen (Nagase
et al., 1995; Premont et al., 2000). The GIT2-short variant in particular was
found at high levels in spleen, testes and leukocytes (Premont et al., 2000).
 GITS: ARF GAPS AND SIGNALING SCAFFOLDS 1657

The presence of native GIT1 protein has only been reported for brain and
testes, as well as the HeLa, COS7 and CHOK1 cell lines (Zhao et al., 2000b;
Manabe et al., 2002). The GIT2-short protein has been detected in several
cell lines (U937, HeLa and NIH3T3 cells), with the highest levels in the
immunoderived Jurkat cell line (Mazaki et al., 2001). Neither a
comprehensive study of GIT1 and GIT2 protein expression in various
tissues, nor the identification of most of the multiple predicted GIT2 protein
variants has been reported so far. The main limitation to such a study has
been the lack of specific antibodies able to distinguish between GIT1 and
GIT2. Specific antibodies are crucial because the full-length forms have the
same molecular weight. Antibodies are commercially available from Santa
Cruz Biotechnology and Transduction Laboratories. The GIT1 and GIT2
antibodies from Santa Cruz do not appear to be very avid and give only faint
signals in immunoblots, although they do distinguish recombinant GIT1 and
GIT2. The GIT1 monoclonal antibody from Transduction Labs has good
specificity for GIT1 versus GIT2, but only weakly recognizes endogenous
levels of protein. The PKL monoclonal antibody from Transduction Labs
(raised against chicken GIT2) is fairly avid but not at all specific for
PKL/GIT2 versus GIT1 (R. T. Premont, unpublished observations). Thus,
for example, it remains unclear which GIT protein(s) is present in the Jurkat
T cell line, as detected with the PKL monoclonal antibody (Ku et al., 2001).

4. GIT STRUCTURE AND DOMAINS

4.1 GAP domain

In GIT1, the GAP domain was the first functional domain characterized. It
was found by similarity to the two known Arf GAPs at the time, rat Arf
GAP1 and yeast Gcs1. All proteins that have been shown to have Arf GAP
activity share a conserved domain containing a CX2CX16CX2C zinc finger
(Figure 1). A second feature essential for GAP activity is an arginine
residue that has been suggested to serve as the ‘arginine finger’ that
enhances the rate of GTP hydrolysis by several orders of magnitude
(Scheffzyk et al., 1998). Mutation of either of the first two cysteine residues
of the zinc finger abolished GAP activity of Arf GAP1 (Cukierman, et al.,
1995). Mutation of this arginine residue in other Arf GAPs (Arf GAP1,
ASAP1/PAPα, ACAPs, ARAP1) results in ablation of GAP activity
(Mandiyan et al., 1999; Randazzo et al., 2000; Jackson et al., 2000a; Szafer
et al., 2000; Miura et al., 2002).
The GAP domain of GIT1 is located at the very N-terminus of the protein
and contains both the cysteine residues of the zinc finger followed by the
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8 SCHMALZIGAUG AND PREMONT

essential arginine residue five amino acids downstream of the zinc finger
(Figure 2). The GAP domain of GIT2 has the conserved cysteines and
arginine at the exact same positions as in GIT1 (Premont et al., 2000).
Purified recombinant GIT1 and GIT2 contain stoichiometric amounts of zinc
(Vitale et al., 2000), and the X-ray crystal structure of the GAP domains of
rat Arf GAP1 and human β-PAP show a single zinc atom bound by the four
cysteine residues (Goldberg, 1999; Mandiyan et al., 1999).
Arf GAP1, GIT1 and GIT2 have very similar GAP activities, with GIT1
being slightly more potent than Arf GAP1 and GIT2, using Arf1 as a
substrate (Vitale et al., 2000). GITs promote GTP hydrolysis in all three
subtypes of Arfs with similar efficiency and thus do not show any obvious
enzymatic specificity. In contrast, Arf GAP1 stimulated the GTP hydrolysis
on Arf1-5, but had little effect on Arf6 (Vitale et al., 2000). The distinct
stimulation of Arfs by the different Arf GAP subtypes introduces a level of
specificity within the large GAP family.
The GAP activity of GIT1 is regulated by the phosphatidylinositiol 3,4,5-
trisphosphate (PI(3,4,5)P3) in vitro. The presence of PI(3,4,5)P3 increases
GAP activity of GIT1 3.5-fold and of GIT2 2-fold (Vitale et al., 2000). The
components of the in vitro assay were limited to the Arf1 and GIT proteins
and vesicles carrying PI(3,4,5)P3. Thus, it is likely that the stimulatory
effect of PI(3,4,5)P3 comes from lipid-dependent localization of Arf and GIT
to the vesicles. Stimulation of Arf GAP activity through a direct effect on
the Arf itself by PI(3,4,5)P3 is unlikely, as the GAP activity of Arf GAP1 is
not promoted by PI(3,4,5)P3 under identical assay conditions (Vitale et al.,
2000). However, GIT proteins do not have an obvious PH domain to bind to
PI(3,4,5)P3 (or PIP2) like many other Arf GAPs (Figure 1) (Jackson et al.,
2000b). The mechanism of regulation of the GIT GAP activity by
PI(3,4,5)P3 remains elusive. Perhaps GITs can interact with PI(3,4,5)P3
through sequences distinct from the PH domain, as reported for neurogranin
(Lu et al., 1997). It is interesting to note that various Arf GAPs are
stimulated by distinct sets of phosphoinositides and their metabolites; e.g.
Arf GAP1 activity is stimulated by PIP2 and diacylglycerol, ASAP1, β-PAP
and ACAPs by PIP2, and ARAPs by PI(3,4,5)P3 (Antonny et al., 1997;
Randazzo, 1997, Vitale et al., 2000; Jackson et al., 2000a; Krugmann et al.,
2002, Miura et al., 2002). The regulation of GAPs by distinct
phosphoinositides adds another level of specificity to the regulation of the
Arf GTPases by their GAPs.
The first GAP-dead mutant of GIT1 was made by deleting the 45 N-
terminal residues, including the cysteine residues of the zinc finger and the
conserved arginine residue. GIT1-¨45 failed to activate GTPase activity of
Arf1 (Premont et al., 1998). The single mutation of the conserved arginine
39 (to alanine) abolished the Arf GAP activity of GIT1 and GIT2 as well (R.
 GITS: ARF GAPS AND SIGNALING SCAFFOLDS 1679

T. Premont, unpublished observations). In addition, the first cysteine residue

of the zinc finger in GIT2-short has been mutated to alanine. This mutation
abolished the Arf GAP activity of GIT2-short (Mazaki et al., 2001).
Therefore, both the arginine and the zinc finger are required for the GAP
activity of GIT proteins.

4.2 Ankyrin repeats

GITs contain three ankyrin repeats immediately adjacent to the GAP

domain. In the Arf GAP1 and β-PAP structures, the zinc finger and the
immediately downstream region (which is the ankyrin repeats in PAP) form
a single structural domain, with the downstream region serving as a
foundation for the zinc finger (Goldberg, 1999; Mandiyan et al., 1999). This
suggests a structural role in GIT proteins as well. To date, no precise
function has been described for these motifs in GITs, although they may also
function as protein - protein interaction sites. A predicted coiled-coil motif
partially overlapping this region has been suggested to be a secondary
paxillin binding site, particularly in the GIT2-short splice variant (Turner et
al., 1999; Mazaki et al. 2001), but this has not been verified through
mutagenesis.

4.3 Spa2 homology domain

In the yeast Spa2 and Sph1 proteins, two short, near repeat units that also are
predicted to form a coiled-coil appear to mediate binding to the yeast MEK
proteins Mkk1, Mkk2 and Ste7 (Sheu et al., 1998; Roemer et al., 1998).
GIT1 and GIT2 contain Spa2 homology domains (SHD), consisting of two
30 amino acid near-repeat units separated by 30 residues. This region
appears to be the site by which GITs bind tightly to α- and β-PIX (Zhao et
al., 2000b), guanine nucleotide exchange factors (GEFs) for Rac1/Cdc42 as
well as major binding partners for the Rac1/Cdc42 p21-activated protein
kinases (PAKs) (Manser et al., 1998).
GIT1 fragments containing the SHD bind to β-PIX in protein overlay
assays (Zhao et al., 2000b). The GIT binding site was identified as the C-
terminal region of β-PIX using a variant of β-PIX (p50-Cool1) lacking the
C-terminal 200 residues, which failed to bind (Bagrodia et al., 1999), and in
two-hybrid assays (Premont et al., 2000). The GIT binding site was later
mapped to a 60 residue sequence within the C-terminus of β-PIX by direct
binding in overlay assays (Zhao et al., 2000b). Recently, a valine to alanine
point mutation within this region of β-PIX was reported to decrease GIT1
binding dramatically (Feng et al., 2002). In addition to PIX binding, the
SHD of GIT1 has been reported to interact directly with focal adhesion
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kinase (FAK) (Zhao et al., 2000b). The direct demonstration that these
SHDs mediate specific protein-protein interactions is currently lacking and
will make an important contribution to our understanding of signaling by
GIT1 and other SHD-containing proteins.

4.4 Coiled-coil dimerization domain

A predicted coiled-coil region is found following the Spa2 homology

domain in GIT1, and is conserved in GIT2. Recent experiments have shown
that GITs dimerize (R. T. Premont, unpublished observation), although an
earlier report suggested that they do not (Zhao et al., 2000b). Flag-tagged
GIT1 is able to efficiently co-immunoprecipitate 6xHis-tagged GIT1 or
GIT2, and GIT1 interacts directly with GIT1 or GIT2 in yeast two-hybrid
assays. The smallest GIT1 fragments able to co-immunoprecipitate 6xHis-
tagged GIT1 or endogenous GIT2 contained this coiled-coil domain. GIT1
fragments containing the coiled-coil region also interacted with endogenous
PIX proteins, independently of the Spa2 homology region, suggesting that
GIT proteins contain two distinct PIX binding sites (R. T. Premont,
unpublished observations).
β-PIX has been reported to dimerize through the interaction of a coiled-
coil region (Kim et al., 2001). Indeed, α-PIX and β-PIX can form homo-
and heterodimers (R. T. Premont, unpublished observation). GITs also
homo- and heterodimerize, and bind tightly to PIX proteins through two
distinct sites, the SHD and coiled-coil region (R. T. Premont, unpublished
observation). Therefore, there is the potential for GIT-PIX complex
oligomerization rather than simple heterodimerization. Knowing that all
binding sites for GIT1 are contained in the final 186 amino acids of β-PIX,
we prepared a β-PIX C-terminal (CT) fragment as a GIT–PIX interaction
inhibitor. Although Flag-tagged β-PIX CT co-immunoprecipitated GIT1 as
expected, HA-tagged β-PIX CT failed to inhibit the interaction of Flag-
tagged GIT1 with β-PIX, but simply added into the immunoprecipitated GIT
– PIX complex (R. T. Premont, unpublished observation). Multiple recent
studies have suggested models for regulated GIT – PIX interaction where
these two proteins are assumed to dissociate (Turner et al., 1999; Zhao et al.,
2000b; Di Cesare et al., 1999; Nishiya et al., 2002). Our model for GIT –
PIX interaction, in contrast, suggests that GIT and PIX proteins are tightly
associated oligomers, and form the non-dissociable core of a complex of
signaling proteins. Indeed, separation of cellular proteins on a gel filtration
column reveals that endogenous GIT1 and β-PIX proteins co-migrate at an
extremely high molecular size, far larger than even a GIT/PIX
heterotetramer. We hypothesize that GIT and PIX proteins are always
associated in this large complex, which is critical to both GIT and PIX
 GITS: ARF GAPS AND SIGNALING SCAFFOLDS 169
11

functions, and suggest that other multidomain Arf GAPs might also behave
similarly with their own specific partners.

4.5 Paxillin binding site

Another prominent feature of GIT proteins is binding to paxillin. Paxillin

binding in vitro and in vivo was initially discovered with chicken PKL/GIT2
(Turner et al., 1999). Comparisons to the paxillin binding sites of FAK and
vinculin revealed two putative paxillin binding sites in GIT2. One putative
site (PBS1) was predicted between the GAP domain and the first ankyrin
repeat, while the second site (PBS2) was predicted in the C-terminal half of
GIT2, within another coiled-coil region. Crude mapping suggested that the
C-terminal half of PKL contained the binding site, and deletion of each
putative site identified the C-terminal PBS2 as the major paxillin-binding
domain (West et al., 2001). The deleted C-terminal site included 35 amino
acids and represents the smallest paxillin-binding site reported in GIT
proteins so far. Surprisingly, human GIT2-short, which lacks this major
paxillin-binding region, was also found to bind paxillin (Mazaki et al.,
2001). Deletion mutants of GIT2-short identified a region around the C-
terminal end of the GAP domain and the first ankyrin repeat, which
correlates with the PBS1 site described above (Mazaki et al., 2001). In rat
GIT1, the paxillin-binding site was mapped to the 120 C-terminal residues,
and deletion of this region abolished paxillin binding to GIT1 (Zhao et al.,
2000b). However, the paxillin binding site in GIT1 has not been further
refined. A direct comparison of mammalian GIT1 and GIT2 suggested that
GIT1 binds more tightly to paxillin than does GIT2 (Premont et al., 2000).
It has been reported that GIT1 binding to paxillin can be enhanced by co-
expression of β-PIX, leading to the suggestion that GIT-PIX interaction
modulates the interaction of GIT proteins with paxillin (and thus with focal
adhesions) (Zhao et al., 2000b). However, in our hands, co-expression of
either PIX protein with GIT1 or GIT2 has no apparent effect on the amount
of co-immunoprecipitated paxillin, and we believe that GIT and PIX
proteins are constitutively associated (R. T. Premont, unpublished). The
GIT binding site on paxillin is the LD4 motif (LDXLLXXL), which is also a
binding site for FAK, vinculin and clathrin heavy chain (Tumbarello et al.,
2002). Deletion of the paxillin LD4 motif prevented the paxillin - GIT
interaction and retarded cell migration (Turner et al., 1999). GIT1 (Nishiya
et al., 2002) and GIT2 (Turner et al., 1999) also bind to the paxillin family
member Hic-5 through its cognate LD3 motif. The regulation of GIT
binding to paxillin, and localization to focal adhesions, is an active area of
interest, because of the apparent role of this interaction in cell attachment to
matrix and cell motility (see below).
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4.6 Phosphorylation

GIT1 phosphorylation was first observed when tyrosine-phosphorylated Cat-

1 was pulled down with PAK3 and activated Cdc42 from fibroblast lysates
(Bagrodia et al., 1999). Tyrosine phosphorylation of GIT1 is regulated in a
cell cycle-dependent manner. GIT1 is highly phosphorylated in a random
cycling population of cells, but is not phosphorylated in cells blocked in
mitosis. When cells are released into G1 phase, GIT1 rapidly becomes
phosphorylated (Bagrodia et al., 1999). Cell adhesion through integrins also
regulates GIT1 phosphorylation. Detachment of cells from the substratum
abolishes GIT1 phosphorylation, while plating cells on fibronectin induces
GIT1 phosphorylation (Bagrodia et al., 1999). FAK and Src can each
enhance GIT1 phosphorylation in cells, and have been suggested as the
primary GIT1 kinases (Bagrodia et al., 1999; Zhao et al., 2000b; Kawachi et
al., 2001). In contrast, little is known about GIT2 phosphorylation.
As described above, GIT1 is a substrate of PTPζ (Kawachi et al., 2001).
PTPζ activity is regulated by its ligand, pleiotrophin. Binding of
pleiotrophin to PTPζ inactivates the phosphatase and increases the tyrosine
phosphorylation of its substrates, i.e., β-catenin (Meng et al., 2000).
Neuroblastoma cells expressing GIT1 were treated with pleiotrophin,
leading to increased GIT1 tyrosine phosphorylation and the conclusion that
PTPζ acts on GIT1 in vivo. GIT1 co-localized with PTPζ in cells, e.g., both
GIT1 and PTPζ are expressed in processes of hippocampal and cortical
pyramidal neurons (Kawachi et al., 2001).

5. GIT PROTEIN CELLULAR LOCALIZATION

Subcellular fractionation of cultured cells expressing endogenous GIT2 or

recombinant GIT1 or GIT2 suggests that the GIT proteins are primarily
soluble, with only a small fraction of the protein associated with the
cytoskeleton or cellular membranes (R. T. Premont, unpublished
observations). Endogenous CHO-K1 cell ‘PKL’ (likely GIT1 based on the
use of a GIT1-specific antiserum in these cells by Manabe et al. (2002)) and
3Y1 cell GIT2-short have also been reported to be primarily soluble (Mazaki
et al., 2001; Brown et al., 2002). Expression of green fluorescent protein-
tagged GIT proteins in various cell types leads to a non-specific cytosolic
expression pattern in most of the cells. However, in a subset of cells, GIT
proteins display striking patterns of localization (Figure 3). Thus, under
circumstances that are generally only poorly understood, GITs can be found
associated with distinct cellular structures. This has led to suggestions that
GITs are highly regulated and involved in transiently localizing associated
 GITS: ARF GAPS AND SIGNALING SCAFFOLDS 171
13

proteins to certain cellular structures, and in transport between distinct

cellular compartments (Turner et al., 1999; Di Cesare et al., 1999; Manabe et
al., 2002). These regulated localizations are discussed below.

Figure 3. Cellular localization of GIT1/EGFP in an unusually flattened, migrating HeLa cell.

Three distinct patterns are evident in this cell. GIT is found at the membrane periphery in
membrane ruffles at the leading edge of the cell, in small finger-like focal adhesions just
behind the spreading leading edge, and in a diffuse cytoplasmic distribution in the thicker part
of the cell near the nucleus.

5.1 Focal adhesions

Focal adhesions are integrin-dependent sites linking the extracellular matrix

to the actin-rich cytoskeleton. Their formation is influenced particularly by
the RhoA, Cdc42 and Rac1 GTPases. Integrins form the transmembrane
link to several focal complex proteins, thereby directly or indirectly
recruiting vinculin, talin, α-actinin, focal adhesion kinase and paxillin.
GITs co-localize with paxillin in focal adhesions at the ends of actin
stress fibers (Turner et al., 1999; Zhao et al., 2000b; Manabe et al., 2002)
and provide the link allowing PIX and PAK proteins to transiently associate
with paxillin-containing focal adhesions. PKL/GIT2 interacts directly with
the LD4 motif of paxillin (Turner et al., 1999). In cells expressing a paxillin
mutant lacking the LD4 motif, GIT2 could no longer localize to focal
adhesions (Brown et al., 2002). The GIT2-short isoform, which lacks the C-
terminal paxillin binding site, does not localize to focal adhesions (Mazaki et
al., 2001). GIT1 also binds paxillin and co-localizes with paxillin in focal
adhesions (Premont et al., 2000; Zhao et al., 2000b; Manabe et al., 2002).
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Deletion of the C-terminal 120 amino acids (including the paxillin binding
site) of GIT1 caused the loss of paxillin binding and localization to focal
adhesions, while C-terminal fragments containing the paxillin-binding site
did localize to focal adhesions (Zhao et al., 2000b; Manabe et al., 2002;
Brown et al., 2002).
The interaction between paxillin and GIT1 is weak compared to the GIT-
PIX interaction, as overexpressed GIT1 can only pull-down a small fraction
of endogenous paxillin (Premont et al., 2000; Zhao et al., 2000b). Paxillin
binds GIT2 weaker than GIT1 (Premont et al., 2000), and the GIT2-short
variant even weaker (Mazaki et al., 2001). The weakness of these
interactions likely results from assays being performed on a mixed
population of cells, in which most cells were not spreading or migrating and
thus did not have GIT/PIX/PAK complexes stably bound to focal adhesions.
Co-expression of GIT1 and β-PIX was reported to enhance paxillin binding
to GIT1 (Zhao et al., 2000b). However, studies in my lab cannot confirm
this enhancing effect of PIX on the GIT-paxillin interaction in the same cell
system (R. T. Premont, unpublished observation). This putative interaction-
enhancing effect of PIX on the GIT-paxillin interaction has not been shown
to lead to enhanced localization to focal adhesions in cells. Thus, the effect
of PIX on GIT localization remains unclear.
Enhanced GIT1 localization to focal adhesions has been shown in three
cases. In the first case, expression of the PAK auto-inhibitory domain
peptide, PAK83-149, led to increased localization of PAK to focal adhesions
(Zhao et al., 2000a). The autoinhibitory domain eliminates PAK
autophosphorylation and prevents full activation (Zhao et al., 1998). The
autoinhibitory domain also enhanced the localization of GIT1 to focal
adhesions (Zhao et al., 2000b). The same effect of the PAK auto-inhibitory
domain was observed with GIT2 in CHO cells (Brown et al., 2002). In
contrast, a constitutively active form of PAK prevented GIT2 localization to
focal adhesions (Brown et al., 2002). These experiments provide evidence
that PAK kinase activity is an important regulator of the localization of GIT
proteins to focal adhesions.
In the other two cases, GIT localization to focal adhesions was stimulated
by constitutively active forms of the GTPases Rac1 (Manabe et al., 2002;
Brown et al., 2002) and Cdc42 (Brown et al., 2002). Constitutively active
Rac1 enhanced GIT1 and GIT2 localization to focal adhesions, while
dominant negative Rac1 had no effect on GIT localization (Manabe et al.,
2002; Brown et al., 2002). In addition, constitutively active Cdc42, but not
RhoA, enhanced GIT2 localization (Brown et al., 2002).
Overall, GIT proteins (in complex with PIX and presumably PAK) are
recruited to paxillin in focal adhesions both as the adhesions are forming and
as they are dissociating. Little GIT (or PIX or PAK) appears associated with
 GITS: ARF GAPS AND SIGNALING SCAFFOLDS 173
15

mature, stable adhesions. Cell spreading (after mitosis or replating) or cell

migration, conditions that activate Rac1 and Cdc42, stimulate GIT/PIX/PAK
localization to focal adhesions. These conditions are also associated with
phosphorylation of GIT1 on tyrosine residues, although the relationship
between GIT phosphorylation, GIT binding to paxillin, and GIT localization
to focal adhesions remains unclear. In contrast, activity of PAK appears
associated with disassembly of focal adhesions and loss of GIT localization.

5.2 Membranes and vesicles

GITs have been found associated with distinct plasma membrane domains.
In chicken embryonic fibroblasts, overexpressed GIT1/p95-APP1 localized
to the plasma membrane at sites of ruffling, along with actin, β1-integrin and
Arf6 (Di Cesare et al., 1999). GIT1 also localized to membrane ruffles in
neuroblastoma cells (Kawachi et al., 2001). Treatments stimulating ruffle
formation localized a C-terminal fragment of GIT1 to the plasma membrane,
where it co-localized with actin (Matafora et al., 2001). In COS7 cells,
GIT1 co-localized with paxillin to the plasma membrane (Zhao et al.,
2000b). GIT1 and a fragment consisting of the paxillin-binding site
localized to membrane ruffles at the leading edge of CHO cells (Manabe et
al., 2002). GIT2-short was found with actin and paxillin at the plasma
membrane (Mazaki et al., 2001).
GIT1 was also found in mobile, punctate structures in the cytosol
(Manabe et al., 2002). Localization to these structures required the presence
of the Spa2 homology domain of GIT1. Using specific markers of
intracellular compartments, these structures were shown to be of neither
endosomal nor Golgi nature. In addition to GIT1, these structures also
contained PIX, PAK and paxillin. In migrating cells, the structures moved
from the cytoplasm towards a pre-existing focal adhesion at the leading edge
of the cells. The reverse movement was found at the retracting side of the
cells, where the GIT1-containing structures moved away from adhesions
towards the cell center (Manabe et al., 2002). The authors conclude that this
represents a transport vesicle carrying GIT/PIX/PAK complexes to and from
sites of focal adhesion assembly and disassembly.
Overexpressed GIT fragments have been found associated with large
cytoplasmic vacuoles that are not seen in untransfected cells. Both N-
terminal and C-terminal fragments of GIT1 containing the SHD can be
found in such structures (Di Cesare et al., 1999). The large vacuoles were
later identified as large Rab11-positive vesicles (Matafora et al., 2001).
Rab11 is a marker for pericentriolar recycling endosomes. Deletion or
inactivation of the GAP domain also induced accumulation of GIT1 to
Rab11-positive structures (Matafora et al., 2001). These findings led to a
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16 SCHMALZIGAUG AND PREMONT

model for GIT1 function in membrane recycling from the endocytic

compartment through recycling endosomes (Matafora et al., 2001). Much
like GIT1, C-terminal fragments of GIT2 lacking GAP activity localized to
large vesicles (Paris et al., 2002). In contrast to co-localization of GIT1 with
Rab11 in the large vesicles, GAP-deficient fragments of GIT2 co-localized
with EEA1, a marker for the early endosomal compartment (Paris et al.,
2002). However, it remains unclear whether endogenous GIT proteins
actually traverse or function in these intracellular compartments, and
whether the apparently distinct localizations of GIT1 and GIT2 reflect
distinct roles in these two compartments.

6. GIT FUNCTIONS

6.1 Receptor endocytosis and GRKs

Agonist activation of G protein-coupled receptors (GPCRs) leads first to the

activation of coupled heterotrimeric G proteins, which activate a multitude
of intracellular messenger-mediated signaling pathways (Lefkowitz, 2000;
Pierce et al., 2002). The second result of agonist activation of receptor is the
activation and membrane recruitment of G protein-coupled receptor kinases
(GRKs), which phosphorylate the activated receptors. Phosphorylated
receptors then bind to β-arrestins, leading to uncoupling of the receptor from
G protein signaling. This results in desensitization of the receptor to further
stimulation. GRK phosphorylation of activated receptors and subsequent
arrestin binding also promote internalization of the inactivated receptor
proteins from the cell surface, facilitating their resensitization by
dephosphorylation in an intracellular acidic vesicle compartment, prior to
return to the cell surface (see Chapter 15 for additional details; Lefkowitz,
1998; Pitcher et al., 1998).
As GIT1 was originally isolated as a GRK-interacting protein, its role in
GRK-mediated GPCR regulation was studied first. GIT1 interacts with the
four widely expressed GRKs: GRK2, GRK3, GRK5 and GRK6. GIT1 is not
phosphorylated by GRK2 and the kinase activity of GRK2 is not regulated
by GIT1 binding in vitro. However, agonist-dependent phosphorylation of
the β2-adrenergic receptor (β2AR) was increased in cells overexpressing
GIT1. GIT1 also affected β2AR signaling, as detected by reduced receptor-
stimulated cAMP production, and inhibited the agonist-stimulated
internalization of the β2AR in living cells (Premont et al., 1998). GIT2
overexpression also has the ability to reduce β2AR internalization (Premont
et al., 2000). As GIT1 was unable to increase β2AR phosphorylation by
GRK2 in vitro, the increased phosphorylation in cells is likely due to the
 GITS: ARF GAPS AND SIGNALING SCAFFOLDS 175
17

inhibition of receptor internalization and subsequent dephosphorylation.

Similarly, the inhibitory effect on adenylyl cyclase stimulation might arise
from the desensitized receptors remaining at the plasma membrane and
failing to be resensitized by dephosphorylation (Premont et al., 1998).
Although GIT1 and GRK2 were found to interact in a yeast two-hybrid
screen, GIT1 binding to the β2AR by GRK2 has not been confirmed in vivo.
With the two proteins not affecting each other biochemically, localization is
still the best potential function of the interaction.
GIT1 was tested for its specificity to regulate internalization of various
GPCRs (Claing et al., 2000). GIT1 expression does inhibit the sequestration
of many receptors, but not all. The specificity of the GIT1 effect does not
correlate with the G protein to which a receptor couples, since GIT1
inhibited internalization of some receptors coupled to Gs, Gi or Gq. GPCRs
are sequestered from the cell surface by several distinct pathways. The
predominant pathway is the clathrin-coated pit pathway. Activated receptors
are phosphorylated by GRKs and bind to β-arrestins. In addition to
uncoupling receptors from G proteins, β-arrestins also serve as adaptors to
link receptors to be internalized with clathrin-coated pits, through β-arrestin
interaction with clathrin itself and with the AP-2 adaptor protein complex
(Lefkowitz, 1998, 2000). This pathway regulates β2AR endocytosis, among
others, and is sensitive to mutants of β-arrestin, clathrin and dynamin II, as
well as several chemical inhibitors of endocytosis. Receptors displaying
GIT1-dependent regulation of internalization also turned out to be sensitive
to mutations in β-arrestin and dynamin. Furthermore, a drug known to
stabilize clathrin cage assembly and hence inhibit internalization affected
only the class of receptors sensitive to GIT1. Thus, GIT1 affects only the
class of G protein-coupled receptors that use clathrin-coated pits as their
route to endocytic internalization (Claing et al., 2000). The function of GIT1
on receptor internalization is not restricted to GPCRs, as EGFR
sequestration is also regulated by GIT1 (Claing et al., 2000). In contrast to
GPCRs and the EGFR, which require ligand binding for sequestration, the
transferrin receptor internalizes continuously and independently of ligand
binding (Claing et al., 2000). Because GIT1 overexpression did not affect
the uptake of the transferrin receptor, this suggested that the GIT1-promoted
defect in receptor internalization is specific for recruiting agonist activated
receptors to clathrin-coated pits, rather than inhibiting pit formation or
endocytosis per se.
The functional effects of GIT1 on β2AR (and other receptor)
sequestration and function appear to be a result of its ability to stimulate the
GTPase activity of Arfs or interact with Arfs, as deletion of the Arf GAP
domain ablated the inhibitory effect of GIT1 on β2AR-stimulated cAMP
synthesis and receptor sequestration (Premont et al., 1998). Several other
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Arf GAPs have been tested and do not alter β2AR sequestration (Claing et
al., 2001), suggesting a unique role for GIT proteins (perhaps through GRK
interaction) in regulating receptor trafficking. Arfs function primarily as
GTP-dependent regulators of vesicular traffic through vesicle coat
recruitment. Arfs are involved in regulating plasma membrane endocytosis,
from experiments in which transferrin receptor traffic is inhibited by Arf6
mutants unable to cycle between the GTP and GDP-bound states (D’Souza-
Schorey et al., 1995). The results with GITs suggested that Arfs, and most
likely Arf6, may be involved in regulating clathrin-coated pit-mediated
internalization of many GPCRs. Direct testing of these Arf6 mutants
revealed that they also inhibited the agonist-dependent sequestration of the
β2AR (Claing et al., 2001). Further, overexpression of the Arf GEF, ARNO,
which activates Arf6 in the cell, led to increased sequestration of the β2AR
(Claing et al., 2001). Thus, it appears that GITs and Arf6 are key regulators
of the recruitment of activated receptors (cargo) to clathrin-coated pits on the
cell surface for their subsequent internalization (see Chapter 15).

6.2 Cell motility and focal adhesions

Cell migration requires the continuous formation and disassembly of cellular

adhesion sites at opposing poles of the cell. The basic migration cycle
includes extension of a protrusion, formation of stable attachments near the
leading edge of the protrusion, translocation of the cell body forward and
release of adhesions and retraction at the cell rear. Focal adhesions are
relatively stable structures and tend to inhibit cell migration (Webb et al.,
2002). In addition to their structural role in linking the extracellular matrix
with the actin cytoskeleton, focal adhesions also serve as centers for cellular
signaling. Many important signaling molecules, particularly non-receptor
tyrosine kinases like Src, FAK and Pyk2, are highly localized to focal
adhesions, and participate in both “outside-in” and “inside-out” signaling
through integrins. Through paxillin, GITs link a large complex of proteins
(PIX/PAK/POPX/Rac1/Cdc42) to focal adhesions.
GITs have been reported to affect cell migration. Overexpressed GIT1
promoted cell migration towards a fibronectin and collagen gradient (Zhao et
al., 2000b) and increased both the number of migrating cells and their
migration speed (Manabe et al., 2002). In contrast, the GIT-associated
proteins PIX and PAK were less effective or had no effect on cell migration,
respectively. Myristoylated fragments of GIT1 that lack the GAP and Spa2
homology domains stimulated cell migration to a similar degree as full
GIT1, while a GIT1 mutant lacking the paxillin binding site did not
stimulate migration (Zhao et al., 2000b). Thus, the paxillin binding but not
the Arf GAP activity of GITs appears to drive cell migration.
 GITS: ARF GAPS AND SIGNALING SCAFFOLDS 177
19

Overexpressed GIT1 can also stimulate paxillin dissociation from focal

adhesions but not adhesion disassembly, since cells with decreased levels of
localized paxillin still contain normally localized vinculin, another marker of
focal adhesions (Zhao et al., 2000b). The ability of GIT1 to stimulate cell
migration appears to correlate with its ability to promote the loss of paxillin
from adhesions. Because paxillin dissociation is thought to be a marker for
focal adhesion remodeling, GIT1 has been suggested to promote this process
(Zhao et al., 2000b).
GIT2-short was also able to increase the number of cells migrating and
their speed of migration, even though it lacks the C-terminal, major paxillin
binding site (Mazaki et al., 2001). In contrast to GIT1, expression of GIT2-
short caused the loss of paxillin only from central (perinuclear) but not from
peripheral adhesions (Mazaki et al., 2001). In this case, vinculin staining
was limited to peripheral adhesions as well. GIT2-short expression
abolished the perinuclear accumulation of paxillin and reduced the number
of actin stress fibers (Mazaki et al., 2001). The effects of GIT2-short on
paxillin, vinculin and actin could be reversed by mutation to ablate its Arf
GAP activity. Furthermore, constitutively active Arf1 partially restored the
localization of paxillin to focal adhesions and actin stress fibers (Mazaki et
al., 2001).
The precise mechanism by which GITs affect cell migration remains
unknown, but presumably involves its role in localizing PIX and PAK to
paxillin-containing focal adhesions during new adhesion formation at the
leading edge of the cell and old focal adhesion disassembly at the rear of the
cell, as well as coordinating vesicle trafficking required for leading edge
protrusion.

6.3 Cell spreading and protrusions

Changes in cell shape occur during cell spreading and the formation of
protrusions, and require changes in the cytoskeleton, the formation or
disassembly of focal adhesions and membrane expansion driven by
intracellular vesicle fusion with the leading edge of the cell. Cell spreading
appeared unaffected by GIT1 or a C-terminal fragment consisting of the
coiled-coil domain and the paxillin-binding site, but these same proteins
were found to promote the formation of cell protrusions (Di Cesare et al.,
1999; Manabe et al., 2002). Similarly, GIT2 lacking the PBS2 paxillin
binding region increased cell protrusions (West et al., 2001). However, the
paxillin- and Hic-5-binding fragment GIT1-CT was able to block Hic-5-
induced inhibition of cell spreading (Nishiya et al., 2002). The formation of
protrusions could be prevented by blockade of Rac cycling by dominant
active or negative mutants of Rac (Di Cesare et al., 2000). Co-expression of
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20 SCHMALZIGAUG AND PREMONT

full-length GIT1 with Arf6 also inhibited cell spreading and caused cells to
retract (Di Cesare et al., 1999). Myristoylated GIT1, and particularly in
combination with β-PIX, induced a severe retraction leading to grossly
elongated cells resembling neurons (Zhao et al., 2000b). Overall, these
studies suggest that GITs are important in regulating a Rac- and PAK-
dependent pathway that requires paxillin-dependent localization, to initiate
cell protrusions. Although cell spreading appears to use the same
mechanisms, it is much less grossly affected by GIT proteins.

6.4 Signaling

PIX and PAK interact with each other but bind paxillin indirectly (Manser et
al., 1998). PKL/GIT2 was then discovered to bind directly to paxillin,
allowing PAK and PIX to indirectly link to paxillin through GITs (Turner et
al., 1999). This GIT/PIX/PAK complex has been found in several situations
and cell lines, and can contain GIT1 or GIT2, α-PIX or β-PIX, and PAK1,
PAK2 or PAK3 (Bagrodia et al., 1999; Premont et al., 2000, Di Cesare et al.,
1999; Ku et al., 2001; Turner et al., 1999). This GIT/PIX/PAK complex can
bind to active Rac1•GTP but not Rac1•GDP (Di Cesare et al., 1999, Paris et
al., 2002). One report, however, suggests that PAK binds directly to paxillin
(Hashimoto et al., 2001), although a mutant of PAK that cannot interact with
PIX also fails to co-immunoprecipitate paxillin (R. T. Premont, unpublished
observation).
The GIT/PIX/PAK complex provides a versatile signaling module to
cells. This complex contains a GAP for Arfs, a GEF for the Rac1/Cdc42
GTPases, and a serine/threonine kinase regulated by Rac1/Cdc42. Activated
Rac1/Cdc42 can also associate with the complex, and the complex can
activate these small GTP-binding proteins. This suggests that recruitment of
this complex to sites within the cell will lead to a local shift from Arf- to
Rac1/Cdc42-dependent pathways, as Arf6 would be deactivated while
Rac1/Cdc42 would be activated. As mentioned earlier, our recent
experiments indicate that GIT and PIX proteins form a tightly associated
oligomeric complex containing multiple GIT and PIX monomers (R. T.
Premont, unpublished observation). This oligomer appears to be at least a
heterotetramer, and may well be far larger. One GIT monomer does not
bind to only one PIX monomer, and the tight association of this oligomeric
GIT–PIX complex suggests that these proteins likely do not dissociate in the
cell but form a stable multiprotein complex. In any case, one clear result of
this oligomeric model is that GIT–PIX complexes should function by
spatially augmenting the activity of the GIT (Arf GAP) and PIX
(Rac1/Cdc42 GEF) proteins through the colocalization of multiple enzyme
 GITS: ARF GAPS AND SIGNALING SCAFFOLDS 179
21

units, as well as function to facilitate the co-localization of multiple copies

of the various GIT and PIX binding partners.
Activated PAKs can bind the SH2-SH3 adaptor protein Nck, allowing
PAK association with other signaling and adaptor proteins (McCarty, 1998).
α-PIX binds the p85 regulatory subunit of phosphatidylinositol 3-kinase,
itself an SH2-SH3 adaptor protein, in response to PDGF activation, and
associates with the activated PDGF receptor itself (Yoshii et al., 1999).
Since PIX also binds to the POPX protein phosphatases, that
dephosphorylate and inactivate PAKs (Koh et al., 2002), these phosphatases
may also be found in a GIT-containing PIX/PAK/POPX complex, although
this has not yet been formally demonstrated. Other GIT and PIX binding
partners currently being identified will only add to the complexity. This
suggests that the confluence of these multiple signaling pathways, and
multiple copies of each molecule, play an important role in regulating
crosstalk among these ‘distinct’ pathways.

7. FUTURE PROSPECTS

GITs have been identified in several distinct cellular contexts. The major
challenge is in understanding how these apparently distinct functions are
integrated. The realization that GIT and PIX proteins are tightly associated
in oligomeric complexes raises many questions about the role of this
association in both what had been thought to be the distinct functions of the
GIT and PIX proteins, and in how this association regulates and integrates
the separate activities of these two GTPase regulators. Further experiments
are necessary to understand the many facets of this complex, including:
defining the structure of the complex, identifying the full set of associated
proteins, understanding the regulation of the localization of the GIT–PIX
complex and its associated proteins, and defining the crosstalk among the
many signaling pathways that intersect within or around this complex.
With regard to regulation of Arf proteins, it seems clear that GITs mainly
regulate Arf6 in the cell. This stems primarily from co-localization, rather
than any enzymatic specificity of the GAP domain for any particular Arf
subtype. The GAP activity is activated in vitro by PI(3,4,5)P3, which is an
intracellular messenger activated by many signaling pathways. The cellular
regulation of GITs by PI(3,4,5)P3 and its role in GIT function in vivo
remains to be shown. It seems clear that GIT and Arf6 are important in
regulating the formation of vesicles containing activated receptors from cell
surface clathrin-coated pits. Whether GITs have other roles in transport
processes is unknown, although recent video microscopy of GIT complexes
shuttling about in cells (Manabe et al., 2002) suggests that this is true.
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22 SCHMALZIGAUG AND PREMONT

A major open question is the role of Arf regulation in other activities of

the GITs. It appears that the GAP activity is required neither for cell
motility effects, nor for binding to paxillin at focal adhesions. On the other
hand, one study suggests that Arfs directly promote paxillin recruitment to
focal adhesions through an unknown mechanism (Norman et al., 1998).
Whether the Arf GAP activity of GITs is important for cellular localization
and signaling roles of GITs, or whether GITs play a primarily scaffolding
function, remains to be determined. It is also interesting to consider whether
GITs function in part as Arf effectors, by transiently localizing associated
proteins to active Arf6 at the plasma membrane or elsewhere.
In a broader view, it will be interesting to determine whether other
members of the Arf GAP family also share functional and structural
similarity with the GITs. In this regard, we have been able to demonstrate
that several other Arf GAP proteins also appear to dimerize, suggesting that
dimerization is an important aspect for the proper function of the
multidomain Arf GAPs. The tight association of GIT and PIX proteins also
hints that other multidomain GAPs might also have tightly-associated
protein partners (preliminary evidence suggests that PIX does not play such
a role for several other GAP proteins), and form the core of distinct
complexes of signaling and scaffolding proteins. Much further work will be
required to fully elucidate the answers to these questions.

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Chapter 9
PAXILLIN-ASSOCIATED ARF GAPS
Their Isoform Specificities and Roles in Coordination

HISATAKA SABE
Department of Molecular Biology, Osaka Bioscience Institute, Osaka, Japan

Abstract: Cell migration is a multifactorial process in which a number of distinct events

occur simultaneously. Paxillin is an integrin-assembly adaptor protein. Here,
properties of Arf GAPs bearing paxillin-binding capacity are described with
their isoform specificity. Roles in linking Arf function with other intracellular
events all need to be coordinately regulated during integrin-mediated cell
adhesion and migration.

1. INTRODUCTION

Integrin-mediated cell movement is a multifactorial process in which a

number of distinct intracellular events must occur simultaneously and
coordinately. These include signal transduction, cytoskeletal remodeling,
and membrane traffic and remodeling. These are required for proper
adhesion, protrusion, traction and retraction, physical force generation and
polarity formation. Cell migration is initiated by a variety of extracellular
cues, such as by growth factors, cytokines, chemoattractants and
extracellular matrix components. The intracellular biochemical events that
are evoked by these stimuli (such as ion fluxes, protein activation, protein
phosphorylation, and gene expression) have been extensively studied during
the last two decades, and each has been well documented. The existence of
cross talk between these events is also well known. However, several
fundamental questions still remain largely unsolved. Prominent among these
are the molecular mechanisms involved in the temporal and spatial
coordination of distinct processes as well as the orchestration of distinct
events occurring at different parts within a single cell, all of which result in a
unified, efficient, directional migration. What is the principal or primordial
185
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2 SABE

mechanism employed in such coordination and orchestration in integrin-

mediated migration?
Arf GTPases are important factors regulating intracellular vesicle traffic
and membrane remodeling. They are also important for actin-cytoskeletal
remodeling. Recently, several Arf GTPase activating proteins (GAPs) have
been found to bind paxillin, an integrin-assembly scaffolding/adaptor
protein. In this chapter, the roles of a subset of cellular Arf GAPs in
integrin-mediated cell adhesion and migration are discussed. We have
focused on aspects of the mechanisms by which Arf activities coordinate the
remodeling of membrane and actin-cytoskeleton in motile cells, together
with the isoform specificity of the Arf GAPs involved. We will describe
how paxillin-associated Arf GAPs differ structurally and functionally, and
discuss the model in which different Arfs work with different subsets of Arf
GAPs to effect changes in the actin cytoskeleton and membrane remodeling.
The specificity of Arf GAPs for different Arfs, assessed both biochemically
and in cell biological assays, will also be addressed. A hypothesis that
explains the dual regulation of substrate specificities toward the different
Arfs in vivo is proposed.

2. INTEGRINS, PAXILLIN AND CELL MIGRATION

Integrins are bi-directional, cell surface α/β heterodimeric signaling proteins.

Integrins require assembly of several different types of proteins at their
cytoplasmic tails to transmit their signals (Hynes, 2002). These integrin-
assembly proteins also play a role in transmitting intracellular information
into changes in the ability of integrins to bind extracellular ligands.
Integrins exhibit dynamic properties during cell adhesion and migration.
Newly synthesized integrins are transported to the plasma membrane via
intracellular membrane/vesicle traffic. In actively migrating cells, subsets of
integrins (including, e.g., fibronectin receptors) are recycled actively and can
be brought to the leading edges of the cell periphery, perhaps coupled with
their endocytosis at the trailing edge of the cell (Bretscher, 1989; Bretscher,
1992; Lawson and Maxfield, 1995). However, it appears that not all types of
integrins can enter recycling endosomes (Bretscher, 1992; Roberts et al.,
2001). In contrast, in cultured fibroblasts most of the β1 integrin molecules
adhering to the extracellular matrix (ECM) at the rear of the cell are left
behind as the cell body moves forward (Regan and Horwitz, 1992).
Most integrin-assembly proteins are recruited to integrins by the binding
of integrin to extracellular environments. A hierarchical and step-wise
regulation exists in the recruitment of integrin-assembly proteins (Miyamoto
et al., 1995a; Miyamoto et al., 1995b). How then do such proteins assemble
 PAXILLIN-ASSOCIATED ARF-GAPS 1873

at the cytoplasmic tails of integrins at the cell surface, while dynamically

entering cycling endosomes in motile cells?
Paxillin is an integrin-assembly adaptor protein (Turner, 2000). Paxillin
is not constitutively associated with the cytoplasmic tails of integrins, but is
recruited to these sites upon integrin engagement (Miyamoto et al., 1995b).
Paxillin was one of the first integrin assembly proteins to be successfully
tagged with EGFP (Mazaki et al., 1998). Viewed by video recording,
EGFP-paxillin is swiftly recruited to the leading edges of lamellipodial
structures in migrating NMuMG epithelial cells (Nakamura et al., 2000) and
in Chinese hamster ovary cells (Laukaitis et al., 2001). Macroaggregates of
paxillin-containing focal adhesions, connected to stress fibers, formed as a
cell body extended forward, apparently as a consequence of the
accumulation of paxillin molecules formerly localized laterally along
lamellipodia (Nakamura et al., 2000). Further, paxillin was not diffuse in the
cytoplasm but accumulated at the perinuclear regions, including the Golgi
apparatus and ER structures (Mazaki et al., 1998; Mazaki et al., 2001).
Perhaps closely related to this, Norman, et al. (1998) have shown that Arf1 is
involved in the recruitment of paxillin to focal adhesions. It was later shown
that all three classes of Arfs affect the subcellular localization and focal
accumulation of paxillin (Kondo et al., 2000; Mazaki et al., 2001).

3. PAXILLIN-ASSOCIATED ARF GAPS

3.1 Structures and Nomenclatures

Protein pull-down assays have demonstrated that a large number of different

proteins can associate with paxillin (Mazaki et al., 1997; Turner et al., 1999).
These proteins include those bearing the Arf GAP domain; e.g., p95PKL
(Turner et al., 1999), PAG3/KIAA0400 (Kondo et al., 2000),
PAG2/KIAA1249 (clone 43 and 81; Kondo et al., 2000), p95APP1 (Di
Cesare, 2000), Git2 and its alternatively spliced variants (Mazaki et al.,
2001). The current nomenclature of these proteins is confusing, primarily
due to their simultaneous but independent identification by different groups
as binding proteins for different proteins. The Arf GAPs discussed here, and
alternative names, are summarized in Table 1.
p95PKL was affinity-purified using GST-paxillin as a probe, and turned
out to be orthologous to human Git2. PAG1 and PAG1-short were also
identified by affinity-purification from HeLa lysates, and turned out to be the
same molecules as Git2 and Git2-short, respectively. The mouse ortholog of
PAG1/Git2, Cat2, was originally identified as a Cool/Pix binding protein
(Bagrodia and Cerione, 1999). PAG1-short/Git2-short is a C-terminal
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4 SABE

truncated isoform of PAG1/Git2. Git2 and p95PKL have two paxillin

binding sequences (PBS; Turner et al., 1999), only one of which binds
strongly to paxillin (Mazaki et al., 2001). Git2-short only contains the
weaker PBS, suggesting the possibility of a cellular role independent of
paxillin binding. p95APP1 was originally isolated from chicken embryonic
brain lysates as a binding partner of activated Rac1A and Rac1B (Di Cesare
et al., 2000), and is an ortholog of human Git1 (Premont et al., 1998), a close
relative of Git2.

Table 1. Names of paxillin-associated ArfGAPs

Original Names Names as Proposed Standard
PAGs Names
GIT1 (human), CAT-1 (mouse), p95APP1 (chick) ⎯ APAP1
GIT2 (human), CAT-2 (mouse), p95APP2 / p95PKL (chick) PAG1 APAP2
ASAP1 (mouse), DEF-1 (bovine) PAG2 AMAP1
Pap α (human) PAG3 AMAP2

Integrins are activated upon maturation of monocytes into adherent

macrophage-like cells, at which time paxillin expression is augmented
(Mazaki et al., 1997). PAG2 and PAG3 were identified from human
monocyte U937 cells, by far-western screening of a λgt11 cDNA library
with the N-terminal half of paxillin as a probe. PAG2 is the human ortholog
of mouse ASAP1 (Brown et al., 1998) and bovine DEF1 (King et al., 1999).
PAG3 is identical to Papα, which was first identified as a protein tyrosine
kinase, Pyk2, binding protein (Andreev et al., 1999). ASAP1 and PAPα
each contain a src homology 3 (SH3) domain at their C-terminus, to which
paxillin binds (Kondo et al., 2000).
More than a dozen Arf GAP domain-containing proteins exist in humans
(see Figure 1), though not all have been demonstrated to exhibit Arf GAP
activity. Based on structural similarity, they can be classified into several
distinct classes, with the paxillin-binding Arf GAPs falling into two groups,
Git and ASAP1/PAPα proteins (Figure 1). The Git group all have the Arf
GAP domain at the N-terminus, followed by ankyrin repeats. The
ASAP1/PAPα group bear a single PH domain for putative lipid binding,
followed by the Arf GAP domain, ankyrin repeats, the proline-rich domain
(PRD), and the SH3 domain at the C-terminus. Predicted proteins that
contain Arf GAP domains were found in the Kazusa database
(http://www.kazusa.or.jp/en/database.html); including KIAA0050/ACAP1,
KIAA0167/centaurin γ1, KIAA0580/ARAP2/centaurin δ1 and
KIAA0782/ARAP1/centaurin δ2. These were examined for paxillin
binding, but none exhibited such an activity (H. Uchida and H. Sabe,
unpublished observations). Thus, binding to paxillin is an activity that is
specific to only two groups of Arf GAPs.
 PAXILLIN-ASSOCIATED ARF-GAPS 1895

In this chapter, PAG1 is called APAP2, and PAG2 and PAG3 are called
AMAP1 and AMAP2, respectively, following the proposed unified naming
of ArfGAPs. Other ArfGAPs are also called accordingly.

Figure 1. Arf GAP proteins in human genome. The class I and class II paxillin-associated
Arf GAPs are indicated on the right, with the paxillin-binding regions underlined.

3.2 Isoform Specificities of Paxillin-associated Arf GAPs

3.2.1 Biochemical Arf GAP Assays

Arf GAP activity can be measured in vitro using GTP-loaded Arfs as

substrates. Both APAP1 and its short isoform exhibit comparable activities
towards Arf1, 2, 3, 5 and 6 in vitro (Vitale et al., 2000). Their activities
towards Arf1 were augmented several fold by the presence of
phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3), but not by PIP,
PI(4,5)P2, or diacylglycerol. On the other hand, both AMAP1 (Brown et al.,
1998) and AMAP2 (Andreev et al., 1999) exhibit PI(4,5)P2-dependent GAP
activity for Arf1 and Arf5, with much less activity for Arf6 in vitro. Neither
AMAP1 and AMAP2 exhibit much GAP activity in the absence of
PI(4,5)P2, and this effect is specific to PI(4,5)P2 as other lipids (i.e.,
phosphatidic acid or phosphatidylinositol) were almost ineffective in the
augmentation of their GAP activities. Phospholipids added in these assays
190
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may interact with the N-terminus of Arfs or with the PH domains of the Arf
GAPs, when present (see Chapter 3 for details).
Biochemical assays were also performed in vivo. Mammalian cells,
overexpressing epitope-tagged Arfs and Arf GAPs were labeled with
radioactive orthophosphate. Tagged Arfs were subsequently pulled-down
and bound radiolabeled nucleotides analysed by thin layer chromatography.
With this assay, AMAP1 again appeared to exhibit GAP activity for Arf1,
but not for Arf6 (Furman et al., 2002).

3.2.2 Biological Arf GAP Assays

Biochemical assays have verified the presence of Arf GAP activity in every
paxillin-associated Arf GAP. However, in vitro GAP assays often show
relatively broad specificity that can be contradictory to results obtained from
in vivo studies (e.g., see Settleman et al., 1992 for p190RhoGAP; Ridley et
al., 1993 for rhoGAP-C). Moreover, given the known abilities of other
proteins (e.g., COPI, see below) or lipids (see Chapter 3) to dramatically
alter Arf GAP activities and the potential loss of specificity inherent in the
use of purified proteins, it seems to be important to define Arf GAP
activities and specificities in a cellular context whenever possible. Of
course, the value of such data from intact cells must be weighed against the
dangers of using modified and overexpressed proteins. In this section I
describe several cell biological approaches to assay Arf GAP activities for
the paxillin binding Arf GAPs. The in vivo specificity of these Arf GAPs
was verified using mutants within the GAP domains (Cukierman et al.,
1995) as negative controls in each experiment.
A cell based approach to assaying the GAP activity for Arf1 involved
analyzing the subcellular localization of β-COP, a subunit of the COPI coat
that is involved in shuttling of vesicles between the Golgi and ER
compartments. Activated Arf1 specifically recruits COPI to the appropriate
intracellular membrane. Thus, β-COP can be an excellent marker for the
intracellular activity of Arf1; it is diffusely redistributed in the cytosol when
Arf1 is inactivated (Peters et al., 1995; Ooi et al., 1998). This redistribution
is specific to Arf1, and is not seen by inactivation of Arf5 or Arf6.
Overexpression of APAP2-short caused the redistribution of β-COP in
fibroblasts (Mazaki et al., 2001). This redistribution was suppressed by co-
expression of the GTP hydrolysis-deficient form of Arf1, Arf1-L67. On the
other hand, overexpression of AMAP2 did not exert such an effect on β-
COP (Andreev et al., 1999; Kondo et al., 2000).
A second cell biological assay involved analysis of the GAP activity for
Arf6. The class III Arf, Arf6, but not class I or II Arfs, is involved in Fcγ
receptor-mediated phagocytosis in macrophage cells (Zhang et al., 1998).
 PAXILLIN-ASSOCIATED ARF-GAPS 1917

Dominant Arf6 mutants, one defective in GTP hydrolysis (Arf6-L67) and

the other defective in GTP binding (Arf6-N27), each inhibit Fcγ receptor-
mediated phagocytosis, while mutants at homologous sites of the other Arfs
do not exert such inhibitory effects. Moreover, brief treatment of
macrophages with brefeldin A had no effect on Fcγ receptor-mediated
phagocytosis, despite the disruption in the organization and function of the
Golgi apparatus (Zhang et al., 1998). Brefeldin A inhibits the activity of
several guanine nucleotide exchange factors (GEFs) for Arfs and thereby
inhibits the function of Arfs, with the exception of Arf6 (Peters et al., 1995;
Radhakrishna et al., 1996; Cavenagh et al., 1996; Chardin and McCormick,
1999). Thus, this experiment also indicates that brefeldin A-sensitive Arf
GEFs and their target Arfs appear to be nonessential for phagocytosis. We
originally identified human AMAP1 and AMAP2 from mature monocytes of
macrophage-like cells (Kondo et al., 2000). Paxillin has also been shown to
accumulate at phagocytic cups, though its function in phagocytosis is still
unclear (Greenberg et al., 1994; Allen and Adrem, 1996). Both AMAP1 and
AMAP2 accumulate at phagocytic cups in P388D1 mouse macrophage cells,
while APAP2 and its short isoform do not (Uchida et al., 2001; H. Uchida
and H. Sabe, unpublished observations). Such accumulation was also seen
with EGFP-tagged AMAP2 that was exogenously expressed at moderate
levels (2-3 fold higher than endogenous AMAP2). Overexpression of
AMAP1 or AMAP2 inhibited phagocytic activity, while overexpression of
APAP2 or APAP2-short did not (Uchida et al., 2001). In these assays, the
levels of overexpressed Arf GAPs were more than twenty fold higher than
the endogenous levels, assessed optically with each plasmid-transfected cell.
The phenotypes induced by AMAP2 overexpression were essentially the
same as those seen with expression of the Arf6 mutants (Zhang et al., 1998).
Moreover, co-overexpression of Arf6, but not the other Arfs, with AMAP2
restored phagocytic activity (Uchida et al., 2001). These results are all
consistent with roles for AMAP1 and AMAP2 in Arf6-mediated Fcγ
receptor phagocytosis, and further suggest that these two Arf GAPs have
specificity for Arf6 in this context.
A third assay involved the addition of aluminum fluoride to COS-7 cells
that over express different Arf proteins. AlF (a mixture of 30 mM NaF and
50 µM AlCl3; (Radhakrishna et al., 1996)), is an activator of heterotrimeric
G proteins (Sternweis and Gilman, 1982; Al-Awar et al., 2000; Boshans et
al., 2000). AlF treatment of Arf cDNA transfected cells gave rise to distinct
cell phenotypes depending on the cDNAs transfected; it increased the
number and size of Arf1-associated punctuate structures in the Arf1-
transfected cells (Ooi et al., 1998) and induced membrane protrusion in the
Arf6-transfected cells (Radhakrishna et al., 1996). Overexpression of
APAP2 or APAP2-short (Mazaki et al., 2001) suppressed the Arf1-mediated
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phenotype in AlF treated cells, while it was ineffective in suppressing the

Arf6-mediated membrane protrusions. In contrast, overexpression of
AMAP2 suppressed the Arf6-induced membrane protrusions, but not the
Arf1-mediated phenotype (Kondo et al., 2000).
The three different in vivo assays described above revealed that the
APAP group of paxillin-associated Arf GAPs act as GAPs for Arf1, and
those of AMAP group act as GAPs for Arf6. However, it is also possible
that a single Arf GAP acts on different isoforms of Arf, as suggested by in
vitro biochemical assays. Therefore, although these assays indicate
specificity for Arfs exists in certain cellular contexts for each Arf GAP in
vivo, they do not exclude the possibility that these Arf GAPs also act on
other Arfs in different cellular contexts. For example, chicken APAP1
colocalizes with Arf6 at the cell periphery in cultured neuron cells, and was
hence proposed to act as a GAP for Arf6 in vivo (Di Cesare et al., 2000). In
Arf6-transfected COS-7 cells treated with AlF, a significant fraction of
APAP2-short was also recruited by and colocalized with Arf6 at the plasma
membrane, without significant suppression of Arf6 activity even when
overexpressed (Mazaki et al., 2001).

3.3 Selective and Stable Binding of the Arf GAP Domain

to Arf•GTP

The results described above from biochemical and cell biological assays are
not consistent with each other. One of the critical questions is why AMAP2
does not exhibit GAP activity for Arf6 when measured in vitro, although it
acts as an efficient GAP for Arf6 in vivo. This issue is discussed further
below.
Protein pull-down assays have demonstrated that the GEF domains of
several GEF proteins, such as intersectin, can selectively and stably bind to
their substrate GTPases in their GDP bound forms (Hart et al., 1994;
Hussain et al., 2001). In the case of the GAP proteins, on the other hand,
assessment of their substrates by a similar method has not been applicable,
because the binding sites for the GAPs and effectors on Ras family GTPases
have been shown to largely overlap (Goldberg, 1999), which may result in
large areas of their GAP binding domains being occluded by effectors, and
therefore unavailable for being pulled-down efficiently by exogenously
expressed GAP domains. Moreover, GTP bound to GTPases may quickly
be hydrolysed upon binding to their GAPs in vivo so that GAPs cannot bind
stably to the GTPases. However, it has been proposed that the binding sites
for the GAPs and coatomers or other effectors do not overlap in Arf1
(Goldberg, 1999). These observations may explain the success in the use of
pull-down assays with at least one Arf GAP and Arfs. AMAP2 was found to
 PAXILLIN-ASSOCIATED ARF-GAPS 1939

bind stably to wild type Arf6 in vivo, but not to other Arfs, in cells
overexpressing all of these proteins (S. Hashimoto and H. Sabe, unpublished
observation). The presence of divalent cations did not affect the binding.
Moreover, the Arf GAP domain of AMAP2 selectively bound Arf6-L67, but
not Arf6-N27 in vitro. Consistent with this, AMAP2 was efficiently
recruited to the plasma membrane by activated Arf6, but not by other Arfs,
and both proteins colocalize stably at the plasma membrane of these
transfected cells (S. Hashimoto and H. Sabe, unpublished observation).
Therefore, it is conceivable that this 'non-catalytic' and stable binding of
AMAP2 to Arf6 prevents AMAP2 from exhibiting efficient catalytic activity
against Arf6, when measured using the above mentioned biochemical
method.

3.4 Dual Regulation of the Arf GAP activities towards

Arfs in vivo-a Hypothesis

The fact that AMAP2 can bind stably to Arf6•GTP without hydrolyzing the
GTP appears to be consistent and analogous to a current model for the
interaction of Arf GAP1 with Arf1. Arf1 is the best studied of the Arf
isoforms, and may provide some insight into the mechanism of regulation of
the other Arf isoforms. Arf1 mediates transport between the ER and Golgi
by employing coat protein complexes, such as COPI (Roth, 1999). GAP
molecules for Arf1 are found as components of a priming complex induced
by Arf1-GTP in the COPI system (Schekman and Orci, 1996). The GTPase
activity of other small GTPases, such as Ras and Rho, is accelerated
dramatically upon binding to their cognate GAPs. With these protein
complexes, the ‘arginine finger’ model has been proposed, in which the
corresponding GAP supplies a critical arginine, which hydrogen bonds to the
γ-phosphate of GTP and stabilizes the transition state (Scheffzek et al.,
1998). In contrast to this model, Goldberg (1999) has proposed that
Arf1/ArfGAP1 constitutes only part of the usual protein-binding interface,
and that the key residue(s) required for maximal rates of GTP hydrolysis,
like the 'arginine finger' in the case of other GAPs, may not be provided by
ArfGAP1. Rather, further association with the coatomer and/or
accumulation of the coatomer with the Arf1/ArfGAP1 complex may be
required for the maximal catalytic activity of the Arf1/ArfGAP1 complex.
This model may explain why ArfGAP1 by itself does not function as an
efficient catalytic GAP upon binding to Arf1•GTP (Roth, 1999; Springer et
al., 1999). It should be noted, however, that there are several reports
apparently contradictory to this model, which suggest that the coatomer may
simply aid the productive association of Arf and ArfGAP (Mandiyan et al.,
1999; Szafer et al., 2000; Eugster, et al., 2000). Nevertheless, a recent
194
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extensive study of the 'interface array' of protein interactions of the small

GTPases also indicates that the protein-protein contacts in the Arf-ArfGAP
complex are significantly different from those in other GAP complex, and
supports the finding that the Arf/ArfGAP structure may represent a complex
with only low catalytic activity (Corbett and Alber, 2001). Therefore, it is
also possible that coatomer proteins may act as allosteric activators of the
catalytic activity of the Arf1/ArfGAP1 complex. Further scrutiny is needed
to clarify the picture of ArfGAP action, and roles of the coatomer or other
proteins regarding their possible involvement in the temporal-spatial
determination of the onset of the GTP-hydrolysis activity of Arf1/ArfGAP1
complexes. On the other hand, structural analysis of AMAP1 (Papβ)
suggests that the critical residues for catalysis are directly provided by the
Arf GAP (Mandiyan et al., 1999), consistent with in vitro results showing
that AMAP1 exhibits higher basal GAP activity for Arf1 than does Arf
GAP1.
Considering that AMAP2 exhibits efficient GAP activity for Arf1 and
Arf5 in the presence of PI(4,5)P2 in vitro (Andreev et al., 1999), I propose
that AMAP2 may function as a GAP for different Arfs in at least two
different ways: one mechanism requires other protein(s) or modification(s)
for AMAP2 to be active for Arf6, in the other mechanism AMAP2 is
'constitutively' active as a GAP for Arf1 and Arf5, as long as phospholipids
such as PI(4,5)P2 exist at the required levels. This putative dual regulation
of the GAP activities of AMAP2 may be important for the intracellular
regulation of Arfs. The former mechanism may be necessary for priming
complex formation of Arf6 on the donor membrane, similar to that proposed
for the interaction of Arf GAP1 with Arf1 on Golgi or ER membranes (Roth,
1999; Springer et al., 1999). Subsequent association with additional
proteins, such as with coat or cargo proteins, as proposed in the case of Arf
GAP1 (Goldberg, 1999; Springer et al., 1999), or some modification of
AMAP2, may be necessary for the full activation of the GAP activity of the
Arf6/AMAP2 complex. On the other hand, unlike other members of Ras-
superfamily GTPases, Arfs do not have intrinsic GTPase activity (Kahn and
Gilman, 1986). Hence, it is possible that at least some Arf GAPs retain a
relatively “non-specific” GAP activity that may be used to silence any Arf
that was inappropriately activated while the same Arf GAP may retain a
highly specific Arf GAP function that is context and possibly co-factor
dependent. This putative “non-specific GAP” function of AMAP2 may be
important, for example, after the mitosis of cells where proper intracellular
compartmentalization of membrane components could be lost, at least
transiently. Such function of AMAP2 may also contribute excluding
inappropriate activities of the class I and class II Arfs from areas where Arf6
has to function.
 PAXILLIN-ASSOCIATED ARF-GAPS 195
11

A previous structural study was performed of the Arf1•GDP/Arf GAP

complex (Goldberg, 1999). However, since we have shown that the Arf
GAP domain of AMAP2 binds stably to Arf6•GTP (S. Hashimoto and H.
Sabe, unpublished observation), structural analysis of the complex of
Arf6•GTP and the Arf GAP domain of AMAP2 might be informative for
further analyses of the regulation of the catalytic activity of Arf GAPs.
Moreover, this analysis will also help to assess our hypothesis regarding the
possible dual regulation of the GAP activity of AMAP2, described above.

3.5 BIOCHEMICAL CIRCUITRY OF PAXILLIN-

ASSOCIATED Arf GAPS IN MOTILE CELLS

3.5.1 Links between Arf1-mediated membrane traffic and actin-

cytoskeletal remodeling -roles for paxillin-associated
Arf1GAPs

The paxillin-associated Arf GAPs of the APAP group, Git1, Git2, and Git2-
short, are orthologs of murine Cat-1 and Cat-2 and their splice variants,
identified as Cool/Pix binding proteins (Bagrodia and Cerione, 1999).
Cool/Pix proteins were identified as binding to Pak (p21-activated
serine/threonine kinase; Manser et al., 1998; Bagrodia et al., 1998); a kinase
activated by GTP-bound Cdc42 or Rac1 (Manser et al., 1994). Binding of
Cool/Pix to Pak also activates its kinase activity, either by cooperating with
the binding of activated Cdc42 or Rac or by altering Pak conformation
directly. There are several isoforms of Cool/Pix proteins, p50Cool-1,
p85Cool-1/βPix and Cool-2/αPix, and all contain the Dbl-homology (DH)
domain that can activate Rho family members by stimulating guanine
nucleotide exchange (Bagrodia and Cerione, 1999). p85Cool-1/βPix
increases GTP binding to Rac but not Cdc42 (Manser et al., 1998) and Cool-
2/αPix may increase the levels of GTP-bound Cdc42 (Daniels et al., 1999).
On the other hand, in vitro measurements of Cool/Pix-catalyzed GDP release
from small GTPases have failed to detect any exchange activity (Bagrodia et
al., 1998), suggesting that additional factors may be required for this
exchange activity (Bagrodia and Cerione, 1999).
Chicken APAP2, p95PKL, was identified as a paxillin-binding protein at
the same time that Cat proteins and Cool/Pix proteins were identified, and
was originally proposed to be a protein kinase-linker (PKL). p95PKL plays
a role in connecting paxillin to Pak through Pix: i.e., paxillin binds to
p95PKL, p95PKL to Pix, and Pix to Pak in a linear configuration (Turner et
al., 1999). APAP2 and its short isoform in other species also bind to both
Cool-2/αPix and p85Cool-1/βPix (Premont et al., 2000; Hashimoto et al.,
2001; Mazaki et al., 2001). Given that the binding of Pix to Pak activates its
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kinase activity, the above model implies that the connection of Pak with
paxillin may be activated together with activation of the kinase activity of
Pak. Paxillin can associate with both the kinase-inactive and the Cdc42-
activated forms of Pak3 in vivo, in which the kinase activities were assayed
by phosphorylation of myelin basic protein in vitro (Hashimoto et al., 2001).
Nck, an adaptor protein, binds directly to Pak1 to link Pak1 to growth factor
receptors (Galisteo et al., 1996; Bockoch et al., 1996; Lu et al., 1997; Zhao
et al., 2000). Nck binding to Pak1 had no stimulatory effect on Pak1 kinase
activity, and it did not alter the ability of Pak1 to be stimulated by activated
Rac or Cdc42. Pak1 can phosphorylate Nck. Hashimoto, et al. (2001) have
shown that the biochemical properties of the binding of paxillin to Pak are
similar to those ascribed to Nck: paxillin can bind directly to Pak without
affecting its kinase activity, and can be phosphorylated by Pak3. Moreover,
paxillin and Nck exhibit comparable affinities and bind competitively to
Pak3. Therefore, paxillin appears to function as a template linking integrin
to Pak, as Nck links growth factors to Pak (Hashimoto et al., 2001).
Therefore, there seem to be at least two different ways in which paxillin and
Pak can associate: one is through the aid of APAP2 , in which Paks may be
activated by binding to Pix, and the other is direct, in which Paks can either
remain inactive or be activated by Cdc42 or Rac1.
Chicken APAP2 and paxillin colocalize to focal adhesions, and are
connected to stress fibers, at the edges of CHO.K1 fibroblast cells (Turner et
al., 1999; Brown et al., 2002). On the other hand, human APAP2-short
primarily localize to perinuclear areas in 3Y1 and NIH3T3 fibroblasts,
largely overlapping with paxillin as well as β-COP (Mazaki et al., 2001).
However, AMAP2-short does not accumulate at focal adhesions in 3Y1 and
NIH3T3 fibroblasts, while a fraction colocalizes with paxillin at focal
complexes of lamellipodia (Mazaki et al., 2001). The cell morphology
induced by overexpression of Git2-short is similar to that induced by active
Rac1, exhibiting active membrane ruffles with concomitant substantial loss
of actin stress fibers and focal adhesions (Mazaki et al., 2001). Consistently,
rat p78Pix, a homolog of human p85Cool-1/βPix, and paxillin colocalize to
Cdc42 and Rac1-induced focal complexes in HeLa cells (Manser et al.,
1998). In CHO.K1 cells, on the other hand, APAP2 was recruited to focal
adhesions connected to stress fibers, by the expression of activated Cdc42
and Rac1, but not RhoA (Brown et al., 2002). These data suggest that focal
adhesions may be regulated differently in CHO.K1 cells compared to 3Y1 or
NIH3T3 cells. Accumulation of APAP1 to adhesion-like structures, which
apparently resemble focal adhesions, was also observed in REF52 fibroblast
cells (Manabe et al., 2002). It has also been shown that paxillin plays a role
in recruiting APAP2 and Pak to focal adhesions in CHO.K1 cells (Brown et
al., 2002). On the other hand, it is yet to be clarified as to whether APAP1
 PAXILLIN-ASSOCIATED ARF-GAPS 197
13

and APAP2 localize to the Golgi or ER to regulate their structure and

function, as reported with the APAP2-short isoform.
Arf1 is involved in the regulation of ER-Golgi membrane/vesicle traffic.
Besides the perinuclear areas, Arf1 also functions at the cell periphery and is
involved in the recruitment of paxillin to focal adhesions (Norman et al.,
1998). Given that the Git group of paxillin-associated Arf GAPs act as
GAPs for Arf1 (as discussed above), a molecular complex between paxillin,
these Arf GAPs, βPIX, and Pak may be important for the temporal and
spatial coordination of the Arf1-mediated processes with several
Cdc42/Rac1-mediated processes, resulting in efficient transport and
secretion of newly synthesized proteins into areas where focal complexes
and focal adhesions are formed in motile cells (see later).

3.5.2 Links between Arf6-mediated processes and membrane

remodeling at leading edges - roles for paxillin-associated Arf6
GAPs

3.5.2.1 Link to membrane protrusion: a lesson from Fcγ receptor-

mediated phagocytosis in macrophages
One of the interesting features of Arf GAPs of the AMAP group, PAG2 and
PAG3, is that their overexpression in epithelial or monocyte cells blocks
haptotactic migratory activities towards different extracellular matrices,
measured using modified Boyden chambers in the absence of serum (Kondo
et al., 2000; H. Uchida and H. Sabe, unpublished). In contrast,
overexpression of APAP2 and its short isoform did not exhibit such
inhibitory effects. Rather, each has a slightly stimulatory effect on
migratory activities (Zhao et al., 2000; Mazaki et al., 2001). Cell migration
proceeds in several discrete stages, in which membrane protrusion is one of
the earliest. There are striking similarities between the processes involved in
the remodeling of the cytoskeleton and membrane, and the formation of
leading edges during cell migration and extension of pseudopods during Fcγ
+receptor-mediated phagocytosis in macrophages (Aggeler and Werb, 1982;
Aderem and Underhill, 1999). Moreover, engulfment of even large numbers
of IgG-opsonized particles by macrophages is not accompanied by a
reduction in surface area, indicating that membrane mass is supplied and
maintained during phagocytosis, possibly by exocytosis of intracellular
vesicles or endosomal membranes (Greenberg, 1999; Cox et al., 1999; Bajno
et al., 2000).
To address whether AMAP2 is involved in the maintenance of membrane
mass and/or formation of membrane protrusion, Fcγ receptor-mediated
phagocytosis in macrophages was used as a model system. Overexpression
of AMAP2 was found to inhibit phagocytosis at very early stages, and the
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formation of membrane protrusions was almost completely blocked (Uchida

et al., 2001). The focal accumulation of F-actin beneath the test particles
was also blocked. This result is essentially the same as that observed by
inhibition of Arf6 (Zhang et al., 1998), as also mentioned earlier. A GAP-
inactive mutant of AMAP2, as well as APAP2 and its short isoform, did not
exert such inhibitory effects on membrane protrusion. Therefore, the
inhibitory effects on membrane protrusion were specific to the AMAP
group, PAG2 and PAG3, and did not extend to the APAP group of Arf
GAPs. The inhibition of membrane protrusion may be one of the
mechanisms by which overexpression of AMAP1 and AMAP2 block cell
migration. As Arf6 is the only Arf that participates in Fcγ receptor-mediated
phagocytosis, it is conceivable that Arf6 is involved in this membrane mass
supply. Effects of AMAP overexpression in this system could result either
from its activity as a GAP for Arf6 or as a result of its stable binding to Arf6,
without GTP hydrolysis, as described above.

3.5.2.2 Link between Arf6-mediated membrane recycling and Arf

GAPs
By what mechanism do the paxillin-associated Arf GAPs participate in the
processes of membrane protrusion? Arf6 primarily functions in membrane
recycling at the cell periphery, probably through its GTPase cycle (D'Souza-
Schorey et al., 1995; Peters et al., 1995; Radhakrishna et al., 1996;
Radhakrishna and Donaldson, 1997; D'Souza-Schorey et al., 1998; Al-Awar
et al., 2000; Brown et al., 2001). The majority of the GTP-hydrolysis
defective mutant Arf6-Q67L localizes to plasma membrane invaginated pits
or accumulates in intracellular vacuoles, where it acts to block endocytosis
of several cell surface molecules. On the other hand, the majority of the
GTP-binding defective mutant Arf6-T27N localizes to intracellular
tubulovesicular structures (thought to represent the pericentriolar recycling
compartment) and blocks cell surface molecules from recycling back to the
cell surface. It has therefore been proposed that activated Arf6, may regulate
the outward flow of the recycling endosomes. Hydrolysis of the GTP is then
thought to signal the return of Arf6 to the tubular endosome. There are
several distinct processes for endocytosis of different types of cell surface
molecules, including clathrin-dependent endocytosis and a variety of
clathrin-independent processes. In which types of endocytosis Arf6 is
involved has not been well established and appears to be rather cell type-
specific. In HeLa cells, for example, it has been reported that Arf6 might
not act in clathrin-dependent endocytosis, but is involved in recycling of Tac
and MHC class I molecules, which apparently do not contain cytoplasmic
tails conferring clathrin/AP-2 localization (Radhakrishna et al., 1996;
Radhakrishna and Donaldson, 1997; Brown et al., 2001). β1 integrin also
 PAXILLIN-ASSOCIATED ARF-GAPS 199
15

colocalized with Arf6-L67-positive intracellular vacuoles (Brown et al.,

2001), although it is not yet established whether β1 integrin is under the
control of Arf6 activity.
Arf GAPs of the AMAP group contain protein interaction domains (i.e.,
the PH, PRD and SH3 domains) that are not present in those of the APAP
group. As already mentioned, the SH3 domain of human AMAP2 binds to
Pyk2 (Andreev et al., 1999) and paxillin (Kondo et al., 2000). Further
identification of the interaction partners of these Arf GAPs may help reveal
the molecular mechanism by which these Arf GAPs act in vivo. The PRDs
of AMAP1 and AMAP2 bind to SH3 domains of several proteins, most of
which are components of the endocytic machinery, such as amphiphysin-II
and intersectin (S. Hashimoto and H. Sabe, unpublished observation; Y.
Onodera and H. Sabe, unpublished observation). The PRDs of AMAP1 and
AMAP2 contain several distinct proline-rich sequences, and the binding to
endocytic proteins largely overlap in specificity, but are not identical. The
SH3 domains of AMAP1 and AMAP2 also bind POB1 (Oshiro et al., 2002),
which is a binding partner for RalBP1, a Ral effector that is also involved in
receptor-mediated endocytosis (Nakashima et al., 1999). These results are
consistent with the conclusion that the AMAP group of paxillin-associated
Arf GAPs act to regulate Arf6-mediated endocytosis. However, it still
remains established how and when these Arf GAPs attack Arf6 to down
regulate its activity, during the Arf6-mediated membrane recycling back to
the cell surface or the endocytosis. It also remains to be established which
cell surface molecules are involved in the membrane recycling regulated by
AMAP.

3.5.2.3 Links to Cell Migratory Activity

The intracellular circuitry of paxillin-associated Arf GAPs has been
described above and shown to be related to the machinery that regulates cell
motility. Overexpression of APAPs enhances cell migration, while
overexpression of the AMAPs inhibits it. Cell adhesion was not
significantly affected by the overexpression of any of these Arf GAPs.
These results do not necessarily imply that each of the Arf GAPs have a role
either in enhancing or in inhibiting cell migratory activities under
physiological conditions. AMAP1 and AMAP2, for example, are induced
during monocyte maturation and accumulate at phagocytic cups, implying
that these Arf GAPs might be necessary for, rather than inhibitory to,
monocyte maturation and phagocytic activity. Because cell migration does
not operate by a simple linear cascade of signaling events but is a
multifactorial process in which a number of distinct, linked events take
place, the results of the overexpression of Arf GAPs might give rise to either
enhancing or blocking effects depending on the cellular context,
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experimental conditions, and the presence/expression of other proteins. It is

also possible that Arf GAP overexpression gives rise to indirect effects,
depending on the degree of their overexpression. Therefore, effects caused
by the overexpression of Arf GAPs, regardless of whether the wild type or
mutant proteins are used, should be interpreted as evidence for their
involvement in the processes examined. Indeed, unlike our results of
AMAP2 overexpression, moderate levels of its exogenous expression have
recently been shown to slightly enhance haptotaxis of NIH3T3 cells towards
a gradient of fibronectin plus platelet derived growth factor (Furman et al.,
2002).

4. PROSPECT

In this chapter, we have focused on the Arf GAPs that bind paxillin, and
described their isoform specificity and roles in cytoskeletal remodeling and
membrane remodeling. The latter two are coordinately regulated during
integrin-mediated cell adhesion, migration and intracellular signaling. An
important distinction that emerges from the results described is that the
paxillin binding Arf GAPs fall into two groups. One, the APAPs, has
specificity for class I Arfs (e.g., Arf1) and cellular roles in membrane traffic
and ruffling at the leading edge of motile cells. The other, the AMAPs, is
active on Arf6 in cell-based Arf GAP assays and coordinates effects on
endocytosis and recycling at the plasma membrane and on membrane
protrusions in motile cells (see Figure 2).
Migrating cells secrete proteins, such as cytokines and membrane
metalloproteases, at the leading edges of cells. The APAPs, exhibiting a
GAP activity for Arf1, localize both at the cell periphery and perinuclear
areas, and bind Pix proteins, that contain Rac/Cdc42 GEF activity and bind
Paks. Arf1 plays important roles in the secretion of newly synthesized
proteins, and Cdc42 and Rac1 are important in polarity and membrane
transport. I speculate that these Arf GAPs may be able to influence the
determination of the cell polarity or movements, as mentioned earlier.
Moreover, Cdc42 and Rac1 appear to be activated at the perinuclear areas, in
addition to the cell periphery (Stowers et al., 1995; Erickson et al., 1996;
Kroschewski et al., 1999; Wu et al., 2000; Kraynov et al., 2000; Del Pozo et
al., 2001; Ridley, 2001). It will thus be interesting to explore whether the
APAPs, as well as their interaction with Pix or Pak, are involved in the
perinuclear activation of Cdc42 and Rac1. Possible relationships between
the perinuclear activation of Cdc42 and Rac1, and activity of Arf1 may also
provide interesting results.
 PAXILLIN-ASSOCIATED ARF-GAPS 201
17

Figure 2. Biochemical circuitry of paxillin-associated Arf GAP proteins. See text for details.

Given that Arf6 mediates recycling of some integrins via endosomes, it is

possible that overexpressed AMAPs inhibit integrin recycling by shutting off
Arf6 activities, resulting in the inhibition of cell migration. However, it
remains to be determined whether Arf6 regulates the recycling of integrins
through endosomes. Moreover, not all isoforms of integrins recycle
(Bretscher, 1992). It is not yet established whether recycling of subsets of
integrins is necessary for and contribute to cell migratory activity, rather
than merely associate with it. Membrane protrusion is also inhibited by the
overexpression of these Arf GAPs. Membrane protrusion must be
coordinated with cytoskeletal remodeling, and there are a number of reports
indicating that Arf6 is involved in actin-cytoskeletal remodeling at the cell
periphery. At least in the early stages of protrusion, membranes may be
pushed forward from the inside by polymerizing F-actins (Mitchison and
Cramer, 1996; Borisy and Svitkina, 2000). On the other hand, the supply of
membrane mass from the inside is clearly demonstrated in the case of
phagosome protrusions, in which Arf6 activity is likely to be required, as
already mentioned. The source of membrane components supplied for the
protrusions involved in cell migration and the precise mechanism how Arf6
activity is involved in the protrusive process remain uncertain (Bretscher,
1996; de Curtis, 2001). It will also be important to clarify the molecular
mechanism by which membrane protrusions are tightly coupled with actin
cytoskeletal remodeling, occurring underneath the protrusions. Furthermore,
it remains to be determined how the polarity of integrin traffic to the cell
surface and endosomal recycling are coupled to the direction of cell
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18 SABE

movement. The AMAP Arf GAPs remain excellent tools to explore these
issues.
Paxillin is found at all subcellular areas where the APAPs and AMAPs
exist. Paxillin is tyrosine phosphorylated in most of these areas, and linked
to the regulation of Rho-family GTPases (Nakamura et al., 2000; Tsubouchi
et al., 2002). Binding of paxillin to these Arf GAPs may be necessary for
the recruitment of paxillin to certain subcellular areas where paxillin is
subsequently phosphorylated and acts as an adaptor linking the upstream
signals to the activities of Rho GTPases. This model, however, also needs
further experimental scrutiny. Lastly, paxillin-binding Arf GAPs are all
tyrosine phosphorylated (Andreev et al., 1999; Bagrodia et al., 1999), though
the biological significance of tyrosine phosphorylation awaits description.
Although the discussion in this chapter has been restricted to those Arf
GAP that were identified by their binding to paxillin, most of the other Arf
GAPs also have multiple protein interaction modules and are thus likely to
act to coordinate multiple signaling events. Arfs are found in all eukaryotes.
Hence, Arf GAPs are great targets for the analysis of the principles of
intracellular multifactorial processes, where membranes have been employed
as primordial superintendents of cell function, perhaps from the very
beginning of the existence of eukaryotes on this planet (http://smart.embl-
heidelberg.de/smart/do_annotation.pl?DOMAIN=Arf&BLAST=DUMMY&
EVOLUTION=Show#Evolution).

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Uchida, H., Kondo, A., Yoshimura, Y., Mazaki, Y., and Sabe, H. (2001).
PAG3/Papα/KIAA0400, a GTPase-activating protein for ADP-ribosylation factor (ARF),
regulates ARF6 in Fcγ receptor-mediated phagocytosis of macrophages. J. Exp. Med., 193,
955-966.
Vitale, N., Patton, W. A., Moss, J., Vaughan, M., Lefkowitz, R. J., and Premont, R. T. (2000).
GIT proteins, A novel family of phosphatidylinositol 3,4, 5-trisphosphate-stimulated
GTPase-activating proteins for ARF6. J. Biol. Chem., 275, 13901-13906.
Wu, W. J., Erickson, J. W., Lin, R., and Cerione, R. A. (2000). The gamma-subunit of the
coatomer complex binds Cdc42 to mediate transformation. Nature, 405, 800-804.
Zhang, Q., Cox, D., Tseng, C.C., Donaldson, J.G., and Greenberg, S.(1998). A requirement
for ARF6 in Fcγ receptor-mediated phagocytosis in macrophages. J. Biol. Chem., 273,
19977-19981.
Zhao, Z. S., Manser, E., Loo, T. H., and Lim, L. (2000). Coupling of PAK-interacting
exchange factor PIX to GIT1 promotes focal complex disassembly. Mol. Cell. Biol., 20,
6354-6363.
Chapter 10
ACTIVATION OF CHOLERA TOXIN AND E.
COLI HEAT-LABILE ENTEROTOXIN (LT) BY
ARF

G. PACHECO-RODRIGUEZ1, NAOKO MORINAGA2, MASATOSHI

NODA2, J. MOSS1, AND M. VAUGHAN1
1
Pulmonary-Critical Care Medicine Branch, NHLBI, NIH, Bethesda, USA;22nd Department
of Microbiology, School of Medicine, Chiba University, Chiba, Japan.

Abstract: Cholera (CT) and heat labile E. coli (LT) enterotoxins are members of the AB5
family of bacterial toxins that contain one catalytically active A subunit
associated with a complex of five B subunits. The B subunits of CT and LT
bind the receptor, ganglioside GM1, at the cell surface as a required first step
in entry of the holotoxin into target cells. The A subunit is processed into the
catalytic A1 and linker A2 segments. The holotoxin-receptor complex is
internalized and transported to the endoplasmic reticulum before release of the
active A1 protein into the cytosol. The ADP-ribosyltransferase activity of A1
is a critical determinant of toxin potency. When bound to GTP, all Arfs are
allosteric activators of CT in vitro. The importance of such interactions in the
intoxication of cells is, however, less clear. Recent reports suggest possibilities
for clinically effective prevention of CT action by interference at the critical
initial step of binding to the GM1 receptor or by inhibition of CTA1 ADP-
ribosyltransferase activity in intestinal mucosa cells.

1. INTRODUCTION

Purification of a protein, termed ADP-ribosylation factor or Arf, which was

required for the demonstration of cholera toxin (CT)-catalyzed ADP-
ribosylation of purified GĮs, the stimulatory guanine nucleotide-binding
regulatory protein of adenylyl cyclase, was reported by Kahn and Gilman in
1984. For several years thereafter, the physiological function(s) of Arfs
remained obscure. Possible roles in eukaryotic ADP-ribosylation reactions
were considered, as was the possibility that by defining the mechanisms of
Arf activation of cholera toxin something might be learned about Arf action

209
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2 PACHECO-RODRIGUEZ, MORINAGA, NODA, MOSS, AND VAUGHAN

in animal cells. Identification of a critical role for Arf in protein secretion in

Saccharomyces cerevisiae (Botstein et al., 1988) initiated a remarkable and
continuing expansion of understanding diverse aspects of vesicular traffic at
a level of complexity, molecular and regulatory, not anticipated at the time
that Palade first established the mechanisms of transport of newly
synthesized secretory proteins from the site of synthesis in the endoplasmic
reticulum to the plasma membrane (Palade, 1975).
This chapter deals mainly with the possible involvement of Arf in the
action of CT or E. coli heat-labile enterotoxin (LT) on intact cells. After
summarizing information regarding the structure and function of the toxins,
we consider briefly: 1) the role of Arf in toxin internalization and processing
by cells, and, 2) the mechanism of Arf activation of LT and CT in vivo and
in vitro.

2. STRUCTURE AND FUNCTION OF CT AND LT

CT and LT are members of the AB5 family of bacterial toxins that contain
one catalytically active A subunit associated with a complex of five B
subunits. The latter are responsible for toxin binding to specific receptors on
the cell surface and the subsequent entry of toxin into the cell. In a given
toxin, the five B subunits may or may not be identical, but all bind to
glycoproteins or ganglioside oligosaccharides. In particular, CT and LT
each have a single type of B subunit and the pentamer associates with the
monosialoganglioside GM1 (galactosyl-N-acetylgalactosaminyl-[N-
acetylneuraminyl]-galactosylglucosylceramide), the oligosaccharide of
which is responsible for toxin binding. A subunits of the AB5 toxins can
have different enzymatic activities, although several of them are mono-ADP-
ribosyltransferases (reviewed by Merritt and Hol, 1995). CT and LT are
very similar in structure (ca. 80% amino acid sequence identity in both A
and B subunits) and function, including enzymatic properties, ganglioside
specificity, and immunoreactivity. However, they differ significantly in
toxicity for human cells (Rodighiero et al., 1999). Systematic comparison of
the effects of chimeric CT/LT molecules on secretion of Cl¯ ion by human
T84 epithelial cells grown in culture demonstrated that differences in the
receptor binding B subunits, or in the catalytic A1 fragment were not
responsible for the differences in toxicity. However, when the holotoxins
were bound to GM1 the ability of CT to withstand denaturation by acid or
urea was greater than that of LT. This was accounted for by sequence
differences in a ten-residue segment of the A2 fragments. Stability of the
holotoxin-receptor complex as it is internalized and transported to the
 ACTIVATION OF CT AND LT BY ARF 2113

intracellular site where release of A1 will have the greatest toxicity is critical
to toxin potency (Rodighiero et al., 1999).

Figure 1. Structure of LT (reproduced by permission from Merritt and Hol, 1995). In LT, the
A subunit is positioned above a ring of five identical B subunits with the A2 domain
extending through the center of the B pentamer. In this image, the catalytic A1 subunit is at
the top and connected to the pentameric B subunit ring (bottom) by the long alpha helix that
comprises most of the A2 subunit.

The crystal structure of LT was first reported in 1991 (Sixma et al., 1991)
and that of the CTB complex, with GM1 oligosaccharide bound, in 1994
(Merritt et al., 1994). Information from these and other structural studies is
all consistent with a model in which the B pentamer that binds GM1 on the
cell surface forms a circular base on which the single A subunit rests, with
its C-terminal 5 kDa A2 segment extending into the B subunit ring (see
212
4 PACHECO-RODRIGUEZ, MORINAGA, NODA, MOSS, AND VAUGHAN

Figure 1). The KDEL (RDEL in LT) sequence at the C-terminus marks the
toxin for transport to the endoplasmic reticulum (Lencer et al., 1995).
Additional critical sequence in A2 is that required for appropriate release of
active A1 from the A2 fragment. Proteolytic cleavage (nicking) of the
holotoxin at R192 can occur even before toxin entry into the cell, but the A1
and A2 fragments remain covalently linked until reduction of the single
disulfide bond between them releases A1. This is probably catalyzed by a
protein disulfide isomerase in the endoplasmic reticulum (Moss et al., 1980;
Orlandi, 1997; Tsai et al., 2001).
In the mono-ADP-ribosyltransferase reaction catalyzed by CT or LT, the
ADP-ribose moiety of NAD is transferred to a specific arginine in an
acceptor protein (Moss and Vaughan, 1977; Moss and Richardson, 1978).
These toxins also catalyze a reaction (NAD glycohydrolase) in which water
serves as the ADP-ribose acceptor (Moss et al., 1976). Both activities are
intrinsic to the A1 fragment of CT (Moss et al., 1979a). In addition, CTA
can ADP-ribosylate itself (auto-ADP-ribosylation) and Arf (Tsai et al.,
1988). In the stereospecific reaction catalyzed by CT, the Į-anomer of
ADP-ribosylarginine is produced from ȕNAD+ (Moss et al., 1979b). The
major ADP-ribose acceptor in CT- and LT-catalyzed reactions in eukaryotic
cells is GĮs, but in vitro, CT and LT can use several guanidinium compounds
as ADP-ribose acceptors. Agmatine is one of those used in a simple assay
(Moss and Vaughan, 1977) for quantification of CT ADP-ribosyltransferase
activity (and that of other transferases that use arginine as an ADP-ribose
acceptor).

3. CELL BINDING AND PROCESSING OF CT AND

LT

Ganglioside GM1 is the cell surface binding site/receptor for the B subunit
pentamer through which CT or LT is internalized (Fishman et al., 1978). CT
bound also to GM1 oligosaccharide that had been covalently attached to
proteins on the cell surface (neoganglioproteins), but was not internalized,
indicating that the toxin-GM1 interaction contributes to intoxication beyond
the initial cell-surface binding (Pacuszka and Fishman, 1992). It also
suggests that toxin internalization should be evaluated along with cell
binding to assess the biological relevance of the interaction (Pacuszka and
Fishman, 1992). The CT receptor, GM1, appears to be concentrated in
caveolae or lipid rafts (Tran et al., 1987). The toxin was localized at non-
clathrin-coated invaginations of fibroblast membranes by electron
microscopy (Tran et al., 1987). In the intestine, CT binds to and is
internalized via ganglioside GM1 on the apical membrane of the mucosal
 ACTIVATION OF CT AND LT BY ARF 2135

cells. Cell polarity may be, in fact, an essential property of a CT (or LT)
target cell in vivo, although many studies of toxin action are performed in
non-polarized cells, e.g., CHO cells, that are also susceptible to CT
(Morinaga et al., 2001). In intestinal mucosa cells, it is the GĮs subunit
located on the basolateral membrane that is the main CT substrate. Covalent
modification of GĮs and the resulting essentially irreversible activation of
adenylyl cyclase leads to sustained elevation of cellular cAMP content,
efflux of Na+ and water into the intestinal lumen, and eventual dehydration
of the host.
The complex of CT with GM1 ganglioside is internalized by receptor-
mediated endocytosis, which is selective, regulated, and carried out via
either clathrin-coated or non-clathrin-coated invaginations of the plasma
membrane (e.g., caveolae and lipid rafts). Clathrin-coated pits are lined on
the cytoplasmic surface with light and heavy clathrin chains plus the adaptor
proteins required for assembly of the clathrin coat (for review see Higgins
and McMahon, 2002). The process includes: 1) binding and localization of
the ligand (toxin)-receptor complex in a nascent clathrin-coated vesicle, and,
2) accumulation of other specific molecules to complete vesicle formation.
Caveolae are flask-shaped invaginations of the plasma membrane
characterized by their size (50-100 nm), morphology, and biochemical
constituents, which include cholesterol, sphingomyelin, and ceramide among
other lipids (Anderson, 1998). Protein components include caveolin,
glycosylphosphatidylinositol-anchored proteins, and receptors (Anderson,
1998). Lipid rafts or caveolae appear to be the main site of holotoxin entry,
although access to some cells can occur via clathrin-coated pits or non-
clathrin-coated invaginations. The site of entry can depend on the cell type,
as was shown by studies of the Caco-2, HeLa, and BHK cell lines
(Torgersen et al., 2001). CT-induced morphological changes of CHO cells
were not affected by over-expression of dynamin, but were suppressed
significantly by over-expression of the mutant dynamin in which K44 is
replaced by alanine, resulting in an impaired capacity to hydrolyze GTP
(personal communication, N. Morinaga). Dynamin functions in
internalization via caveolae (Henley et al., 1998; Oh et al., 1998). As
chlorpromazine, an inhibitor of clathrin-dependent endocytosis (Sofer and
Futerman, 1995; Orlandi and Fishman, 1998), did not affect CT-induced
cAMP accumulation in CHO cells, the entry of CT into those cells may be
via caveolae. The mutant of Eps15, a component of clathrin-coated pits,
should clarify which is involved. Independent of the membrane site of
receptor interaction or mechanism of internalization, specific binding of
CTB is the critical initial step in CT action. It represents, therefore, an
important potential site for intervention with drugs that could prevent the
CT-GM1 interaction (Merritt et al., 2002).
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6 PACHECO-RODRIGUEZ, MORINAGA, NODA, MOSS, AND VAUGHAN

Fishman reported more than 20 years ago that CT binding and

internalization had occurred well before the increase in cell cAMP was
detected. The delay was considered to be the time required for intracellular
transport and processing of the holotoxin (Fishman, 1982). Detailed studies
by several groups, plus the greatly improved understanding of intracellular
vesicular traffic have clarified the processes involved in intoxication of
different types of cells by CT and LT. In the human T84 intestinal cell line,
the A and B subunits separate as a result of cleavage by a serine protease in
the endoplasmic reticulum (Lencer et al., 1997). The B subunit complex
was returned to the plasma membrane via a secretory pathway, while the A
subunit was further processed to generate A1 and A2 peptides by a protein-
disulfide isomerase in the endoplasmic reticulum (Moss et al., 1980;
Orlandi, 1997; Tsai et al., 2001). Finally, the A1 subunit was released into
the cytosol, potentially via the Sec61p complex (Schmitz et al., 2000), from
which it moved to its site of action. In polarized cells, that site is on the
basolateral membrane where CTA1 ADP-ribosylates GĮs.
In cultured fibroblasts incubated with CT, detection of the enzymatically
active A1 peptide paralleled the activation of adenylate cyclase, from which
it was inferred that the final transit of A1 to the GĮs substrate is very rapid
(Kassis et al., 1982). Although the molecular interactions that result in toxin
transit from plasma membrane to the Golgi are not completely understood,
the transport appears to be vesicle-mediated. BFA, which disrupts Golgi
formation and structure, prevented the action of CT, suggesting the
requirement for an intact Golgi (Orlandi et al., 1993). Known targets of
BFA in mammalian cells are BFA-inhibited guanine nucleotide-exchange
proteins BIG1 and BIG2 (see Chapter 6), which activate Arf, and the BFA-
stimulated ADP-ribosyltransferase, which is a lysophosphatidic acid: acyl-
CoA transferase (Weigert et al., 1999). All of these proteins are important
for Golgi function. Whether or not all are important for CT and LT
processing remains to be determined.

4. ROLE OF ARF IN CT AND LT ACTION

The interactions of CT and LT with Arfs and resulting effects on ADP-

ribosyltransferase activity have been extensively investigated. The
importance of such interactions in the intoxication of cells is, however, less
clear. The six mammalian Arfs are grouped in three classes: class I (Arfs 1-
3), class II (Arfs 4, 5), and class III (Arf6) (reviewed in Moss and Vaughan,
1995 and 1998). When bound to GTP, each of the Arfs can activate CT in
vitro, and the allosteric nature of this interaction was demonstrated in kinetic
studies (Noda et al., 1990). Questions remain, however, regarding the role
 ACTIVATION OF CT AND LT BY ARF 2157

of Arf in CT effects on cells. Interaction of Arf with CT or LT in the Golgi

could precede the toxin effects that result from elevation of cellular cAMP
concentration.
Arf1 function was shown to be necessary for the morphological effects of
CT on CHO cells (Morinaga et al., 2001). These cultured cells assume an
elongated or bi-polar shape after incubation with CT. Over-expression of
mutant Arf1 proteins indicated that the cyclic activation of Arf, i.e., the
continuing inter-conversion of Arf•GTP and Arf•GDP, was necessary for
CT action. The results were also consistent with the conclusion that Arf1
was critical for the induction of cAMP formation, and probably participated
early in the process of CT intoxication of the cell. The morphological
changes induced by CT were mimicked by treatment of cells with dibutyryl-
cAMP, 8-Br-cAMP, or forskolin, each of which increases cAMP without
activating GĮs. The changes in morphology appeared about one hour later
with CT than with 8-Br-cAMP, corresponding to the difference between the
time required for CT to activate cyclase and increase cAMP and that needed
for the cAMP analogue to reach its site of action. In cells over-expressing
Arf1 mutants that blocked CT actions, 8-Br-cAMP had effects similar to
those seen in cells transfected with wild-type Arf1 or empty vector,
presumably because its action does not depend on Arf function, which was
required for the morphological effects of CT (Morinaga et al., 2001).
To evaluate the specificity of the effect of Arf1, similar over-expression
experiments were carried out with the analogous mutants of Arf5 and Arf6.
Unlike the Arf1 mutants, the over-expressed Arf6 and Arf5 mutants did not
interfere with CT induction of morphological changes, consistent with the
different functions of individual Arfs in vesicular transport. In CHO cells,
COPI-coated vesicles may be involved in CT movement from Golgi to
endoplasmic reticulum. Therefore, the distribution of Arfs and ȕ-COP (one
of the seven protein components of COPI) was investigated. Arf1-L71, a
mutant that is essentially entirely GTP-bound, was largely colocalized with
ȕ-COP, and Arf5-L71 less so. Arf6-L67 was partially concentrated at the
plasma membrane and was not associated with ȕ-COP. The observation that
both GTP- and GDP-bound forms of Arf1 were required for CT action in
CHO cells is consistent with the conclusion that Arf1 influences CT action
in those cells, not via the direct interaction by which all GTP-bound Arfs can
activate CT in vitro, but by its specific function in the vesicular transport
pathway that CT must travel from plasma membrane to Golgi to
endoplasmic reticulum before it can ADP-ribosylate GĮs and alter CHO cell
morphology (Morinaga et al., 2001).
A different kind of study in CHO cells, however, showed that Arf
interaction with toxin in the Golgi, resulting in stimulation of toxin ADP-
ribosyltransferase activity, could be important for the activation of adenylyl
216
8 PACHECO-RODRIGUEZ, MORINAGA, NODA, MOSS, AND VAUGHAN

cyclase and effects on cell morphology. To assess the role of Arf in

localization of the toxin to Golgi and its subsequent action, Zhu and Kahn
(2001) used CHO cells for transient over-expression of LTA1 (the A1
fragment of LT) with myc and his6 sequences at the C-terminus (Zhu and
Kahn, 2001). These experiments eliminated factors related to toxin
internalization, transport from the plasma membrane, and protein processing.
By 16 hr after transfection of cells with myc-his6-LTA1, it had accumulated
throughout the Golgi compartments where an increased amount of
endogenous Arf was also seen. With greater intracellular accumulation of
recombinant LTA1, it became dispersed throughout the cell, along with
endogenous Arf and several Golgi marker proteins. The authors suggested
that this effect of large amounts of LTA1 on Golgi morphology, which
resembled that of BFA, was due to toxin binding of Arf•GTP, leaving
insufficient active Arf to maintain Golgi function, just as results from BFA
inhibition of the guanine nucleotide-exchange proteins required for
formation of Arf•GTP. It was unclear whether the toxin-induced elevation
of cellular cAMP content might also have affected Golgi structure (Zhu and
Kahn, 2001).
Co-expression of Arf3 with LTA1 was used to investigate its role in
Golgi localization and cAMP production. The effects of a variety of Arf
mutants with impaired intrinsic activity and/or capacity for toxin interaction
were evaluated. It was concluded that Arf function was necessary for the
concentration of LTA1 on Golgi membranes and its associated effects on
Golgi structure, as well as for toxin-induced accumulation of cAMP and its
effects on cell morphology (Zhu and Kahn, 2001). Over-expression of LTA1
in the endoplasmic reticulum and transport of holotoxin from the
extracellular medium for processing in the region of the Golgi/endoplasmic
reticulum obviously result in very different numbers of molecules of active
A1 subunit at that site, as pointed out by Zhu and Kahn (2001). In the study
of Morinaga et al. (2001), it seemed clear that the action of Arf1, and not
that Arf5 or Arf6, was specifically important for the internalization and
delivery of active CTA1 from the CHO cell surface to the site of ADP-
ribosylation of GĮs, although the possibility of Arf enhancement of CTA1
ADP-ribosyltransferase activity or participation in effects of CTA1 on Golgi
structure was not evaluated. It is difficult to know how to relate the effects
of relatively massive amounts of LTA1 synthesized in the CHO cell (Zhu
and Kahn, 2001) to the action of much smaller amounts of CTA1
accumulated in the Golgi when it is generated, presumably much more
slowly, from holotoxin internalized after binding to its GM1 receptor on the
cell surface. In studies with intact cells, only a limited amount of the total
bound toxin may actually enter the cell and be delivered to the Golgi. If
CTA1 generated in the Golgi moves rapidly to its site of action, as was
 ACTIVATION OF CT AND LT BY ARF 2179

concluded by Kassis et al. (1982), CTA1 (or LTA1) may not, in fact,
accumulate in the Golgi of intoxicated cells. As the multiple actions of BFA
become better understood, it may be useful in answering some of these kinds
of questions, just as it continues to be useful in elucidating mechanisms of
intracellular vesicular transport (Presley et al., 2002).

5. MECHANISMS OF ACTIVATION OF LT AND CT

BY ARF

In their early experiments, Kahn and Gilman (1986) had demonstrated that
Arf activation of CT-catalyzed ADP-ribosylation of GĮs was dependent on
GTP, and quantification of GTP binding to Arf can often be a more
convenient measure of Arf activation than is measurement of the activated
Arf by its effects on an enzyme activity. Low stoichiometry of GTPȖS
binding to Arf in vitro has been found in many laboratories, and it is known
that the assay conditions, notably Mg2+ concentration and the presence of
specific phospholipids or detergents are of major importance (reviewed in
Moss and Vaughan, 1998). A dramatic effect of recombinant LTA1 on the
stoichiometry of GTPȖS binding to recombinant Arf3 was reported by Zhu
et al. (2000). They observed that LTA1, LTA, and CTA had similar effects;
those of POR1∆N, GGA1, and MKLP1, were analogous but smaller. The
authors interpreted their findings as evidence that these proteins acted as Arf
effectors, which interacted with inactive Arf, to increase its affinity for GTP,
resulting in the formation of stoichiometric effector-Arf•GTP complexes.
For several years, it had seemed plausible that the mechanism by which
Arf activates CT (or LT) might also be the mechanism employed for Arf
activation of its endogenous effectors. It is difficult, however, to compare
quantitatively Arf activity in vitro with its physiological actions in a cell.
Perhaps the most obvious reason is that Arf function in the cell requires the
continuing alternation between GTP- and GDP-bound forms that must be
constantly regulated (temporally and spatially) by the proteins that modulate
GDP release and GTP binding and hydrolysis in response to diverse signals
and molecular interactions. This Arf activity (or its absence) can be
observed or inferred (e.g., in effects of BFA on Golgi morphology). Unlike
a difference in catalytic rate, however, it is not readily quantified.
Following reports that Arf1 and Arf3 activate phospholipase D (PLD) in
animal cells (Brown et al., 1993; Cockcroft et al., 1994), an Arf-activated
PLD in rat brain extract was separated from an oleate-dependent PLD and
shown to be activated by recombinant Arf5 (class II) and Arf6 (class III), as
well as by class I Arfs (Massenburg et al., 1994). It appeared that a
quantitative comparison of Arf effects on the activities of the partially
218
10 PACHECO-RODRIGUEZ, MORINAGA, NODA, MOSS, AND VAUGHAN

purified PLD and the pure CTA1 protein might permit identification of
elements of the Arf structure that are responsible for activation of the two
enzymes. Experiments were facilitated by the production of recombinant
chimeric proteins in which segments of the human Arf1 and Arl1 proteins
were exchanged (Zhang et al., 1995). These two proteins are, overall 53%
identical in amino acid sequence (181 residues), but Arl1 is deficient in Arf
activity, as defined by the usual criteria of 1) ability to stimulate cholera
toxin ADP-ribosyltransferase activity, and, 2) to rescue yeast bearing the
lethal double mutation of both arf1 and arf2 genes (Moss and Vaughan,
1995). On assay of the chimeric molecules, the protein containing the first
73 amino acids of Arf1 (and the remainder of Arl1) activated PLD to the
same extent as did Arf1, but failed to activate the toxin. The latter was
activated by the reciprocal chimera containing Arf sequence in positions 74-
181 and the first 73 amino acids of Arl1. Positions 37-55 in Arf correspond
to the “effector loop” in Ras and Ras-related proteins, through which the 20
kDa GTPases produce many of their effects (Vetter and Wittinghofer, 2001).
Thus, the mechanism of Arf activation of the ADP-ribosyltransferase
activity of the toxin seems unlike that of its phospholipase D activation and
at least some of its actions in cells (Zhang et al., 1995). These observations
do not, of course, rule out the possibility that Arf stimulation of CTA1 and
LTA1 activity proceeds by a mechanism that is employed by the GTPase for
other, as yet unrecognized, biological purposes.

6. CONCLUSIONS

Studies of the mechanism of action of CT continue to extend the

understanding of diverse aspects of biology and medicine. Unfortunately,
however, infection and disease caused by Vibrio cholerae remain serious
worldwide problems that must be addressed. Cholera is especially prevalent
and damaging in those parts of the world in which its prevention, through
improvement of sanitation and other public health measures, is particularly
difficult. When a person is infected, oral rehydration, with or without drugs
that reduce diarrhea, can be effective therapy, only however, when
accomplished in the relatively short time before effects of dehydration are
irreversible. Too often, such seemingly simple measures are unavailable in
the environment of epidemic cholera.
Based on present knowledge of the mechanism of CT action, at least two
events may be potentially useful targets for therapeutic intervention.
Binding of CT to the intestinal mucosal cell might be prevented by the use of
ganglioside GM1 analogues, as suggested by Merritt et al. (2002). This
group synthesized a molecule composed of five m-nitrophenyl-Į-D-
 ACTIVATION OF CT AND LT BY ARF 219
11

galactoside moieties, which interacts in a 1:1 molar ratio with the AB5
holotoxin.
Inhibition of ADP-ribosyltransferase activity is another potentially
efficacious approach. On investigation of herbal medications from several
countries, it was determined that kampo formulations, or the gallate
compounds that they contain, can effectively prevent or reduce the diarrheal
effects of CT. The gallate derivatives inhibited ADP-ribosyltransferase
activity of CT in vitro, when agmatine was used as a model substrate. They
also inhibited the diarrheogenic activity of CT in an ileal loop model of in
vivo action on the intestine (Oi et al., 2002), consistent with a significant role
for this activity in disease. Thus, study of the mechanisms of action of CT
and LT, their cell surface binding and entry into cells, as well as activation
of GĮs by ADP-ribosylation can continue to provide clinically useful
information.

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Chapter 11
PHOSPHOLIPASE D, ARFAPTINS AND
ARFOPHILIN

JOHN H. EXTON
Department of Molecular Physiology, Biophysics and Pharmacology, Howard Hughes
Medical Institute, Vanderbilt University, Nashville, USA n

Abstract: The effects of Arfs on three potential effectors (phospholipase D, Arfaptins

and Arfophilin) are described. Arfs activate mammalian PLD1 in vitro, but
their roles in the regulation of PLD in vivo are not well defined. Direct
binding of Arfs to PLD1 has not been demonstrated in vitro and activation
may involve PI(4,5)P2, which binds to the enzyme. Evidence for a role of
Arf6 in the regulation of PLD in vivo is accumulating and may partly involve
a PIP kinase that is activated by this Arf. The role of Arf-stimulated PLD in
Golgi traffic remains controversial and evidence for and against this is
presented. The properties of the Arf-binding proteins Arfaptins 1 and 2 are
described, and interactions of POR1, a truncated form of Arfaptin 2, with Arfs
and Rac1 are discussed. A hypothesis for Arfaptin 2 as a communicator
between these two small GTPases in vivo is presented. A possible role of
Arfaptin 2 in Huntington’s disease is discussed. Finally, the properties of
Arfophilin, which selectively binds ARFs 4, 5 and 6 are described.

1. PHOSPHOLIPASE D

1.1 Regulation of the PLD1 Isozyme of Phospholipase D

by Arfs in vitro

Arf was recognized as an activator of phospholipase D (PLD) in early

experiments with partially purified preparations of the enzyme (Brown et al.,
1993) or with neutrophils depleted of cytosol (Cockcroft et al., 1994).
Studies with individual members of the Arf family indicated that all
mammalian Arfs were capable of activating PLD in the presence of GTPγS
(Massenburg et al., 1994; Brown et al., 1995; Tsai et al., 1998). The
myristoylated forms of the Arfs were much more effective than the

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unmodified forms (Massenburg et al., 1994; Brown et al., 1995; Tsai et al.,
1998). No large differences were observed in the potency or efficacy of
different Arf members to activate PLD in vitro (Massenburg et al., 1994;
Brown, 1995; Tsai et al., 1998). As noted for all other stimulators of PLD,
phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) was required for the effect
of Arf on the enzyme.
Cloning studies have revealed the existence of two mammalian isozymes
of PLD (PLD1 and PLD2), each of which exist as splice variants. These
isozymes have four conserved sequences (I-IV) and sequences I and IV each
contain the highly conserved HKD motif (Exton, 2002). Searches of the
human genome database have not revealed other PLD isozymes. PLD1 is
strongly stimulated by Arfs 1 and 3 in vitro (Hammond et al., 1995, 1997;
Min et al., 1998; Sung et al., 1999b) whereas PLD2 shows little or no
response (Colley et al., 1997; Kodaki and Yamashita, 1997; Lopez et al.,
1998; Sung et al., 1999a). Marked synergism is observed between Arf and
RhoA or protein kinase C in the activation of PLD1 or partially purified
brain PLD (Hammond et al., 1997; Singer et al., 1996). The site at which
Arf interacts with PLD1 has not been defined, but deletional mutagenesis of
the first 325 amino acids of the N-terminus indicates that the site is not
contained within this sequence (Sung et al., 1999b; Park et al., 1998).
Surprisingly, deletion of the first 308 amino acids of PLD2 decreases the
basal activity of the enzyme, but renders it highly responsive to Arfs 1 and 5
in vitro (Sung et al., 1999a). This suggests that the N-terminus may block
binding of Arf or otherwise interfere with its ability to stimulate catalysis.
This observation, plus the finding that PLD2 does show a weak response to
Arf1 in vitro (Kodaki and Yamashita, 1997; Lopez et al., 1998; Sung et al.,
1999a), suggests that this PLD isozyme could mediate part of the action of
Arfs on PLD.
The domain in Arf1 that interacts with PLD has been defined (Zhang et
al., 1995; Jones et al., 1999). Initial work with chimeric proteins constructed
between Arf1 and the structurally related, but inactive, protein ARL1
indicated that the first 73 amino acids at the N-terminus of Arf1 were
required for activation of PLD (Zhang et al., 1995). The region comprising
residues 35-94 was shown in another study to be required for PLD1
activation and AP-1 recruitment, but not for Arf1 activation by a Golgi-
associated GEF (Liang et al., 1997). A later study utilized substitution and
deletion mutagenesis and knowledge of the crystal structure of Arf1 to map
the PLD interaction site to the α2 helix, part of the β2 strand and the N-
terminal helix and its ensuing loop (Jones et al., 1999). Mutants that showed
increased or decreased ability to activate PLD produced similar effects on
secretion in HL60 cells (Jones et al., 1999). However, another study
utilizing selective mutations showed that amino acid residues (I49T, F51Y)
 PHOSPHOLIPASE D, ARFAPTINS AND ARFOPHILIN 225
3

in the switch I region of Arf3 were required for PLD activation, but only one
of these (F51Y) was needed to promote vesiculation of Golgi (Kuai, et al.,
2000).
The mechanism by which Arf activates PLD is unclear. In in vitro
experiments, recombinant Arf1 can activate recombinant PLD1 in the
presence of GTPγS and PI(4,5)P2, which is included in the PLD assay
mixture (Hammond et al., 1995; Min et al., 1998). Because PI(4,5)P2 is
required for PLD activity (Brown et al., 1993; Hammond et al., 1995; Min et
al., 1998; Sciorra et al., 1999), it is difficult to explore the role of this lipid in
the action of Arf on the enzyme. As stated above, no interaction site for Arf
on PLD has been defined, although binding of PI(4,5)P2 to the enzyme has
been demonstrated (Sciorra et al., 1999; Hodgkin, et al., 2000). The binding
of PI(4,5)P2 to Arf1 has been demonstrated and shown to increase nucleotide
exchange (Randazzo, 1997; Terui et al., 1994). The binding promotes the
interaction of Arf1 with an Arf GAP (Randazzo and Kahn, 1994) and with
the Arf GEF ARNO (Paris et al., 1997), and this may be true for PLD. As
discussed below, indirect effects of Arf family members on PLD isozymes in
vivo are also likely.

1.2 Regulation of PLD by Arf in vivo – Roles of Arf6 and

PIP Kinase

A role for Arf in the regulation of PLD in vivo is not as well defined as the
roles of protein kinase C and Rho family members. Early experiments
showed that depletion of the cytosol of HL-60 cells through detergent
permeabilization caused loss of GTPγS-stimulated PLD activity, which
could be restored by addition of brain cytosol (Geny et al., 1993). The
restorative agent was subsequently shown to be Arf (Cockcroft et al., 1994).
A similar role for Arf in the regulation of PLD in permeabilized neutrophils,
mast cells and HEK cells has been reported (Rumenapp et al., 1995, 1997;
Fensome et al., 1998; Way, et al., 2000). In the studies with neutrophils and
mast cells, Arf also restored the response to FMLP (Fensome et al., 1998) or
antigen (Way, et al., 2000). In other studies, addition of Arf1 or Arf6 to
permeabilized A10 vascular smooth muscle cells or Rat 1 fibroblasts did not
restore the activation of PLD by GTPγS, but restored the ability of
angiotensin II, PDGF or PMA to activate the enzyme (Shome et al., 1998;,
2000). However, these results have not been observed in all cells (Glenn et
al., 1998). Transfection of wild type or constitutively active forms of Arf3
does not activate PLD1 in COS-7 cells (Park et al., 1997), but this may be
due to mislocalization of the expressed proteins or disruption of the Golgi.
Isolated Golgi fractions possess Arf-stimulated PLD activity (Provost et al.,
1996; Ktistakis et al., 1995, 1996), and PLD1 has been localized to the
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4 EXTON

perinuclear region in many studies of intact cells. However, efforts to

localize PLD1 specifically to Golgi in vivo have not given uniformly
positive results (Colley et al., 1997; Chen et al., 1997; Freyberg, et al., 2001;
Brown et al., 1998).
Several studies of the regulation of PLD by Arf in vivo have utilized
brefeldin A and have given divergent results. This is probably because this
compound does not inhibit all Arf GEFs (Kam and Exton, 2001). Thus, a
subclass of Arf GEFs containing certain residues in their Sec7 domains are
targets of brefeldin A, whereas others e.g.. ARNO and cytohesin-1 are not
(Moss and Vaughan, 1998; see Chapters 4-6 for additional details).
Brefeldin A has been reported to inhibit carbachol stimulation of PLD in
HEK cells expressing M3 muscarinic receptors (Rumenapp et al., 1995) and
to impair activation of PLD by PMA and PDGF in Rat 1 fibroblasts (Shome
et al., 1998). Other studies have shown inhibitory activity of this fungal
metabolite on PLD activation by angiotensin II, endothelin 1, carbachol and
PDGF in A10 vascular smooth muscle cells and 1321N1 astrocytoma cells
(Shome, et al., 2000; Andresen, et al., 2001; Mitchell et al., 1998). In
contrast, brefeldin A has no effects on the stimulation of PLD by ATP and
FMLP in HL60 cells (Guillemain and Exton, 1997; Bourgoin et al., 1995) or
by PMA, sphingosine 1-P or bradykinin in A549 adenocarcinoma cells
(Meacci et al., 1999). It is not known whether these differences reflect
different complements of Arf GEFs in these cells or different roles for Arfs
in agonist regulation of PLD.
Arf6 differs from the other mammalian Arfs in that it is associated with
the plasma membrane (Meacci et al., 1999; Cavenagh et al., 1996; Gaschet
and Hsu, 1999; Radhakrishna and Donaldson, 1997; Caumont et al., 1998,
2000; D’Souza-Schorey et al., 1995; Yang et al., 1998; Al-Awar, et al.,
2000) and cycles between an intracellular compartment and the plasma
membrane as a function of its GTP/GDP binding or agonist stimulation
(Gaschet and Hsu, 1999; Radhakrishna and Donaldson, 1997; Caumont et
al., 1998, 2000; Al-Awar, et al., 2000). Stimulation of chromaffin cells with
nicotine causes activation of PLD, and there is a translocation of Arf6 to the
plasma membranes that correlates with an enhancement of GTPγS-
stimulated PLD activity in this fraction (Caumont et al., 1998). In addition,
a myristoylated peptide corresponding to residues 2-13 in Arf6 inhibited
Ca2+-stimulated PLD activity in permeabilized chromaffin cells (Caumont et
al., 1998). In contrast, the corresponding peptide from Arf1 was ineffective.
The introduction of antibodies to ARNO, a brefeldin A-insensitive GEF for
Arfs 1 and 6, into permeabilized chromaffin cells also inhibited Ca2+-
stimulated PLD activity. These findings indicate a role for Arf6 in the
regulation of PLD in chromaffin cells, and this has been related to the
regulation of exocytosis in these cells (Caumont et al., 1998, 2000).
 PHOSPHOLIPASE D, ARFAPTINS AND ARFOPHILIN 227
5

Translocation of Arf1 to a membrane fraction in response to GTPγS, FMLP

or PMA has been reported in HL-60 cells (Fensome et al., 1998; Houle et al.,
1995; Martin et al., 1996), although the nature of the membrane fraction was
not defined. Arf1-stimulated PLD activity has been localized to both the
plasma membrane and endomembranes in subcellular fractionation studies in
HL-60 cells (Whatmore et al., 1996). Thus Arf1 may also be involved in the
regulation of PLD in these cells.
As alluded to earlier, Arf may indirectly regulate PLD activity in vivo.
This is through its effects on the synthesis of PI(4,5)P2, which is a major
regulator of this phospholipase. Phosphatidylinositol phosphate kinase, the
enzyme that synthesizes PI(4,5)P2 from PIP, exists in multiple forms in
mammalian cells (Anderson et al., 1999). Thus Type I comprises several 5-
kinase isoforms that convert PI 4-P to PI(4,5)P2, and Type II comprises
several 4-kinase isoforms that convert PI 5-P to PI(4,5)P2. Members of the
Rho family of GTPases have been demonstrated to activate Type I PIP
kinase (Exton, 2002), and recent work has shown that Arfs 1 and 6 can also
activate this kinase (Martin et al., 1996; Honda et al., 1999; Godi et al.,
1999; Jones et al., 2000; Skippen et al., 2002). As PI(4,5)P2 is absolutely
required for PLD activity, this could represent a cellular mechanism for
activation of PLD by Arf. This idea is supported by the co-localization of
Type I PIP kinase with PLD2 in pulmonary artery endothelial cells co-
expressing both enzymes (Jones et al., 2000) and the demonstration that both
PLD1 and PLD2 interact with the Type I kinase in vivo, as shown by
immunoprecipitation studies (Jones et al., 2000). Arf6 has also been shown
to co-localize with Type I PIP kinase and PI(4,5)P2 in HeLa cells (Honda et
al., 1999; Brown et al., 2001).

1.3 Roles of Arf-stimulated PLD in Golgi Traffic and

Exocytosis

A critical issue is the role of Arf-regulated PLD in membrane traffic. This

remains a controversial area. Early work showing that Arf could activate
PLD and that Golgi preparations possessed Arf-stimulable PLD activity
suggested that PLD was involved in traffic at the Golgi (Ktistakis et al.,
1995; Kahn et al., 1993). This was supported by the observation that, in a
cell line with high constitutive PLD activity, Arf was not required for the
formation of COPI-coated vesicles (Ktistakis et al., 1996). Furthermore,
addition of ethanol, which reduces phosphatidic acid (PA) formation by
PLD, inhibited the formation of coated vesicles from Golgi, whereas
addition of PLD to Golgi membranes induced COPI binding and vesicle
formation (Ktistakis et al., 1996). Ethanol was also found to inhibit
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6 EXTON

transport between the endoplasmic reticulum and Golgi, and the block could
be reversed by addition of PA (Bi et al., 1997).
In permeabilized GH3 pituitary cells, purified PLD was observed to
stimulate secretory vesicle budding from the trans Golgi, whereas this was
inhibited by primary butanol (Chen et al., 1997). Generation of PA through
the combined action of phospholipase C and diacylglycerol kinase also
stimulated vesicle budding (Siddhanta and Shields, 1998). Treatment of
GH3 cells with primary butanol reduced transport from the endoplasmic
reticulum and the Golgi, and also inhibited hormone secretion (Siddhanta et
al., 2000). There was an associated decrease in PI(4,5)P2 synthesis by Golgi
membranes and disassembly and fragmentation of the Golgi (Siddhanta et
al., 2000). Exogenous PLD has also been shown to recruit AP-2 adaptors
onto plasma membranes and thus mimic the effects of GTPγS plus Arf
(West et al., 1997). This suggests a role for PLD in vesicle traffic at other
cellular sites. Although it has been assumed that the effects of PLD on
vesicle traffic are due to the effects of PA per se, they may be due to
changes in PI(4,5)P2. PA has been reported to stimulate Type I PIP kinase
(Skippen et al., 2002; Jenkins et al., 1994; Moritz et al., 1992; Ren et al.,
1996) and there is much evidence that PI(4,5)P2 is required for traffic at
several cellular sites (Skippen et al., 2002; Martin, 1998,, 2001; Simonsen et
al., 2001).
There is other evidence implicating a role for PLD in exocytosis. This
has mainly relied on the ability of primary alcohols to inhibit secretion from
many cell types (for references, see Cockcroft, 1996, and, Jones et al., 1999).
However, it must be recognized that these reagents may not be specific for
PLD when used in intact cells. However, exogenous PLD stimulates
secretion in many cell types (Cockcroft, 1996) and permeabilization of cells
to release Arf decreases GTPγS-stimulated PLD activity and exocytosis,
both of which can be restored by adding Arf (Jones et al., 1999). Additional
evidence for a role of PLD in exocytosis comes from studies of the effects of
overexpressed wild type and catalytically inactive PLD1 in PC12 cells.
These active and inactive enzymes stimulated and inhibited secretion,
respectively (Vitale et al., 2001). On the other hand, wild type and inactive
PLD2 were without effect. Ceramide is known to inhibit the stimulation of
PLD by PMA and agonists in several cell types (Exton, 2002) and Arf may
possibly be involved. Addition of C2 ceramide to HL-60 cells inhibits
membrane translocation of Arf and PLD activation induced by FMLP
(Abousalham et al., 1997). In chromaffin cells, C2 ceramide inhibits both
PLD activity and catecholamine release (Vitale et al., 2001).
In contrast to the preceding evidence supporting a role of Arf-stimulated
PLD in membrane traffic at the Golgi and in exocytosis, there are findings
that do not support this hypothesis. For example, measurements of PA
 PHOSPHOLIPASE D, ARFAPTINS AND ARFOPHILIN 229
7

levels in Golgi during budding of COPI coated vesicles indicated that this
lipid declined rather than increased (Abousalham et al., 1997). Another
study examined the effects of mutations in Arf3 on PLD activity and the
recruitment of COPI to Golgi membranes (Kuai et al., 2000) and observed a
poor correlation. Similarly, an N-terminally deleted form of Arf1 was found
to be without effect on Arf-stimulated PLD activity, but competed with Arf1
to prevent COPI binding to Golgi membranes (Jones et al., 1999). In
another study, exogenous PLD was observed to recruit AP-2 adaptors onto
the plasma membrane, but was unable to recruit AP-1 adaptors onto the trans
Golgi network (West et al., 1997). In summary, it seems very unlikely that
PLD alone mediates the effects of Arf on vesicle traffic at various sites.
However, it probably plays a supporting role through the generation of
PI(4,5)P2 and perhaps other lipids.

2. ARFAPTINS

2.1 Properties of Arfaptins 1 and 2

Arfaptins 1 and 2 were identified as Arf-binding proteins by the yeast two-

hybrid system using constitutively active Arf3 as bait and an HL-60 cDNA
library (Kanoh et al., 1997). The interactions were not seen with wild type
Arf3 and were independent of the myristoylation state of the Arf.
Sequencing revealed that the arfaptins were 60% identical at the amino acid
level and 81% homologous, with the greatest homology being in the C-
terminal half (Kanoh et al., 1997). Northern blot analysis revealed that the
mRNAs for both Arfaptins were ubiquitously expressed, with high levels in
liver, pancreas and placenta (Kanoh et al., 1997). The proteins bound
predominantly to Class I Arfs compared with Class II and III Arfs, and only
when the Arfs were in the GTP-bound form. Arfaptin 1 was recruited to
Golgi membranes in vitro by GTPγS and this was dependent on Arf (Kanoh
et al., 1997). Arfaptin 1 also localized to Golgi when transfected into COS
7cells. Arfaptin 2 was not examined in these experiments.
The function of Arfaptin 1 was subsequently tested by examining its
effects on the activation of PLD and cholera toxin ADP-ribosyltransferase
by Arfs (Tsai et al., 1998). Arfaptin 1 inhibited the activation of both
enzymes by all classes of Arf in a dose-dependent manner. The effects on
Class II and III Arfs (Arfs 5 and 6) were less than those on Class I Arfs (Tsai
et al., 1998). In agreement with previous results (Kanoh et al., 1997),
myristoylation of Arf did not alter the inhibitory actions of Arfaptin 1.
When tested against Arf1 in which the N-terminal 13 residues were replaced
by the corresponding sequence for Arl1, Arfaptin 1 was unable to inhibit the
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action of this Arf mutant on the cholera toxin activity (Tsai et al., 1998).
The corresponding experiments could not be conducted with PLD, because
the Arf mutant was unable to activate this enzyme. These data imply the
existence of an Arfaptin 1 binding site at the N-terminus of Arf1. In studies
of guanine nucleotide binding and release from Arf3, Arfaptin 1 was found
to increase the binding of GTPγS and GDP, but the effects were not large
and may have been due to stabilization of Arf (Tsai et al., 1998). The
release of GTPγS from Arf3 was also only slightly altered by Arfaptin 1.
When tested in the presence of a guanine nucleotide exchange factor, only
slight effects of Arfaptin 1 were observed on GTPγS or GDP binding (Tsai
et al., 1998). These results indicate that Arfaptin 1 has little intrinsic GEF
activity and does not modify the action of GEFs on Arf. The possible effects
of Arfaptin 1 on the inactivation of Arf by GTPase-activating proteins were
not examined.
In binding studies of the interaction of Arfaptin 1 with Arf3, deletional
mutagenesis of Arfaptin 1 indicated that sequences in both the N-and C-
termini were involved (Williger et al., 1999). Both were required for the
inhibitory effect of Arfaptin 1 on Arf3 stimulation of PLD1 (Williger et al.,
1999). Arfaptin 1 was also shown to associate with a 'high speed' fraction
containing Golgi membranes in a GTPγS- and Arf3-dependent manner
(Williger et al., 1999). The functional consequences of the effects of
Arfaptin 1 on Arf interactions with PLD in Golgi were explored in NIH 3T3
cells overexpressing Arfaptin 1 (Williger et al., 1999). In these cells, the
activation of PLD by PMA was suppressed and there was a slowing of
vesicular traffic through the Golgi, as measured by the glycosylation of
vesicular stomatitis virus (Williger et al., 1999). Consistent with an earlier
report (Tsai et al., 1998), Arfaptin inhibited the effect of Arf3 on PLD
activity in Golgi membranes.

2.2 Functions of POR1, a Truncated Form of Arfaptin2

An intriguing development in knowledge of the function of Arfaptin 2 came

when an N-terminally truncated form (POR1) was identified in a yeast two-
hybrid screening of a Jurkat T cell cDNA library using active Rac1 as bait
(Van Aelst et al., 1996). Its sequence was identical to that of Arfaptin 2,
except that it lacked the N-terminal 38 amino acids (Van Aelst et al., 1996).
In studies of the interactions between POR1 and members of the Rho family,
the Arfaptin 2 truncation mutant was found to be selective for Rac1, but
surprisingly, the interaction with wild type Rac1 was as strong as with
constitutively active Rac1-V12. POR1 was localized to membrane ruffles in
cells expressing Rac1-V12, and it was able to synergize with Ras-V12 (but
not Rac1-V12) in the induction of membrane ruffles (Van Aelst et al., 1996).
 PHOSPHOLIPASE D, ARFAPTINS AND ARFOPHILIN 231
9

Furthermore, N-terminally deleted POR1 was able to inhibit the effect of

Rac1 to cause ruffling. In an extension of this work, mutants of Rac1 were
found to interact differentially with POR1 and the protein kinase PAK1
(Joneson et al., 1996). PAK1 is implicated in the activation of the c-Jun N-
terminal kinase (JNK) pathway by Rho family proteins, but not in the actin
cytoskeleton changes (Lamarche et al., 1996). These results implicate POR1
in the actin skeleton rearrangements induced by Rac1. Future experiments
directed toward defining the impact of the N-terminal truncation of
Arfaptin2 on localization and function, as well as comparisons between the
localization and cellular roles for Arfaptin 1 and 2 will help define the
functions of this family of Arf effectors.

2.3 Interactions between Arfaptin 2/POR1 and Arfs and

Rac1

In a later study of the actin cytoskeleton rearrangements induced by

constitutively active Arf6-L67, it was found that the changes were different
from those induced by Rac1-V12 and were not observed with Arf1-L71
(D’Souza-Schorey et al., 1997). The actin rearrangements caused by Arf6-
L67 were inhibited by N-terminally and C-terminally deleted POR1. Co-
expression of wild type Arf6 and POR1 synergistically induced actin
polymerization at the cell periphery (D’Souza-Schorey et al., 1997). The
effects of Arf6-L67 were not blocked by dominant negative Rac1-N17, and
dominant negative Arf6-N27 did not modify Rac1-V12-induced
lamellipodia formation (D’Souza-Schorey et al., 1997). These data indicated
that Arf6 and Rac1 were not in the same pathway to regulate cytoskeletal
architecture, but operated in parallel. Further work showed that POR1
bound directly to the GTP-bound form of Arf6, and also to Arfs 1 and 3
(D’Souza-Schorey et al., 1997). In summary, these results provided
evidence that Arfaptin 2/POR1 mediated the effects of Arf6 on the actin
cytoskeleton, but its role in the actions of Rac1 was unclear.
The role of Arfaptin 1 as a possible mediator of both Arf6 and Rac1
actions was explored further in two studies. The first involved a
crystallographic study of the interaction of Arfaptin 2 and Rac1 (Tarricone et
al., 2001). An N-terminally truncated form of the Arfaptin crystallized as a
dimer encompassing the entire predicted coiled-coil region and dimerization
involved packing of 2 or 3 helices from each monomer (Tarricone et al.,
2001). Binding measurements utilizing isothermal titration calorimetry
indicated that Rac1•GMPPNP and Rac1•GDP bound to Arfaptin 2 with
similar KDs, and with 1:1 stoichiometry per dimer. Efforts to crystallize
Arfaptin 2 in complex with Rac1•GMPPNP were not successful since the
GMPPNP was hydrolyzed during the crystallization leading to a crystal with
232
10 EXTON

Rac1•GDP, i.e., Rac1 bound to GDP with a β-amidophosphate group. The

structure showed that Rac1 sat at the midpoint of the Arfaptin 2 dimer and
that the interaction involved both the switch I and II regions of the GTPase
and helix A of the Arfaptin (Tarricone et al., 2001). It also revealed why
Arfaptin 2 is selective for Rac1 rather than Rho or Cdc42.
Crystallization of Rac1-L61•GMPPNP in complex with Arfaptin 2 was
also undertaken. This GTPase-deficient form of Rac1 did not hydrolyze
GMPPNP. However, the structure of the complex was very similar to that of
Rac1•GDP-Arfaptin 2 i.e. there was little difference in the two switch
regions (Tarricone et al., 2001). In particular, Thr35 of Rac1 did not interact
with nucleotide-associated Mg2+, as in other Rac1•GTP-containing
complexes, but interacted with Arfaptin 2. This is characteristic of GDP-
bound GTPases (Tarricone et al., 2001). It seems that, in contrast to other
GTPases of the Ras superfamily, the switch regions of Rac and other Rho
proteins are able to adopt similar conformations in the GDP- and GTP-
bound forms in complex with certain binding proteins (Tarricone et al.,
2001).
The binding of Arfaptin 2 to Arfs 1 and 6 was also studied (Tarricone et
al., 2001). In contrast to the findings with Rac, and in agreement with earlier
findings (Kanoh et al., 1997) Arfaptin 2 bound only to the GTP-liganded
forms of these GTPases, and binding to Arf6 was weaker than to Arf1.
Binding of Rac1 and Arf to Arfaptin 2 was mutually exclusive, indicating
that they share part of the same binding site. This raises the possibility that
activated Arf could displace Rac1 from its binding to Arfaptin 2.
Subsequent activation of Rac1 could lead to signaling through this GTPase.
In support of this idea (Tarricone et al., 2001), overexpression of EFA6, a
GEF for Arf6, was found to lead to Rac activation and membrane ruffling
(Franco et al., 1999).
In another study of the interaction of Arfaptin 2 with Arfs and Rac1, it
was found that constitutively active Arf1-L71, Arf5-L71 and Arf6-L67 all
interacted with Arfaptin 2 in the yeast two-hybrid system (Shin and Exton,
2001), whereas the dominant negative forms of these GTPases did not. On
the other hand, active Rac1-L61 did not interact with Arfaptin 2, whereas
wild type and dominant negative Rac-N17 did (Shin and Exton, 2001). Pull-
down experiments with GST-Arfaptin 2 also indicated that active Rac1-L61
was not bound, whereas the active forms of Arfs 1, 5 and 6 were. Finally,
there was significant binding of Rac1 to the Arfaptin in the presence of
GDPβS, but little in the presence of GTPγS, and the reverse was true for the
Arfs (Shin and Exton, 2001). The differential interactions of Arfaptin 2 with
the GTP-bound versus GDP-bound forms of Rac1 and Arf6 would provide
for novel forms of communication between these two GTPases, as indicated
above.
 PHOSPHOLIPASE D, ARFAPTINS AND ARFOPHILIN 233
11

In a structural study of the interaction of Arfaptin 2 with Rac1 (Cherfils,

2001), it was found that the dimeric helical domain of Arfaptin 2 could be
superimposed on a monomeric helical domain of Tiam1, which is a GEF for
Rac. This structural mimicry was utilized to explore the mechanism of the
GEF reaction, in which there is low affinity binding of the GDP-bound
GTPase to the GEF, which converts to a high affinity complex with
nucleotide-free GTPase. Entry of GTP then induces a low affinity complex
leading to its dissociation. The Rac1•GDP complex with Arfaptin 2 was
thus considered to mimic the first step of a GEF reaction.

2.4 PICK1 Proteins are Homologous to Arfaptins

Another interesting finding indicating similarity between Arfaptins and other

proteins potentially involved in signaling is the observation that the PICK1
proteins have sequence homology with Arfaptins 1 and 2 (Takeya et al.,
2000). The PICK1 proteins are PDZ domain-containing proteins that were
cloned in a yeast two-hybrid screen using PKCα as bait (Staudinger et al.,
1995). However, PICK1 does not interact with Arfs. On the other hand, its
PDZ domain binds to GTP-bound Arfs 1 and 3, but not Arfs 5 and 6, and not
when Arfs 1 and 3 are GDP-bound. The association is lost if the PDZ
domain is mutated and if the C-terminus of Arf1 is deleted (Staudinger et al.,
1995). It was suggested that PICK1 might participate in Arf1- and 3-
mediated cellular processes.

2.5 Possible Role of Arfaptin2 in Huntington’s Disease

A possible role of Arfaptin 2 in Huntington’s disease is suggested by a

recent report (Peters et al., 2002). This inherited neurodegenerative disease
is associated with nuclear and cytosolic aggregates of the protein huntingtin.
Expression of Arfaptin 2 in PC12 cells resulted in deposition of dense
aggregates of this protein in circular nuclear structures and also around the
centriole (Peters et al., 2002). Because the pattern resembled that seen with
mutant huntingtin, the distribution of endogenous huntingtin was examined,
and it was found to co-localize with expressed Arfaptin 2 in the nuclear
region. Introduction of GFP-tagged mutant huntingtin also showed
colocalization with Arfaptin 2 (Peters et al., 2002). Deletion mutants of
Arfaptin 2 were then tested. A C-terminally truncated form promoted
aggregate function as well as the wild-type protein, but an N-terminally
truncated form had greatly impaired ability, and huntingtin was diffusely
present in the cytoplasm. Immunoprecipitation experiments also revealed
that huntingtin and Arfaptin 2 were able to interact in vitro (Peters et al.,
2002). Involvement of the ubiquitin-proteasome system in the effects of
234
12 EXTON

Arfaptin 2 was suggested by the effects of lactacystin, an inhibitor of this

system. In wild-type cells, this inhibitor enhanced huntingtin-induced
aggregation, whereas in cells transfected with Arfaptin 2, aggregation was
minimal (Peters et al., 2002). It was suggested that Arfaptin 2 promotes
huntingtin aggregation by inhibiting ubiquitin-proteasome function. In
transgenic mice expressing huntingtin, increased levels of Arfaptin 2 were
found in striatum, cortex and cerebellum, and overlapped with huntingtin
aggregates (Peters et al., 2002). These data suggest that Arfaptin 2 can
regulate huntingtin aggregates, possibly by inhibiting proteasome activity.

3. ARFOPHILIN

Arfophilin is an Arf-binding protein discovered in a yeast two-hybrid screen

of a kidney cDNA library using Arf5 as bait (Shin et al., 1999). This 82 kDa
protein was initially shown to be specific for Class II Arfs (Arfs 4 and 5) and
only when these were in the GTP-liganded form. Northern analysis showed
Arfophilin to be expressed at high levels in heart and skeletal muscle (Shin
et al., 1999). The Arf binding site was determined by deletional analysis to
be located in the C-terminus (amino acids 612-756). This contains a coiled
coil structure, but this alone is not enough for Arf5 binding (Shin et al.,
1999). Through the use of Arf5/Arf3 chimeras the binding site for
Arfophilin in Arf5 was localized to the N-terminal 17 amino acids. Because
this sequence is different in Arf6 it was assumed incorrectly that Class III
Arfs would not bind to Arfophilin (see below). Arfophilin could be
translocated to the membrane fraction in CHO-K1 cell lysates in response to
GTPγS or Arf5-L71, suggesting that active Arf5 and arfophilin could
associate in vivo.
More detailed studies of the interactions of Arfophilin with different
classes of Arf confirmed that it did not bind to Arf1, but surprisingly bound
to Arf6 as well as Arf5 (Shin et al., 2001). Using chimeric constructs, the
Arf6 binding site was localized to a sequence towards the N-terminus
(amino acids 37-80). Surprisingly, this is different from the C-terminal
sequence (amino acids 612-756) involved in binding Arf5 (Shin et al., 1999).
Overexpression of Arfophilin in CHO-K1 cells led to its localization to the
perinuclear region, including Golgi, presumably in association with Arf5.
There was also some localization to the periphery of the cell and this may
involve association with Arf6. The function of Arfophilin remains
unknown. However, there is evidence that it is overexpressed in human
pancreatic cancer (Shin et al., 2001).
 PHOSPHOLIPASE D, ARFAPTINS AND ARFOPHILIN 235
13

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Chapter 12
COAT PROTEINS

ANNETTE BOMAN1 AND TOMMY NILSSON2*

1
Department of Biochemistry and Molecular Biology, University of Minnesota School of
Medicine Duluth, Duluth, USA; 2European Molecular Biology Laboratory Heidelberg,
Heidelberg, Germany; * contact person for this chapter

Abstract: A major cellular function of Arfs is the regulation of membrane traffic. Many
studies, using several independent approaches, have shown that activated Arfs
directly recruit coat complexes to membranes. These coats sort cargo and
facilitate vesicle formation. Three classes of Arf-dependent coat proteins or
complexes have been described so far, including COPs, adaptins, and GGAs.
This chapter focuses on aspects of Arf-dependent coat proteins and complexes.
We will discuss the binding between Arfs and coats, with an emphasis on
COPI and GGAs, as well as the localization and function of each coat.

1. ARF AND MEMBRANE TRAFFIC:

BACKGROUND

A long-standing question in the membrane traffic field is how Arf proteins

regulate vesicle formation. Coat recruitment is clearly downstream of Arf
activation. The cycle of coat recruitment and vesicle formation is intimately
tied to the GTP cycle of Arf. Upon activation, Arf•GTP recruits coat
proteins to a membrane surface. Blocking nucleotide exchange causes rapid
dissociation of Arf and coats from membranes. Conversely, preventing GTP
hydrolysis stabilizes Arf and coats on membranes and/or vesicles.
Blocking the GTP cycle of Arf with drugs (e.g., brefeldin A (BFA)),
slowly hydrolyzable GTP analogs (e.g., GTPγS), Arf mutants, or an Arf
peptide affects many transport pathways: ER-Golgi transport, intra-Golgi
transport, nuclear envelope assembly, endosome fusion and endocytosis, and
synaptic vesicle fusion (Balch et al., 1992; Boman et al., 1992; Faundez et
al., 1997; Lenhard et al., 1992; Taylor et al., 1992). These experiments
showed that Arf activity is critical at many steps of the biosynthetic and
241
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endocytic pathways. Interestingly, a distinct coat protein or protein complex

is associated with each distinct transport pathway. A major question is
whether Arf recruits coats directly or indirectly, for example via lipid
modification. The direct interaction hypothesis has gained support in recent
years following reports of direct interaction between Arf and many of these
coats (Austin et al., 2002; Austin et al., 2000; Boman et al., 2000;
Dell'Angelica et al., 2000; Zhao et al., 1999). The major Arf-dependent coat
complexes are described here.

2. ARF-DEPENDENT COAT COMPLEXES

There are currently three different classes of Arf-dependent adaptors that act
in the biogenesis of vesicles and sorting of their cargo: the tetrameric
adaptins (APs), heptameric coatomers (COPs), and monomeric GGAs
(Golgi-localized, γ-adaptin ear homology domain, Arf-binding). Each class
has more than one member in mammals with at least four APs, two COP
complexes, and three GGAs.

2.1 Adaptor complexes and clathrin

Clathrin-coated vesicles were first discovered by electron microscopy in the

mid 1960s. An electron-dense, regular lattice was observed to coat buds and
vesicles formed from the plasma membrane (Roth and Porter, 1964) and the
trans-Golgi network (Friend and Farquhar, 1967). Assembly proteins (APs)
or adaptins were found to recruit clathrin onto sites of vesicle budding and
facilitate clathrin assembly into a triskelion. To date, four adaptin
complexes have been described in mammals (AP-1 through AP-4) and three
are present in S. cerevisiae (AP-1 to AP-3). These adaptin complexes are
each composed of 4 subunits with conserved structures: 2 large subunits
(100 kDa; γ and β1 in AP-1, α and β2 in AP-2, δ and β3 in AP-3, ε and β4
in AP-4), one medium (50 kDa; µ1-µ4), and one small (20 kDa; σ1-σ4).
The heterotetramers assemble into body, hinge, and ear domains. AP-1, AP-
2, and AP-4 bind clathrin; it is yet unclear if AP-3 binds clathrin (Simpson et
al., 1996; Dell'Angelica et al., 1998). The AP complexes each bind the
cytoplasmic tails of transmembrane cargo, but function in distinct pathways,
as described below. Adaptin complexes AP-1, AP-3, and AP-4 were found
to be downstream of Arf activation due to their BFA sensitivity and GTPγS-
stimulated recruitment (Boehm et al., 2001; Ooi et al., 1998; Stamnes and
Rothman, 1993; Traub et al., 1993). Recent studies show cross-linking of
AP-1 and AP-3 to Arf (see below).
 COAT PROTEINS 2433

AP-2 appears to bind membranes independently of Arf. First, membrane

association of AP-2 is insensitive to BFA (Robinson and Kreis, 1992).
Second, expression of Arf6 mutants, which dramatically perturbs the plasma
membrane, has no effect on AP-2 localization (Radhakrishna et al., 1996).
Third, AP-2 was not cross-linked to Arf under conditions where AP-1 and
AP-3 were cross-linked (Austin et al., 2002). However, AP-2 recruitment to
plasma membranes is dependent upon PLD activation (West et al., 1997),
suggesting that an Arf, most likely Arf6, may play a role in the regulation of
the budding of AP-2 coated vesicles. Such a role would be indirect and
distinct from that played by Arfs in recruitment of other coat proteins.

2.2 Coatomer complexes

Like AP/clathrin-coated vesicles, COPI coated vesicles can be visualized as

electron dense coats by electron microscopy (Orci et al., 1986). Assays
designed to reconstitute ER-Golgi and intra-Golgi transport each required
GTP and were blocked by GTPγS (Beckers and Balch, 1989). Coated
vesicles accumulated in the presence of GTPγS (Orci et al., 1989), allowing
their visualization by electron microscopy and later purification. These
Golgi-derived, non-clathrin-coated vesicles (Waters et al., 1991) were highly
enriched for seven proteins, defined as coatomer or COPI, that was later also
found as a stable, cytosolic complex. Arf was also abundant on these
vesicles when purified from cell-free, intra-Golgi transport assays containing
GTPγS (Serafini et al., 1991). The first subunit to be cloned (Duden et al.,
1991), β-COP, has been used extensively as a marker for COPI, due to the
availability of high quality antisera. Binding of β-COP to membranes was
sensitive to BFA; within 1 minute after BFA addition, β−COP dissociated
from the Golgi to the cytosol (Donaldson et al., 1991). This was similar to
the time course of Arf dissociation from membranes, suggesting that β−COP
was downstream of Arf activation. Though genetic studies in yeast (Stearns
et al., 1990) and biochemical studies involving reconstitution of vesicle
budding revealed functional ties between Arf and subunits of COPI, it
wasn’t until 1999 that direct interactions between Arf and COPI were
demonstrated, using cross linking techniques (see below).
A related coat complex, named COPII, functions in ER exit (Barlowe et
al., 1994). COPII comprises five subunits: Sec23 and Sec24 (which form a
stable dimer), Sec13 and Sec31 (which form a stable dimer), and Sar1, a
GTPase related to Arf (Barlowe et al., 1993). Membrane recruitment of the
COPII subunits requires Sar1 rather than Arf.
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4 BOMAN AND NILSSON

2.3 GGAs

The GGAs are a recently identified family of Arf effectors that are
monomeric, clathrin-binding adaptors (for review, see Boman, 2001). Three
GGAs are present in mammalian cells: GGA1, GGA2, and GGA3. Two are
present in S. cerevisiae (Gga1p and Gga2p), and D. melanogaster, C.
elegans, and S. pombe genomes each have at least one GGA gene. GGA
proteins have four domains (Figure 1) that possess many or all the functions

N C
VHS GAT hinge ear
Figure 1. Schematic of GGA domain structure.

of AP complexes. These domains were named based on homology with

other proteins. The VHS domain binds to cargo proteins, the GAT (GGA
and Tom1) domain binds Arf, the hinge domain binds clathrin, and the ear
domain binds accessory proteins. The monomeric nature of GGA proteins
has allowed rapid testing of the model that Arf directly regulates vesicle
coating.
GGAs were originally identified through their direct interaction with Arf
proteins (Boman et al., 2000). This interaction has been studied both in vitro
and in vivo, and all data indicate that GGAs are direct effectors of Arf. All
three human GGA proteins and both yeast Gga proteins interact with
activated Arf in two-hybrid assays. Purified GST-GGA proteins
(mammalian and yeast) interact specifically in vitro with Arf•GTP, but not
Arf•GDP, which confirms the two-hybrid results (Boman et al., 2000;
Zhdankina et al., 2001). These interactions do not require membranes or
cargo. However, binding to Arf may be enhanced by cargo or other protein
interactions; each domain of yeast Ggas contributes to Golgi localization and
function (Boman et al., 2002).
Like most Arf effectors, purified GST-GGA1 stabilizes and increases the
amount of the GTP-bound form of Arf in in vitro nucleotide binding assays
(Zhu et al., 2000). The rate of GTP-binding to Arf is not affected; hence
GGA proteins are not exchange factors. GGAs have no GTPase-activating
protein (GAP) activity (Boman et al., 2000; Puertollano et al., 2001); nor do
they enhance the GAP activity of Arf GAP1 (Puertollano et al., 2001).
Rather, GGAs can compete with Arf-GAPs for binding to Arf (Puertollano
et al., 2001). In the presence of GGAs, therefore, GTP hydrolysis on Arf is
slowed. Arf has no intrinsic GTPase activity. The dual effects of GGAs to
 COAT PROTEINS 2455

stabilize Arf•GTP and to reduce GAP-dependent GTP hydrolysis will likely

act synergistically in vivo.
Several in vivo studies have shown that human GGAs are Arf effectors.
First, stable expression of a dominant activating mutant of Arf, Arf1-L71, in
NRK cells caused dramatic expansion of the Golgi lumen through an
unknown effector (Zhang et al., 1994). Overexpression of GGA1 in these
cells prevented the Arf1-L71-induced expansion of the Golgi apparatus
(Boman et al., 2000), indicating a direct interaction between GGA1 and Arf
in vivo and a function distinct from Golgi expansion. Second, an increased
amount of Arf is associated with Golgi membranes in cells overexpressing
GGA1, as visualized by immunofluorescence (Zhu et al., 2000). Similarly,
overexpression of GGA3 slows the BFA-induced dissociation of Arf1 from
Golgi membranes (Puertollano et al., 2001). These results show that the
membrane-associated, GTP-bound form of Arf is stabilized in vivo, as
expected from the in vitro experiments described above. Finally, a number
of experiments reveal that the localization of GGA proteins at the Golgi is
due to interaction with Arf. First, treatment with BFA causes rapid
translocation of GGA proteins to the cytosol in a time frame
indistinguishable from that of Arf itself (Boman et al., 2000; Dell'Angelica
et al., 2000; Hirst et al., 2000; Poussu et al., 2000). Second, the interaction
between the GAT domain of GGA proteins and Arf is strong enough to drive
a reporter construct (GFP) onto the Golgi, even in the absence of the VHS,
hinge and ear domains (Dell'Angelica et al., 2000). Third, and most
convincingly, point mutations within the Arf-binding domain that cause loss
of Arf interaction also cause loss of Golgi localization (Boman et al., 2002;
Puertollano et al., 2001; Takatsu et al., 2002). Together, these data suggest
that Arf•GTP recruits GGAs from the cytosol onto late Golgi membranes by
interacting with the GAT domain.
Arfs localize to many organelles and can selectively recruit one or more
coat complexes onto a target membrane. Yet each Arf-dependent coat has a
distinct localization and some organelles are the targets of multiple coats
(Figure 2). It is not known how this selectivity is achieved. In two-hybrid
and in vitro binding experiments, each of the human Arfs bind equally well
to GGA proteins (Boman et al., 2000). Similarly, each Arf can recruit COPI
and crosslink to AP-1 in vitro (Austin et al., 2002). These data suggest that
Arf isoform specificity does not account for the specific localization of
coats. Perhaps other proteins, such as cargo, stabilize the interaction
between Arf and coat proteins at the target membrane, whereas interaction at
other locations might be transient. A role for Arf GAP(s) in timing and
proofreading of the Arf-coat-cargo complex has been proposed and is
discussed in more detail in Chapters 7-9. This section summarizes the
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6 BOMAN AND NILSSON

current understanding of determinants of localization and traffic of each

coat, as diagrammed in Figure 2.

Figure 2. Sites of action of each coat protein. COPs function in the early secretory pathway,
mediating transport between the ER, cis-Golgi network (CGN), and Golgi. APs and GGAs
function in later pathways, primarily between TGN, endosomes, and plasma membrane.
However, the specific pathways mediated by each coat are still being debated.

3. LOCALIZATION AND TRANSPORT PATHWAYS

OF EACH COAT

3.1 Adaptin complexes

Each of the adaptin complexes aids in the recruitment of clathrin to nascent

vesicles, though binding of clathrin to AP-3 is still contentious (see Chapter
 COAT PROTEINS 2477

13). Each adaptin functions at a distinct location and in distinct pathways.

AP-2, which appears to be either Arf-independent or indirectly subject to Arf
regulation, is the most abundant in many cell types and functions at the
plasma membrane (for review see Hirst and Robinson, 1998). AP-1 is
localized predominantly to the trans-Golgi network (TGN), endosomes, and
small vesicles (Seaman et al., 1996). It is still unclear precisely which step
(or steps) are mediated by AP-1. AP-1 binds to the cytoplasmic tail of both
the cation-dependent and cation-independent mannose 6-phosphate receptors
(M6PR). Two leucine residues in the M6PR tail and other cargo proteins
define a dileucine motif and are essential for AP binding. In yeast, deletion
of AP-1 has no known phenotype on its own, but has synthetic defects when
combined with a clathrin temperature sensitive mutant or with GGA
deletions (Costaguta et al., 2001; Stepp et al., 1995; Yeung et al., 1999).
These results, and many others from studies using mammalian cells, suggest
that AP-1 functions from TGN to endosomes (Hirst and Robinson, 1998).
However, mice lacking the medium subunit of AP-1 accumulate M6PR at
endosomes, suggesting that AP-1 functions in retrograde transport from
endosomes to TGN (Meyer et al., 2000). AP-3 is also localized
predominantly to the TGN, but the AP-3 pathway is distinct from that of AP-
1. In both yeast and mammalian cells, AP-3 functions in a direct route from
the TGN to the lysosome (Cowles et al., 1997; Dell'Angelica et al., 1997;
Simpson et al., 1997; Stepp et al., 1997). In neurons, AP-3 also forms
synaptic vesicles from endosomes (Blumstein et al., 2001). AP-4 localizes
to the endosomal/lysosomal system, but neither the function nor the pathway
is known (Dell'Angelica et al., 1999; Hirst et al., 1999).

3.2 COPI and COPII

COPI localizes to the Golgi apparatus, and can be seen distributing mainly to
the cis side, including the cis-Golgi network (CGN) (Oprins et al., 1993).
Though COPI has been extensively studied, the role of this coat complex is
still being debated. The debate can be divided into two categories: one
debating the very existence of COPI (and COPII) as a coat for vesicles, the
other what type of cargo goes into COPI vesicles. Though each issue is far
from settled, there is good biochemical, morphological, and functional
evidence in support of COPI vesicles. However, COPI is also implicated in
other processes such as tubulation of membranes (e.g., such as that seen
upon removal of the coat) and regulation (with Arf) of lipid modifying
enzymes, and the sorting process itself. The issue of cargo sorting is also far
from settled. For a long time, it was assumed that anterograde cargo was the
main cargo of COPI vesicles. There is now growing evidence that COPI
vesicles recycle Golgi resident proteins. Whether recycling is the sole
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8 BOMAN AND NILSSON

function of COPI vesicles remains to be seen (for a recent review, see Storrie
and Nilsson, 2002).
COPII localizes to the endoplasmic reticulum (ER) and functions in ER
exit (Barlowe et al., 1994). As with COPI, COPII components have been
implicated in sorting through direct association to cytoplasmic domains of
both newly synthesized proteins as well as to resident proteins that shuttle
between the ER and the Golgi apparatus (e.g., p24 proteins). One of the
COPII components, Sec13, has also been localized close to the cis side of
the Golgi apparatus in mammalian cells (Tang et al., 1997), raising the
possibility of a functional link between COPII and COPI. There is less
controversy regarding the role of COPII vesicles though important questions
remain. For example, large supra-molecular structures (e.g., collagen and
some viruses) too large to fit in the typical 60nm COPII vesicle can still
leave the ER. Does this occur with the help of COPII and if so, how?

3.3 GGA proteins

Several laboratories have examined the localization of GGA proteins in

mammalian cells, using indirect immunofluorescence and immunoelectron
microscopy. Endogenous GGA1, GGA2 and GGA3 localize predominantly
to the trans-Golgi region in NRK, HeLa, Cos7 and human embryonic skin
cells (Boman et al., 2000; Dell'Angelica et al., 2000; Hirst et al., 2000;
Poussu et al., 2000; Takatsu et al., 2000). Mammalian GGAs isolated
following cell lysis are soluble (Boman et al., 2000; Hirst et al., 2000),
which suggests that the membrane-associated proteins rapidly exchange with
a cytosolic pool of GGAs. The three GGAs show overlapping but subtle
differences in staining patterns. In addition to the shared TGN staining,
GGA1 shows a highly punctate pattern within the TGN and late Golgi
region, GGA2 shows diffuse and cytosolic staining, and GGA3 stains larger
puncta in the cytosol. To date, no clear functional distinctions have been
found for the GGA isoforms. Hence, it is not clear if each mediates a
distinct pathway, if each sorts different cargo into the same vesicles, or if
they are functionally redundant. In yeast, Gga proteins also localize to the
late Golgi (Boman et al., 2002), with apparent differences between Gga1p
and Gga2p. GFP-tagged Gga1p shows almost exclusively punctate, Golgi
staining, whereas Gga2p has a higher level of cytosolic staining. Yeast Ggas
also fractionate predominantly with membranes rather than cytosol, even in
the absence of Arf1p (Boman et al., 2002). These data suggest that
membrane association in yeast is stabilized by non-Arf interactions to a
greater extent than in mammalian cells.
Interestingly, at least five of the coats described here localize to and
function at the TGN of mammalian cells: GGA1, GGA2, GGA3, AP-1, and
 COAT PROTEINS 2499

AP-3. Overexpression of GGA proteins can compete for AP-1 binding at

the TGN, suggesting competition for Arf or other binding sites (Puertollano
et al., 2001). In contrast, GGA overexpression does not alter COPI
localization (Boman et al., 2000). It is not known how Arf can recruit each
of these proteins to the TGN and facilitate proper sorting and vesicle
formation. Regulation of coat binding must occur, but the type and
mechanism of such regulation are not yet known.

4. INTRAMOLECULAR INTERACTIONS
BETWEEN ARFS AND COATS

Several experiments have been performed to identify the region of Arf that
binds to coats and vice versa. The similarities and differences reveal
interesting mechanistic insights into coat recruitment.

4.1 Binding site on Arf

The crystal structure of Arf contains two flexible switch regions, comparable
to switch I and II of Ras (see Chapter 2). These regions have different
conformations in the GDP-bound versus GTP-bound states, suggesting that
they are effector binding regions. Using directed and random mutagenesis
approaches, these switch regions were found to be important for coat
binding.
In a directed approach, three residues in switch I of Arf1 were mutated to
allow photo-activated cross-linking to neighboring residues. In the presence
of membranes and GTP, residues Ile46 and Ile49 were cross-linked to COPI
(Zhao et al., 1999). Under the same conditions, residue Val43 was not
specifically cross-linked (Zhao et al., 1999). Similarly, Ile46 was cross-
linked to AP-1 and AP-3 (Austin et al., 2002; Austin et al., 2000). These
data suggest that switch I is in close proximity to COPI, AP-1, and AP-3 at
membrane surfaces. Notably, the Ile46 cross-linking experiments did not
identify GGAs or AP-2 (Austin et al., 2002).
In a random approach to define effector-binding sites on human Arf3,
reverse two-hybrid screening showed that switch I and switch II are each
important for binding to different effectors (Kuai et al., 2000). Several
mutations were identified that eliminated binding to one effector yet retained
binding to other effectors. Two mutations in switch I were found to
eliminate binding to GGA1: I49T and F51Y (Kuai et al., 2000). These
mutants were later shown to fail to interact with the GAT domain of GGA3,
as expected (Puertollano et al., 2001). In contrast, several mutations in
250
10 BOMAN AND NILSSON

switch II had no effect on GGA binding. These data suggest that switch I is
essential for binding to GGA proteins. In the absence of direct binding tests
with COPI, these mutants were also tested for recruitment of COPI to Golgi
membranes. Interestingly, the switch II mutations were much more impaired
in COPI recruitment to Golgi membranes than were the switch I mutations
(Kuai et al., 2000), suggesting that switch II is essential for COPI
recruitment. This suggested that the binding sites for GGAs and COPI/APs
are distinct, though perhaps overlapping.
The binding site for Arf GAP includes switch II, as determined by x-ray
crystallography. The activity of Arf GAP1 is enhanced by a factor of at least
a thousand by COPI. However, this effect has only been observed in
solution using truncated Arf GAP1 and Arf1 devoid of its N-terminal
domain (Goldberg, 1999; Szafer et al., 2000; Szafer et al., 2001). Thus, the
possibility of a stimulatory role of coatomer is open and additional
experiments, preferably including some in vivo, are required. In contrast,
binding and activity of Arf GAP1 is reduced by GGAs (Puertollano et al.,
2001). These data suggest that COPI binds predominantly to switch I and
GGAs bind to switch II. But such a conclusion is inconsistent with the
mutational analyses. The potential differences between the cross-linking,
GAP, and mutant analyses have not yet been resolved, and will likely require
structure determinations of the protein complexes and additional data.

4.2 Binding site on coats

The binding site for Arf on each coat has been mapped to different extents,
with the most detailed analysis performed on GGA proteins. There is no
apparent homology between coats in the Arf-binding regions. Perhaps the
structures are conserved, but the linear sequence is not.
The binding site on GGA proteins was mapped by truncation analysis to
a highly conserved domain corresponding to residues 170-280 in GGA1
(Boman et al., 2000; Zhdankina et al., 2001). This region lies in a domain
that has been named the Arf-Binding Domain (ABD), GGA and TOM1
(GAT) domain, or GGA Homology (GGAH) domain (Boman et al., 2002;
Dell'Angelica et al., 2000; Takatsu et al., 2002). There appears to be
consensus to use the term GAT in the future. A cluster of residues within
this domain (180-200 of GGA1) is extremely conserved between all yeast
and human GGAs. Point mutations within this region of human and yeast
GGA proteins eliminate binding to Arf (Boman et al., 2000; Puertollano et
al., 2001; Takatsu et al., 2002). In human GGAs, these mutations abolish
membrane association, showing that Arf recruits GGAs to the late Golgi.
Interestingly, in yeast these mutations have minor effects on function and no
effect on localization, suggesting that interactions with proteins other than
 COAT PROTEINS 251
11

Arfs are more important for recruitment or stabilization at the membrane

surface (Boman et al., 2002).
Cross-linking experiments show that the binding site for Arf on COPI
lies within the β and/or γ subunits (Zhao et al., 1999). Binding sites on AP-1
and AP-3 have been mapped to the ‘body’ (Austin et al., 2002) but have not
been narrowed further.

4.3 Cargo binding and vesicle formation

The mechanisms of coat recruitment, cargo selection, vesicle formation,

timing, and coat dissociation are still unclear, though a model for Arf’s
involvement in cargo selection and vesicle formation is emerging. Some
cargoes have been defined for each coat protein. Analyses of coordinated
binding between Arf, coats, and cargoes show that Arf may play an
important role in cargo selection. This was most clearly demonstrated in the
Golgi with COPI. When GTP was used in vesicle formation assays,
accumulated vesicles contained cargo proteins; either newly synthesized
proteins (Ostermann et al., 1993) or resident proteins of the Golgi (Lanoix et
al., 1999). Addition of GTPγS or activating Arf mutants caused
accumulation of empty vesicles (Austin et al., 2002; Pepperkok et al., 2000).
This suggests that the GTP cycle of Arf is critical for sorting and packaging
of cargo into vesicles. As COPI can also interact directly with the
cytoplasmic domains of resident proteins (Cosson and Letourneur, 1994) and
perhaps stimulate Arf GAP activity, this may provide a level of regulation
and cross talk between coats and the GTP cycle on Arf. It is not yet known
if similar cargo selection mechanisms will occur at other sites of Arf action.
Also, the cargo itself has been suggested to influence Arf GAP activity,
effectively down-regulating its activity as more cargo accumulates
(Goldberg, 2000; Lanoix et al., 2001; Springer et al., 1999). This couples
coat recruitment and vesicle formation to cargo sorting ensuring that vesicles
can only form in the presence of cargo. Though an attractive scenario, more
work is needed to fully understand the details of both COPI and COPII coat
proteins and how they recruit their cargo and form vesicles.
The known cargoes for mammalian GGA proteins are transmembrane
receptors that contain an acidic-cluster dileucine motif in their cytoplasmic
tails. To date, these include M6PR, sortilin, sor-LA, LRP3, and yeast
Vps10p. Other proteins contain this motif and will likely join the ranks of
GGA-sorted cargo. The VHS domain of GGA proteins binds these motifs,
regulated by phosphorylation of the cargo tail (Doray et al., 2002; Kato et
al., 2002) and phosphorylation of GGAs at an internal regulatory site in the
hinge domain (Doray et al., 2002). The crystal structure of the VHS domain
bound to the M6PR tail has been solved by two groups (Misra et al., 2002;
252
12 BOMAN AND NILSSON

Shiba et al., 2002). These models show that 12 residues in two α helices (α6
and α8) are involved in the interaction (Shiba et al., 2002). Only three of
these residues are conserved in yeast Gga1 and Gga2, none of which make
contact with the dileucine motif. Consistent with this, human GGAs bind to
yeast Vps10p tail (Dennes et al., 2002), but yeast Ggas do not (A. Boman,
unpublished observation). These data suggest that yeast Gga proteins bind
cargo with a different motif than reported for human GGA proteins.
However, the VHS domain of yeast Gga proteins is required for localization
and function, strongly suggesting that this domain binds to cargo or other
important regulatory proteins (Boman et al., 2002). The VHS and GAT
domains of yeast Ggas work synergistically for Golgi localization,
suggesting that Arf activation will lead to efficient cargo packaging by Gga
proteins. We speculate that Arf binding to yeast or human GGAs may allow
kinases or phosphatases to act on GGA proteins, thus regulating cargo
binding.
It is still unclear how long Arf and coats remain associated with vesicles
in vivo. Based on early experiments with GTPγS, it was proposed that
hydrolysis of GTP on Arf triggered uncoating, just prior to vesicle fusion.
More recent data show that vesicles formed with GTP do not retain their
coats, suggesting that GTP hydrolysis occurs during or shortly after coat
recruitment (Lanoix et al., 1999). Unlike COP and AP complexes, GGA
proteins are not stabilized on membranes by GTPγS (A. Boman, unpublished
observations), nor are they cross-linked to Arf (Austin et al., 2002). These
data suggest that distinct mechanisms are involved in vesicle formation by
each of the different coat proteins.

5. FUTURE QUESTIONS

Many intriguing questions remain, including: What specifies coat

localization? What regulates coat binding and cargo recruitment? What are
the recognition sites on coats, and how does each bind to Arf? Do Arf GEFs
and Arf GAPs have functions in membrane traffic distinct from those of
activating or inhibiting Arf activity? The models for Arf and coat function
presented in this chapter will provide a basis for addressing each of these
questions in the future.
 COAT PROTEINS 253
13

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Chapter 13
HETEROTETRAMERIC COAT PROTEIN-ARF
INTERACTIONS

M. L. STYERS AND V. FAUNDEZ

Department of Cell Biology and Center for Neurodegenerative Diseases, Emory University
School of Medicine, Atlanta, USA

Abstract: The mechanisms by which COPI and the heterotetrameric adaptor complexes
interact with Arf are analyzed from a comparative evolutionary perspective.
Phylogenetic, biochemical, and structural analyses support a common ancestry
of the heterotetrameric adaptors and COPI. Consistent with a structural
conservation between COPI and adaptors, the functional interactions between
Arf and adaptors possess similarities with the way COPI and Arf associate.
However, these interactions have become increasingly complex and appear to
have reached a summit with the adaptor AP-2. A common theme across
different carrier-forming machines is the requirement of interacting networks
of functionally diverse Arf effectors to recruit adaptor complexes to
membranes.

1. INTRODUCTION

The exocytic and endocytic routes are organized as a series of

interdependent membrane-enclosed compartments, each one possessing its
own morphological and functional identity (Palade, 1975; Lippincott-
Schwartz et al., 2000). These compartments communicate via a continuous
flow of membrane carriers of a tubulovesicular nature. This raises the
question of how the specific protein and lipid compositions of compartments
are maintained. A steady state composition is maintained in each organelle
by controlling both the molecules that exit from it as well as the fusion of
incoming membrane carriers (Lippincott-Schwartz et al., 2000; Wickner and
Haas, 2000; Chen and Scheller, 2001). Tight control over the formation and
fusion of these carriers defines the identity of an exocytic or endocytic
organelle.
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To analyze the complex process of membrane carrier formation we will

divide the process into several steps: 1) the formation of membrane
microdomains, or ‘hot spots’, 2) cargo recognition and concentration in
discrete regions of the donor membrane, or sorting, 3) imposition of
membrane curvature and bud formation, 4) bud severing, or pinching off,
and, 5) dissociation of the budding machinery from the vesicle, or uncoating
(Springer et al., 1999). In this sequence of events, Arf GTPases and
coat/adaptor molecules play a central role in the formation of membrane
microdomains and sorting of membrane components into the nascent bud.
The roles of GTPases and coats/adaptors in sorting and vesiculation have
been explored to different extents, depending upon: 1) the coat and GTPase
involved, 2) the particular pathway analyzed, and, 3) the cell model system.
The complexity of these mechanisms is daunting as five different Arfs
functionally interact with a diverse set of effectors, among them monomeric
and multimeric coats/adaptors. An even greater complexity emerges when
coat/adaptor isoforms and splicing variants are considered, as well as their
corresponding interacting molecules.
We will attempt to formulate unitary principles that govern multiple
vesiculation processes that require Arf and a coat. We will approach this
question from a comparative evolutionary perspective in order to define the
mechanisms by which Arf and coat/adaptor regulate the formation of
membrane carriers. Our hypothesis is that the primordial coat-Arf
interaction defines a basic mechanism upon which selective pressures have
acted to diversify the membrane carrier formation machinery.
We propose that Arf and its effectors involved in membrane traffic can
be fitted to a “molecular mutualism model”. We have borrowed and adapted
the mutualism concept as used in ecology. Mutualism is defined here as a
network of interactions of two or more elements that have a positive net
impact on the fitness of those elements to perform their functions.
Mutualistic interactions between organisms control growth and/or density of
the network components. Moreover, there is no need for the elements of the
network to physically interact or for the interactions to be permanent or
obligatory. In our particular case, the lipids and proteins that take part in the
formation of membrane carriers constitute the network. We will concentrate
on the relationship between Arf GTPases and their coat and non-coat
effectors. As we will discuss, and similar to the consequences of mutualism
between organisms, the interactions between Arf and facultative effectors
control the density or growth rate of membrane-associated components like
coats.
We will first describe the evolutionary relationships among the coatomer
(COPI) and the adaptor complexes as a way to define conserved features
 TETRAMERIC COAT PROTEIN-ARF INTERACTIONS 2613

with regard to the coats and their interaction with Arf that could be
obligatory properties of the network.

2. PHYLOGENETIC ANALYSIS OF COPI AND

OTHER ADAPTOR COMPLEX STRUCTURES

If mechanisms common to all coats and adaptors have been maintained

throughout evolution, it is reasonable to expect structural similarities
between coats and adaptor subunits. The first indication of the common
evolutionary origins of the COPI complex and other adaptors came from the
sequence comparisons between subunits that form the COPI complex and
the heterotetrameric adaptors AP-1, AP-2, AP-3 and AP-4 (Lewin and
Mellman, 1998; Schledzewski et al., 1999; Boehm and Bonifacino, 2001)
(See Figure 1).

Figure 1. Proposed evolutionary relationships of the heterotetrameric coat subunits.

Asterisks denote rounds of gene duplication. Diagram has been adapted from Lowe and
Kreis, 1998; Schledzewski et al., 1999; Boehm and Bonifacino, 2001.

Phylogenetically, COPI is the oldest coat complex (Lewin and Mellman,

1998; Schledzewski et al., 1999; Boehm and Bonifacino, 2001). COPI is
organized as a heptamer made up of two biochemically and functionally
distinguishable sub-components, the heterotetrameric F-subcomplex,
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containing β-COP, γ-COP, δ-COP and ζ-COP, and a trimer consisting of α-

COP, β’-COP and ε-COP (Lowe and Kreis, 1998; Kirchhausen, 2000). The
subunits of the F-subcomplex possess sequence similarity to the different
adaptor subunits (Schledzewski et al., 1999; Eugster et al., 2000). Like the
F-subcomplex, the tetrameric adaptors contain two large subunits β1-4 and
γ, α, δ, ε; a medium chain (µ1-4) and a small chain (σ1-4) (Boehm and
Bonifacino, 2001) (see Figure 2).

Figure 2. The pair of dimers model for the assembly of the heterotetrameric coat complexes.
Diagram represents a putative evolutionary sequence for the formation of heterotetramers
from a set of dimers (see Figure 1). The putative proto-dimer pair has been inferred from the
hierarchy of subunit interactions obtained for COPI as well as the adaptor complexes (see
text). Diagram of the canonical heterotetramer structure was derived from the crystal
structure of the AP-2 core complex (Collins et al., 2002).

Heterotetrameric adaptors have likely evolved from the COPI F-

subcomplex through successive rounds of gene duplication (Lewin and
Mellman, 1998; Schledzewski et al., 1999; Boehm and Bonifacino, 2001;
Chow et al., 2001). However, whether this tetrameric organization emerged
as an assembly of independently evolved monomers or as coevolved dimers
is an open question. If a pair of dimers generated the primordial COPI F-
subcomplex it could be possible to observe preferential interaction between
some subunits while others could serve as bridges between subunits that do
not interact extensively. Two-hybrid analysis has revealed that the β-COP
interaction with the medium chain δ-COP is privileged over all the other
possible molecular links. Alternatively, γ-COP forms a stable complex with
 TETRAMERIC COAT PROTEIN-ARF INTERACTIONS 2635

the small ζ-COP subunit (Eugster et al., 2000; Faulstich et al., 1996; Takatsu
et al., 2001). Triple-hybrid analysis has further shown that ζ-COP acts as a
bridge between the β and γ subunits because negligible interaction occurs
between β and γ-COP in the absence of ζ-COP (Takatsu et al., 2001). This
hierarchy of contacts detected by yeast hybrid analysis can also be
biochemically defined, as it is possible to disassemble purified coatomer into
stable β−δ and γ−ζ sub-complexes (Pavel et al., 1998). In particular, the
isolated β−δ sub-complex is functional, as this isolated sub-complex can be
recruited to membranes in an Arf1•GTP-dependent manner (Pavel et al.,
1998). As we will see, the β subunit of the COPI F-subcomplex is involved
in Arf1 recognition. These results suggest that the most primitive interaction
between Arf1 and a coat occurred between the GTPase and a β−δ dimer.
This molecular analysis is supportive of the phylogenetic model proposed by
Schledzewski, et al. (Schledzewski et al., 1999), who suggested that the
primordial COPI precursor emerged by the fusion of two proto-dimers (see
Figure 2).
Does adaptor subunit organization fit the proto-dimer model? The
crystal structure of the AP-2 adaptor core complex (Collins et al., 2002),
which phylogenetic studies indicate is the most recent heterotetramer
addition to the tetrameric adaptor family (Lewin and Mellman, 1998;
Schledzewski et al., 1999; Boehm and Bonifacino, 2001), has provided
insight into this question. The core of AP-2 is organized as a rectangular
structure with its outside flanked by curved α and β2 chains. Nestled in the
elbow of α and β2 are the σ2 and the N-terminus of the µ2 subunits,
respectively. Each pair, α−σ2 and β2-µ2, forms a tight heterodimer, which
creates a flat platform where the C-terminal end of µ2 sits. Remarkably, the
crystal structure of σ2 and the N-terminal end of µ2 are almost identical
(Collins et al., 2002) (see Figure 2). Also, the large subunits α and β2 share
substantial structural homology (Collins et al., 2002). These results are in
agreement with the phylogenetic prediction that the large subunits as well as
the σ and µ-type chain originated by gene duplication from common
ancestor genes that encoded a primitive dimer (Lewin and Mellman, 1998;
Schledzewski et al., 1999; Boehm and Bonifacino, 2001).
Genetic and biochemical evidence regarding other tetrameric adaptors
also support a “proto-dimer” model”. Two-hybrid analysis has revealed the
existence of preferential dimer associations within AP-1 (Takatsu et al.,
2001; Page and Robinson, 1995; Aguilar et al., 1997), AP-3 (Peden et al.,
2002) and AP-4 (Takatsu et al., 2001; Hirst et al., 1999), in addition to AP-2
(Page and Robinson, 1995). Stable AP-3 dimers are also found in vivo.
Naturally occurring AP-3 mutations in mice, either in δ or β3A subunits,
lead to degradation of the non-mutated subunits to different extents
(Kantheti et al., 1998; Feng et al., 1999; Dell’Angelica et al., 1999a; Zhen et
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al., 1999). A small amount of undegraded β3-µ3 dimers can be

immunoprecipitated from δ-deficient cytosol despite the complete absence
of σ3. Conversely, a δ−σ3 dimer can be immunoprecipitated from β3-
deficient cytosol in which all µ3 is degraded (Peden et al., 2002; Zhen et al.,
1999). These results suggest a preferential association of undegraded
subunits following a pair of dimers rule. Each subunit of these dimers
cannot exist in the absence of the other, suggesting that the dimer is the
minimal stable assembly of monomers. These results imply that the dimer
organization is an obligatory mutualistic interaction.
Phylogenetic, biochemical, and structural analyses support a common
ancestry of the heterotetrameric adaptors. The pair of dimers feature of the
most primitive coat, the COPI F-subcomplex, is conserved in one of the
most recent evolutionary additions to the heterotetrameric adaptor family,
AP-2. This core structural organization likely represents the minimal
scaffold where other molecules, like the Arf GTPases, assist the adaptor
complexes in their sorting, coating, and vesiculation functions.

3. COMPARATIVE ANALYSIS OF THE

MOLECULAR INTERACTIONS BETWEEN
ARFS AND HETEROTETRAMERIC ADAPTORS.

With the exception of Arf6 (Cavenagh et al., 1996), all Arfs cycle between
the cytosol and membranes, depending upon whether GDP or GTP is bound,
respectively (Moss and Vaughan, 1998). Cycling of Arf between cytosol
and membranes brings its effectors in close proximity to membranes.
Among those effectors are COPI and other adaptors. These coat complexes
cycle between membrane-bound and cytosolic states in a mechanism that is
functionally linked to the Arf GTPase cycle.
Three non-exclusive models have been proposed that differ in the nature
of the Arf effector. These models include the hypotheses that:
a) Arfs interact directly with COPI and other adaptor complexes (Zhao et
al., 1997).
b) Arfs interact with or modify a putative docking site, inducing a change
that would allow the docking site to bind the coat molecule (Zhu et al.,
1998). The docking site could be either a lipid (Arneson et al., 1999) and/or
a protein (Zhu et al., 1998).
c) Arfs interact with lipid modifying effectors that create membrane
microdomains favorable for adaptor binding (Ktistakis et al., 1996).
It is likely that in a single cell, different membranes organize networks of
Arf effectors that build vesiculation machines that differ in the extent that
 TETRAMERIC COAT PROTEIN-ARF INTERACTIONS 2657

these three mechanisms are used. Variations in the relative amounts of

different effectors present on membranes, differences in lipid composition,
the presence of different adaptors, and the use of different model systems
might explain the emergence of these different models.

3.1 Arf interactions with coat molecules: the direct link

The first indication that Arf was involved in the recruitment of adaptors to
membranes came from the use of the fungal metabolite brefeldin A (BFA),
an inhibitor of some Arf GEFs (Klausner et al., 1992; Jackson and
Casanova, 2000). Shortly after drug treatment COPI (Klausner et al., 1992;
Lippincott-Schwartz et al., 1991; Orci et al., 1991); AP-1 (Seaman et al.,
1993; Robinson and Kreis, 1992), AP-3 (Simpson et al., 1996; Dell’Angelica
et al., 1997; Ooi, et al., 1998) and AP-4 (Hirst et al., 1999; Dell’Angelica, et
al., 1999b) are redistributed to the cytosol. Similar effects can be triggered
using Arf mutants that mimic the GDP-bound state of the GTPase (Hirst et
al., 1999; Ooi et al., 1998). Demonstration of direct binding between coat
molecules and Arf1 was first obtained in an in vitro reconstituted Golgi
membrane system (Zhao et al., 1997; Zhao, et al., 1999). Purified COPI can
be recruited to Golgi membranes by in vitro transcribed-translated-Arf1.
Using tRNA loaded with photo-reactive amino acids it is possible to label
selected positions on in vitro transcribed-translated Arf1. Loading the
GTPase with a photo-reactive phenylalanine in position 82 leads to a cross-
linked product with β-COP (Zhao et al., 1997). In contrast, Arf1 loaded
with photo-reactive residues either at position 46 or 49 forms cross-linked
products with each large coatomer subunit, β-COP and γ-COP (Zhao et al.,
1999). The GTP•Arf1-COPI interaction has been confirmed for the β-COP
subunit using two-hybrid analysis (Eugster et al., 2000). The exact regions
in the β-COP and γ-COP subunits where Arf1 binds are presently not
known. However, if we draw a parallel between the AP-2 core crystal
structure and COPI, these results suggest that Arf1 is wedged in the interface
of the two large COPI subunits in close proximity to the membrane (see
Figure 3).
In light of the structural conservation observed between COPI and the
tetrameric adaptors, a likely prediction is that Arf1 labeled on positions 46,
49 or 82 should be cross-linked to the large subunits of the adaptor
complexes known to be recruited by Arf1. Immature secretory granules
purified from adrenomedullary cells recruit AP-1 and AP-3 in a GTP•Arf-
dependent fashion (Dittie et al., 1996). In this in vitro adaptor recruitment
system, photoreactive Arf1•GTP or Arf5•GTP, labeled at position 46, can
be cross-linked to the AP-1 complex (Austin et al., 2000; Austin et al.,
2002). Immunoprecipitation with β1 and γ antibodies has confirmed that the
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large subunits of the AP-1 complex interact with Arf1. Moreover, removing
the ear domains of both large subunits by controlled proteolysis does not
abrogate the formation of β1/γ-Arf1 cross-linked products (Austin et al.,
2002). These results suggest that the membrane-facing region of AP-1,
composed of the amino-terminal part of β1 and γ, interacts with Arf1.
Similar results have been obtained with AP-3, although the identity of the
high molecular weight cross-linked products could not be defined by
immunoprecipitation with β3 or δ antibodies. However, native
immunoprecipitation with antibodies against the small σ3 subunit brings
down Arf1 cross-linked to polypeptides that correspond to the molecular
weight of the large AP-3 subunits (Austin et al., 2002).

Figure 3. Proposed molecular interactions of the ARF GTPases with heterotetrameric coats.
See text for details.

Like other adaptor complexes, AP-4 is recruited to membranes by a

mechanism linked to the GDP-GTP cycle of Arfs. AP-4 remains cytosolic
when cells are either briefly exposed to brefeldin A or transfected with the
mutants of Arf1 or Arf3 that bind guanine nucleotides poorly. Similar
effects are obtained by introducing Arf GAP or the GGA1 VHS-GAT
domain into cells (Hirst et al., 1999; Dell'Angelica, 1999b). The interaction
of Arf1•GTP and AP-4 has been mapped, using two-hybrid analysis, to the
first 260 amino acids of ε-adaptin, a putative membrane–facing region of the
large subunit, and the switch I region of Arf1 (Boehm et al., 2001). These
results are in agreement with the hypothesis that the structure and
localization of the Arf-binding region in the COPI F-subcomplex and
tetrameric adaptors is evolutionarily conserved. However, no interactions
were detected with the other large AP-4 subunit, β4-adaptin (Boehm et al.,
2001). This might reflect an intrinsic limitation of the two-hybrid technique,
 TETRAMERIC COAT PROTEIN-ARF INTERACTIONS 2679

which cannot detect interactions dependent on binding to membranes and

assembly of a heterotetramer.
Is a simple model of an Arf molecule wedged between the two large
subunits of a multimeric adaptor enough (see Figure 3)? It is possible that
this model represents the primordial mechanism to which all Arf-coat
interactions conform, though perhaps to different extents. If these
interactions have remained constrained to the large coat subunits with the
conserved switch I and II regions of Arf; we can predict that the interactions
between adaptors and Arfs should be indistinguishable among Arf isotypes
known to cycle between cytosol and membranes (Arf1-5). Conversely,
modifications of the putative core mechanism by demands imposed by new
coat-dependent traffic pathways might be reflected through differential
interactions between adaptors and Arf isotypes. Detailed analysis of the AP-
3 and AP-4 interactions with Arfs supports the latter. Expression of an Arf1
mutant that mimics the inactive state induces detachment of coats from
membranes, including AP-3 (Ooi et al., 1998). However, the expression in
cells of the homologous mutant of Arf5 does not release AP-3 from
membranes (Ooi et al., 1998). Notably, in vitro photoreactive Arf5 does not
generate detectable cross-linked species with AP-3; yet photoreactive Arf1
can be cross-linked to AP-3 (Austin et al., 2002). An even more extensively
studied example is AP-4. Expression of the dominant negative mutants of
Arf1 and Arf3 leads to a cytosolic redistribution of AP-4 (Aguilar et al.,
2001). However, neither a similar mutation in Arf4 nor Arf5 modifies the
membrane distribution of AP-4 (Aguilar et al., 2001). Overall, these results
indicate that selectivity determinants further control the mechanism by
which Arfs and adaptor effectors interact. Switch I and II regions of Arfs
are conserved, suggesting that other contacts between Arf and adaptors
might define specificity. In support of this idea, the C-terminal region of µ4
is capable of interacting with Arf1, in GTP or GDP-bound forms, through
regions other than switches I and II of Arf (Aguilar et al., 2001). Similar
analysis performed with the COPI subunits has not led to a detectable
interaction of Arf1 with the µ-adaptin homolog, δ-COP (Eugster et al.,
2000).
In summary, these results suggest that although the primary mechanism
of adaptor-Arf associations is highly conserved and involves the large
subunits, the medium and small subunits may have diverged to add further
specificity to the adaptor-Arf interactions.
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3.2 Arf interactions with coat molecules: the indirect

link

3.2.1 Is a direct Arf-coat interaction sufficient to control coat

membrane recruitment?

A major indication that other factors are required to define the binding of an
adaptor to membranes has come from the reconstitution of Arf-dependent
coat recruitment onto protein-free synthetic membranes. AP-1 and clathrin
can be recruited to liposomes with a similar efficiency to that achieved on
natural membranes, such as Golgi-enriched fractions (Zhu et al., 2001a; Zhu
et al., 1999a). However, AP-1 recruitment to Golgi membranes requires
cytosolic factors other than Arf1 fractions (Zhu et al., 2001a; Zhu et al.,
1999a; Seaman et al., 1996). Moreover, the AP-1-liposome interaction is
significantly influenced by the liposome lipid composition. Acidic
phospholipids, such as phosphatidic acid or phosphatidylinositides (PIs),
notably increase the adaptor recruitment to liposomes (Zhu et al., 1999a).
Phosphatidic acid is generated from phosphatidylcholine by phospholipase D
(PLD). This enzyme is an Arf effector (Brown et al., 1993; Cockcroft et al.,
1994) that is found in association with the Golgi complex, to which it is
recruited by a GTP•Arf-dependent mechanism (Ktistakis et al., 1995;
Freyberg et al., 2001). In addition, PI-4-kinase β is recruited to the Golgi
complex by an Arf-1 dependent mechanism and once on the membrane it
stimulates the production of PI(4,5)P2 (Godi et al., 1999). Thus, it is likely
that lipid-modifying enzymes, recruited to membranes by Arf-dependent
mechanisms, control the dynamics of the Golgi complex (Sweeney et al.,
2002) by creating membrane microdomains favorable for adaptor and coat
recruitment.
AP-1 and AP-3 recruitment to liposomes differ in the specificity of lipids
required. AP-3 recruitment to synthetic liposomes occurs irrespective of the
presence of acidic phospholipid (Drake et al., 2000). Moreover, AP-3, but
not AP-1 or AP-2, can be recruited to synaptic vesicles in an Arf1-dependent
manner (Faundez et al., 1998). Interestingly, synaptic vesicles do not
possess detectable levels of PIs (Micheva et al., 2001). Additionally, the
ATP-requirement to recruit AP-3 to membranes can be completely replaced
by ATPγS (Faundez and Kelly, 2000), a poor substrate for lipid modifying
enzymes (Hay and Martin, 1992). In this regard, AP-3 is closely related to
COPI, which can also be recruited to synthetic liposomes irrespective of the
presence of acidic phospholipids (Spang et al., 1998; Bremser et al., 1999).
These observations agree with the close evolutionary relationship between
AP-3 and COPI (Scheldzewski et al., 1999; Boehm and Bonifacino, 2001).
In addition, they suggest that the interaction of adaptors with acidic
 TETRAMERIC COAT PROTEIN-ARF INTERACTIONS 269
11

phospholipids is a recent evolutionary addition superimposed onto the

general mechanism constituted by a direct interaction of Arf and the large
subunits of the tetrameric adaptors.

3.2.2 What controls AP-2 membrane recruitment?

Initially, adaptor recruitment to membranes was considered to be primarily

governed by interactions with membrane protein cargo. This model was
supported by observations that adaptors could bind the cytosolic tails of
membrane cargo proteins (Glickman et al., 1989; Pearse, 1989; Pearse,
1988; Ohno et al., 1995), that AP-1 recruitment to Golgi membranes was
controlled by the abundance of mannose 6-phosphate receptors (Le Borgne
and Hoflack, 1997; Pearse, 1985), and by the identification of synaptotagmin
I and other isoforms as high affinity, membrane receptors for AP-2 (Haucke
et al., 2000; Zhang et al., 1994; Ullrich et al., 1994; von Poser et al., 2000).
However, the role of M6PRs as a receptor for AP-1 has been recently
challenged (Zhu et al., 2001b; Zhu et al., 1999b; Puertollano et al., 2001).
Moreover, increasing or decreasing the cargo concentration available for
AP-2 at plasma membrane domains, either by ligand induced receptor
clustering or by overexpression, did not affect the recruitment of AP-2 to
plasma membrane (Santini et al., 1998; Santini and Keen, 1996). How then
can we understand AP-2 recruitment to membranes?
The synapse has provided a useful model system to understand the
mechanisms controlling adaptor recruitment. Synaptojanin is a 5’ PI-
phosphate phosphatase; known to control the levels of PI(4,5)P2 in plasma
membrane (McPherson et al., 1996) and to play a central role in uncoating
AP-2/clathrin coated vesicles. Neurons from synaptojanin I -/- mice possess
a substantial increase in the number of AP-2/clathrin coated vesicles,
suggesting that the levels of PIP2 phospholipids could control AP-2
recruitment to membranes (Cremona et al., 1999; Harris et al., 2000). In
fact, AP-2 possesses two PI-binding surfaces containing conserved lysine
residues (Collins et al., 2002). These regions lie on the µ2 and α chains of
AP-2. The crystal structure of the AP-2 core reveals that these lysines
generate positively charged surfaces on the N-terminal region of α-adaptin
facing the membrane and the C-terminal half of µ2. Mutating these residues
in either adaptin completely abrogates the binding of AP-2 to membranes
(Gaidarov and Keen, 1999; Rohde et al., 2002). Replacing the N-terminus
of α-adaptin with the equivalent domain of γ-adaptin also abrogates plasma
membrane AP-2 recruitment (Page and Robinson, 1995). Consistently,
neomycin (West et al., 1997) and the pleckstrin homology domain of PLD-δ
(Jost et al., 1998), molecules known to bind to PI(4,5)P2, hinder AP-2
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binding to membranes in cell free assays that recapitulate adaptor binding to

membranes or AP-2-dependent plasma membrane vesiculation.
The critical residues for phosphatidylinositols binding in α-adaptin,
lysines 55-57 (Gaidarov and Keen, 1999), and µ2-adaptin, lysines 344-346
(Rohde et al., 2002) are absolutely conserved among AP-2 chains from
invertebrates to mammals. These residues are not present in similar regions
of the subunits of other adaptors, suggesting that quantitative and qualitative
differences distinguish the lipid requirements to recruit different adaptors to
membranes. The PI(4,5)P2-AP-2 interaction and functionality has been
nicely demonstrated using reconstituted liposomes. In this experimental
system the presence of negatively charged lipids, like PI(4,5)P2, in the donor
membrane is sufficient to trigger adaptor recruitment and vesicle formation
even in the absence of GTP (Takei et al., 1998).
Although the most recently diverged adaptor, AP-2, maintains the pair of
dimers structure, the mechanism of its recruitment may have diverged from
being dependent upon a direct interaction with Arf to a mechanism
controlled by a network of Arf-effectors. These Arf effectors control the
lipid composition of the membrane and consequently, AP-2 recruitment to
the membrane.

3.2.3 Does Arf control AP-2 membrane recruitment?

Early indications that GTPases could modulate AP-2 came from studies
using perforated cells and cell-free assays (Seaman et al., 1993; West et al.,
1997). AP-2 can be recruited to a variety of membranes by mechanisms that
differ in their nucleotide requirement (Arneson et al., 1999; Zhang et al.,
1994; Traub et al., 1996). The best characterized example is the AP-2
recruitment to synaptic membrane preparations (Takei et al., 1996). In this
system, AP-2 binding to membranes requires ATP and is stimulated by the
addition of GTPγS. GTPγS-dependent AP-2 recruitment to membranes has
been initially regarded as dependent on the GTPase dynamin. However, as
we will discuss, it could reflect the involvement of other GTPases.
If PIP2 lipids are involved in the recruitment of AP-2, then a critical
regulatory step is likely to reside in the localized synthesis of PIP2. Among
those proteins responsible to control PIP2 levels, for this discussion we will
concentrate on the enzymes that regulate AP-2/clathrin recruitment. PIP-5-
kinase Iγ (Wenk et al., 2001) and the class II PI 3-kinase C2α (Gaidarov et
al., 2001) control the production of different PIP2’s at the plasma membrane.
Both enzymes are necessary to recruit AP-2 to either liposomes or
membranes. As expected, the effects of the PIP-5-phosphatase
(synaptojanin I) and the PIP 5-kinase (PIP kinase Iγ) are antagonistic with
regard to AP-2 recruitment to membranes (Wenk et al., 2001).
 TETRAMERIC COAT PROTEIN-ARF INTERACTIONS 271
13

PIP-5-kinase Iγ and the class II PI 3-kinase C2α are both regulated by

proteins involved in vesicle formation. The class II PI 3-kinase
C2α activity is regulated by clathrin binding to the enzyme (Gaidarov et
al., 2001). Importantly, PIP 5-kinase activity is regulated by Arf6 (Honda et
al., 1999; Jones et al., 2000) and phosphatidic acid (Arneson et al., 1999;
Honda et al., 1999), a product of the Arf6 effector, PLD. Arf6, the PI(4)P 5-
kinase Iγ, and PLD are present in plasma membrane, suggesting a
coordinated role in regulating endocytosis. Indeed, dominant negative
mutants of PLD have been described recently with the ability to inhibit EGF
receptor endocytosis (Shen et al., 2001). If Arf6 controls two effectors at the
plasma membrane that are known to regulate AP-2 recruitment, then a
logical prediction would be that Arf6 should modulate the formation of AP-
2-containing vesicles.
Dominant Arf6 mutants perturb the AP-2-dependent endocytosis of
transferrin (D’Souza-Schorey et al., 1995) and β2-adrenergic receptors
Claing et al., 2001). In polarized epithelial MDCK cells, Arf6 is selectively
targeted to the apical plasma membrane where it regulates pIgA receptor-
mediated endocytosis and the levels of plasma membrane-bound clathrin
(Altschuler et al., 1999). These effects are restricted to the apical pole in
polarized cells, without affecting pIgA receptor-mediated endocytosis at the
basolateral domain. Differential sorting of transferrin receptors and β2-
adrenergic receptors in distinct clathrin-coated plasma membrane domains
support the proposal that specialized types of clathrin-dependent endocytosis
exist in a single cell type (Cao et al., 1998; Prasad et al., 2002). This argues
in favor of evolutionary divergence of the mechanisms that control the
recruitment of coats and adaptors to membranes.
The synapse is a highly specialized region of the neuron where
endocytosis contributes to the retrieval of synaptic vesicle components after
regulated secretion (Hannah et al., 1999; Brodin et al., 2000; Gad et al.,
2000; Shupliakov et al., 1997). Deficiencies in the synaptic endocytic
machinery lead to modifications in synaptic transmission (Brodin et al.,
2000; Gad et al., 2000; Shupliakov et al., 1997; Stimson and Ramaswami,
1999). Microinjection of the Arf6-GEF msec7-1, a rat homolog of human
cytohesin I, to the neurons of embryonic Xenopus neuromuscular
preparations increases excitatory post-synaptic potentials, consistent with a
putative role of Arf6 in pre-synaptic membrane traffic (Ashery et al., 1999).
Similarly, expression of a dominant activating Arf6 mutant increases the
targeting of selected membrane protein markers to synaptic-like
microvesicles in PC12 cells, a model neuroendocrine cell line (Powelka and
Buckley, 2001).
So far, there is no evidence of a direct interaction between Arf6 and AP-
2. However, β-arrestin, a clathrin adaptor involved in G protein coupled-
272
14 STYERS AND FAUNDEZ

receptor traffic, can bind Arf6•GDP and ARNO, forming a complex that
catalyzes the exchange of GDP for GTP (Claing et al., 2001). Because β-
arrestin possesses binding sites for AP-2 (Laporte et al., 2000; Miller and
Lefkowitz, 2001) as well as PIP2 (Gaidarov and Keen, 1999) it is possible
that β-arrestin brings together Arf6 and AP-2 in a PIP2 membrane patch.
Alternatively, AP-2 may interact with the by-product of an Arf-effector
interaction, such as PIP2, which is generated by activating a PIP 5-kinase. In
either case, the nature of the interaction is indirect. As previously discussed
for COPI, AP-1, AP-3 and AP-4, it is likely that Arf is wedged between the
membrane facing regions of the two large subunits of the heterotetrameric
coat complexes. Instead, in AP-2, α-adaptin and the µ2 subunit, by virtue of
tight binding to PI(4,5)P2, dock AP-2 to membrane lipids, likely obliterating
an Arf-binding pocket. PI(4,5)P2 binds to Arfs with affinities on the order of
-5 -7
10 M (Randazzo, 1997) whereas it binds α-adaptin with a KD of 10 M
(Gaidarov and Keen, 1999). Thus, it is unlikely that an Arf molecule could
co-exist with AP-2 on membrane patches rich in AP-2 and PI(4,5)P2. The
-7
simultaneous binding of arrestin to AP-2 and PI(4,5)P2 (KD=10 M) could
provide a selective means to generate clathrin-coated vesicles preferentially
enriched in β-adrenergic receptors.

4. MUTUALISM IN ARF-EFFECTOR
INTERACTIONS INVOLVED IN MEMBRANE
CARRIER BIOGENESIS

The concept of mutualism, interactions with a positive net impact on the

fitness of the elements to perform their functions, is not restricted to either
the nature of the interaction (direct or indirect) or to the number of elements
involved. The spectrum of mutualistic interactions extends from that in
lichens, algae living inside of the body of a fungus that are not facultative
and require permanent direct contact between the organisms, to interactions
where each organism lives independently. We propose that a similar
spectrum of interactions operates in the mechanisms that control the
formation of membrane carriers. First, at a simple organizational level, it
appears that the pair of dimers model is an obligatory feature in the
organization of adaptor heterotetramers. As we discussed, those subunits
that are not dimers in vivo cannot exist independently. This core interaction
provides a basis for the interactions with Arf GTPases. The binding of
COPI, AP-1, AP-3 and AP-4 with Arf, though transient, is at some point
direct. Finally, as in the case of AP-2 and AP-1, intermediaries of the
network (such as lipid-modifying enzymes), that are themselves Arf
 TETRAMERIC COAT PROTEIN-ARF INTERACTIONS 273
15

effectors, either completely (AP-2) or partially (AP-1), modulate the

recruitment of the vesiculation machine to the membrane.
Depending upon the adaptor, single or multiple Arf effector interactions
appear to dominate the coating reactions. The simplest vesiculation systems,
those generated by liposomes, purified coat, and GTPase, suggest that
simple interactions dominate primordial coats like COPI (Bremser et al.,
1999) while multiple and indirect mechanisms prevail in the case of AP-2
(Takei et al., 1998), an evolutionarily younger coat. Thus, in the absence of
any other Arf1 effector but COPI, it is possible to bud COPI vesicles from
liposomes by an Arf1•GTP-dependent mechanism, irrespective of the lipid
composition of the artificial membranes (Bremser et al., 1999). On the other
extreme, AP-2/clathrin coats and buds can be generated in brain lipid-
derived liposomes with clathrin coated vesicle enriched mixtures in the
absence of any nucleotide; suggesting that the nature of the membrane
changes the requirement for GTP and therefore the GTPase (Takei et al.,
1998). However, it does appear that the GTPase controls the generation of
lipid microdomains to which adaptors can be recruited.
How can we understand the interactions between a GTPase and adaptors?
Does AP-2 follow a completely different mechanism from other adaptors?
As we have previously discussed, Arf1-5 are recruited to membranes where
they interact with effectors only when they bind GTP. In contrast, Arf6
remains constitutively associated with membranes, irrespective of the
nucleotide bound. In addition, at steady state, a great proportion of AP-2 is
bound to membranes (Pearse and Robinson, 1990; Keen, 1990). Thus, a
strategy based on Arf-adaptor interaction after membrane recruitment seems
unlikely. Instead, an interaction between Arf6 and possibly AP-2 could be
mediated by arrestin and/or lipid modifying Arf effectors; such as PLD or
PI(4)P 5-kinase. This mechanism opens the possibility to control AP-2
recruitment by extracellular signals capable of controlling arrestins, PLD,
PIP-kinase and lipid-phosphatases. These considerations are likely not only
restricted to AP-2, because the recruitment of AP-1 to membranes can also
be controlled by acidic phospholipids or lipid modifying enzymes. These
modifications on an ancient mechanism add new levels of regulation that
likely improve the system in response to new selective pressures.
The “mutualistic network” vision is compatible with an effector
molecule, such as an adaptor, having a life separate from its interactions with
Arf. Rather than Arf-effector interactions being seen as a linear sequence of
cause-effect relationships, we prefer to describe their organization as a
collection of Arf-effector intermediaries that branch and can feed back into
the network. Each effector could bring different inputs to generate a similar
outcome without necessarily invoking the same sequence and/or extent of
events. The mutualistic network idea can account for observations such as
274
16 STYERS AND FAUNDEZ

the recent description that β-arrestin can bind Arf6•GDP and induce the
GDP-GTP exchange (Claing et al., 2001). These results suggest the
counterintuitive idea that an adaptor (arrestin) could arrive at the membrane
first and then activate the GTPase (Zhu et al., 2000).
If Arf activation is the primary input to a branched network with
feedback regulation, it is not unreasonable to expect that even after
shutdown of Arf function, other network components (Arf-activated
effectors or membrane cargo) could maintain the network functional status
(such as an adaptor bound to membranes). We predict that the kinetic
parameters that govern the activation or membrane recruitment of Arf as
well as distinct effectors should become independent, as the initial Arf input
is disseminated in the network. Indeed, in vivo Arf1 and COPI membrane
association at the Golgi complex obey different kinetic and thermodynamic
parameters; indicating that Arf and coat recruitment to membranes can be
dissociated (Presley et al., 2002). Simultaneous in vivo analysis of the
dynamics of Arfs and their effectors on membranes will provide the
evidence required to test the mutualism model.
The interactions between Arf and adaptors have become increasingly
disparate over time and appear to have reached a summit with the adaptor
AP-2. This divergence may have been selected as the cell-cell
communication and intracellular traffic demands have progressively
increased from a free-living unicellular organism to a multi-cellular
organism comprised of highly specialized cells and tissues. However, the
common theme across different levels of complexity in carrier forming
machines is the formation of networks that interact in a mutualistic manner.

5. ACKNOWLEDGEMENT

This work was supported by grants from the Basil O’Connor March of
Dimes Foundation, The Melanoma Research Foundation, and the Emory
University URC. M. L. Styers is a member of the Biochemistry, Cell and
Developmental Biology Graduate program from the Graduate Division of
Biomedical Sciences, Emory University School of Medicine.

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23

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Mol. Biol. Cell, 10, 537-549.
Chapter 14
ARF6

JAMES E. CASANOVA
Department of Cell Biology, University of Virginia Health Sciences Center, Charlottesville,
USA

Abstract: Arf6 is the sole representative of the class III Arfs and is the most divergent
(both structurally and functionally) of the Arfs. Its localization to the plasma
membrane and endosomal system also distinguishes it from the other five
Arfs. Both the localization of Arf6 and its functional roles in endocytosis,
secretion, and desensitization of G-protein coupled receptors exhibit cell type-
specificity, making comparative studies difficult and often confusing. Finally,
models for Arf6 in actin organization and phospholipid signaling are
discussed.

1. INTRODUCTION

Arf6 was first identified in 1991 by low-stringency hybridization with a

bovine Arf2 probe (Tsuchiya et al., 1991). It is the sole representative of the
Class III Arfs and is the most divergent in terms of both structure and
function. In this case, “divergent” is a relative term; human Arf6 is 68%
identical to human Arf1 at the amino acid level, and the sequences of their
effector binding “switch regions” are virtually identical. Like the other Arfs,
Arf6 functions in the regulation of membrane traffic. However, in contrast
to the class I and II Arfs, which operate primarily at the Golgi apparatus,
Arf6 localizes to the plasma membrane and a subset of endosomes, where it
regulates the recycling of internalized membrane proteins. In addition to this
function, and perhaps integrated with it, Arf6 also modulates assembly of the
cortical actin cytoskeleton, a role not ascribed to the other Arfs. In this
chapter, we will critically examine the (often contradictory) Arf6 literature
and attempt to distill some unifying principles that can be used to define the
role of this intriguing protein in eukaryotic cells.
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2 CASANOVA

2. BIOCHEMICAL PROPERTIES

As noted above, human Arf6 is 68% identical to human Arf1, yet the two
proteins differ in important ways. First, the pI of Arf6 is significantly higher
than that of Arf1 (8.5 vs 5.9; Maranda et al., 2001) and modeling of surface
electrostatic potential indicates that it has an overall cationic appearance
(Amor et al., 2001). In particular, the region of the protein surrounding and
including the N-terminal helix is strongly basic, while in Arf1 the primarily
basic helix is surrounded by an acidic ring or “cushion” (Amor et al., 2001).
The strong cationic character of Arf6 may promote electrostatic interactions
with acidic head groups of membrane lipids, contributing to its observed
higher affinity for membranes even in the GDP-bound state. A second key
difference is the presence of a four-residue deletion near the N terminus of
Arf6 relative to Arf1. Crystal structures indicate that this deletion shortens
the loop between the N-terminal helix and the first beta strand, resulting in
subtle changes in the nature of the nucleotide-binding pocket (Menetrey et
al., 2000). In Arf1, E54 helps coordinate the bound Mg++ ion, while in Arf6,
the corresponding residue, E50, instead forms a hydrogen bond with S38,
thus weakening the interaction with Mg++ (Menetrey et al., 2000). The
overall effect of these changes is that Arf6 exhibits a higher rate of
spontaneous nucleotide exchange, particularly in the presence of acidic
lipids (Terui et al., 1994).
As mentioned above, the effector-interacting domains of Arf6 are
virtually identical with those of the other Arfs, and it is therefore not
surprising that, so far, all of the effectors that have been described for Arf6
also interact with at least one other Arf. Because effectors are discussed in
detail in the preceding chapters, we will describe them here only in the
specific context of Arf6 function.

3. LOCALIZATION

Unlike Arf1, which localizes to the Golgi complex, the subcellular

distribution of Arf6 is complex and can vary from one cell type to another.
Using epitope-tagged constructs, Peters and coworkers were the first to
demonstrate the presence of Arf6 in the plasma membrane/endosomal
system (Peters et al., 1995). In cells expressing relatively high levels of
exogenous Arf6, immunostaining was observed at the plasma membrane and
a population of perinuclear endosomes. In cells expressing low levels of the
tagged protein, plasma membrane staining was reduced and the predominant
staining was endosomal. This distribution has been subsequently confirmed
for the endogenous protein using isoform-specific antibodies (Song et al.,
 ARF6 2853

1998). Colocalization studies in CHO cells (D'Souza-Schorey et al., 1998)

revealed that the Arf6-positive endosomes contained transferrin receptors
and the v-SNARE cellubrevin, but were poorly accessible to fluid-phase
markers, such as horseradish peroxidase. These data suggest that they
represent recycling endosomes, a compartment through which many
internalized membrane constituents pass before returning to the plasma
membrane. Unlike Arf1, whose association with Golgi membranes is
readily perturbed by brefeldin A (BFA), the binding of Arf6 to endosomal
membranes was BFA-insensitive (D'Souza-Schorey et al., 1998). This is in
agreement with subsequent work demonstrating that most of the Arf GEFs
found in the cell periphery are insensitive to BFA (Jackson and Casanova,
2000).
Further evidence of a unique localization for Arf6 came from the
expression of constitutively active (Arf6-L67) or dominant inhibitory (Arf6-
N27) mutants. While an activated Arf1 mutant (Arf-L71) localized to Golgi
membranes and stabilized the membrane association of coat proteins (Zhang
et al., 1994) (Peters et al., 1995), the corresponding Arf6 mutant was
typically found at the plasma membrane (Peters et al., 1995). In HEK293
cells, expression of Arf6-L67 induced the formation of numerous, deep
plasma membrane invaginations and sheet-like extensions (Peters et al.,
1995). Importantly, electron microscopy revealed no concentration of the
mutant protein in clathrin-coated pits or vesicles, and the number and
appearance of these structures was unchanged (Peters et al., 1995). These
observations suggested that Arf6 does not become incorporated into clathrin
coated vesicles at the cell surface and thus is less likely to play a direct role
in the recruitment of AP-2.
The distribution of a mutant predicted to be in the GDP-bound
conformation, Arf6-N27, also differed from that of the corresponding Arf1
mutant and from the activating mutant, Arf6-L67. While Arf1-N31 is
predominantly cytosolic, Arf6-N27 was found primarily on endosomal
membranes (Peters et al., 1995). This observation revealed two important
characteristics of Arf6: it is capable of interacting with membranes while in
the GDP conformation, and its localization within the plasma
membrane/endosomal system is regulated by its GTPase cycle.
Interestingly, in some cells (HEK293) but not others (e.g., CHO, HeLa;
D'Souza-Schorey et al., 1998; Peters et al., 1995; Peters et al., 2001),
expression of the Arf6-N27 mutant induced the formation of coated
endosomal structures. Recent biochemical characterization of the protein
coats revealed that they do not contain components of other well-
characterized coats involved in vesicular transport, including clathrin,
adaptor complexes AP-1, AP-2, AP-3 or AP-4, or the coatomer complexes
COP-I or COP-II (Peters et al., 2001). However, it should be noted that the
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4 CASANOVA

ability of Arf6•GDP to stimulate coat formation runs counter to the

paradigm for class I Arfs, in which it is the GTP-bound form that nucleates
coat assembly. Clearly it will be important to determine the nature of this
novel coat, and whether its assembly is a general feature of Arf6 function in
all cells.
The nature of the endosomal compartment(s) containing Arf6 appears to
vary from one cell type to another. For example, in Chinese hamster ovary
(CHO) cells Arf6 is present on endosomes that contain transferrin receptor
and cellubrevin (D'Souza-Schorey et al., 1998). However, in HeLa cells
Arf6 and transferrin appear in distinct endosomal structures and Arf6
mutants do not perturb transferrin traffic in these cells (Radhakrishna and
Donaldson, 1997) (see below). Moreover, in renal tissue, endogenous Arf6
is concentrated at the apical pole of epithelia, on the apical plasma
membrane, in apical endosomes, or both (Londono et al., 1999; Maranda et
al., 2001). Finally, in adrenal chromaffin cells, the majority of endogenous
Arf6 was found on the membranes of secretory granules rather than
endosomes, and translocated to the plasma membrane in response to
secretagogues (Caumont et al., 1998). Such diversity in the subcellular
localization of Arf6 suggests an equally diverse set of functions, however, it
is likely that Arf6 utilizes a small number of effectors to perform similar
functions at multiple cellular sites (see below).

4. FUNCTIONS

4.1 Endocytosis

The localization of Arf6 at the plasma membrane and in endocytic

compartments suggested a potential role in endocytosis. Considering that
Arf1 regulates the binding of the clathrin-associated AP-1 adaptor complex
to membranes of the trans-Golgi network (Stamnes and Rothman, 1993;
Traub et al., 1993; Seaman et al., 1996), it seemed plausible that Arf6 may
perform the same function for the AP-2 adaptor complex at the plasma
membrane. However, as noted above, early morphological studies
determined that wild-type Arf6 did not concentrate in clathrin-coated pits or
vesicles, nor did expression of Arf6 mutants perturb the number or
distribution of these structures (Peters et al., 1995), implying that any effect
of Arf6 on clathrin-mediated endocytosis is likely to be indirect. Moreover,
several studies have determined that expression of either wild-type or
constitutively active Arf6, or the Arf6 exchange factor EFA6 in CHO cells
actually inhibits the clathrin-dependent endocytosis of transferrin receptor
(D'Souza-Schorey et al., 1995; Franco et al., 1999). This ability of Arf6 to
 ARF6 2875

affect clathrin-mediated endocytosis without actually localizing to clathrin-

coated pits suggested that it might regulate endocytosis through an indirect
mechanism.
An alternative hypothesis for Arf6 function in the endocytic pathway is
that Arf6 controls the recycling of internalized membrane components from
endosomes back to the plasma membrane. A prediction of this model is that
expression of the dominant inhibitory mutant, Arf6-N27, should induce the
accumulation of normally recycling proteins in an endosomal compartment
and reduce their levels at the plasma membrane. In general, available
experimental data support this model, although the specific proteins affected
vary with cell type. For example, the endosomal distribution of transferrin
receptor (which is often used as a model for recycling receptors) overlaps
significantly with that of Arf6 in CHO cells (D'Souza-Schorey et al., 1998),
and expression of Arf6-N27 inhibited recycling of internalized transferrin in
these cells (D'Souza-Schorey et al., 1995). In contrast, Arf6 exhibited little
overlap with transferrin receptors in HeLa cells, and instead appeared to
localize to a distinct endosomal compartment, enriched in proteins
internalized by clathrin-independent mechanisms (e.g., MHC class I or IL-2
receptor α chain [also known as Tac] (Radhakrishna and Donaldson, 1997)).
Perhaps not surprisingly, Arf6-N27 has little effect on transferrin recycling
in these cells, and instead dramatically inhibited the recycling of Tac
(Radhakrishna and Donaldson, 1997). While the observations in CHO and
HeLa cells were superficially disparate, they share important similarities. In
both cases, Arf6 regulates the flow of membrane from an endosomal
compartment to the cell surface. In both cases, the compartment on which
Arf6 resides is perinuclear, exhibits tubular extensions and is relatively
inaccessible to fluid-phase markers such as HRP. All of these are defining
characteristics of “recycling endosomes”. Maxfield and colleagues have
shown in CHO cells that the default transport pathway for internalized
“bulk” membrane components (i.e., lipids or membrane proteins lacking
cytoplasmic sorting signals that would divert them from this route) passes
through the recycling endosome (Mayor et al., 1993). The enrichment of
MHC class I and Tac in the Arf6-labeled compartment in HeLa cells
suggests that it is functionally similar to the recycling endosome in CHO.
The major difference between the two cell lines therefore appears to be that
Arf6 regulates transferrin recycling in CHO but not HeLa cells. This may
simply reflect a difference in the site at which transferrin receptor is sorted
following endocytosis. In CHO cells, a large fraction of the receptor passes
through the recycling endosome, whereas in HeLa it is possible that most of
it is returned to the plasma membrane directly from more peripheral sorting
endosomes. A direct comparison between the two cell lines, using the same
parameters, will be required to definitively resolve this issue.
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6 CASANOVA

4.2 Recycling of the GLUT4 Glucose Transporter

Insulin stimulates glucose uptake into adipose and muscle tissues by

triggering translocation of the glucose transporter GLUT4 from an
intracellular storage pool to the plasma membrane. This shift in distribution
is accomplished by an insulin-induced increase in the rate of GLUT4
recycling, coupled with a decrease in endocytic rate. However, the
transporters continue to cycle through the endocytic and recycling pathways
even in the presence of insulin, indicating that GLUT4 transport is a form of
regulated recycling (Pessin et al., 1999).
The effects of Arf6 mutants on the recycling of other membrane proteins
suggested that Arf6 may regulate GLUT4 traffic in insulin-responsive cells.
However, initial tests of this hypothesis produced somewhat contradictory
results. In one report, addition of myristoylated peptides, corresponding to
the Arf6 N-terminal helix, to permeabilized adipocytes significantly
impaired insulin-induced GLUT4 translocation, while corresponding
peptides from Arf1 or Arf5 had little effect (Millar et al., 1999). However,
in a second report, expression of a nucleotide-free, dominant inhibitory Arf6
mutant (Arf6-N125) in intact adipocytes had no effect on either GLUT4
translocation or glucose uptake (Yang and Mueckler, 1999). More recent
data from two other laboratories suggest that the latter report is correct;
expression of the more widely used dominant inhibitory form, Arf6-N27,
failed to inhibit insulin-induced GLUT4 translocation (Lawrence and
Birnbaum, 2001; Bose et al., 2001). However, glucose uptake can also be
stimulated by a different agonist, endothelin-1, and it was discovered that
Arf6-N27 did inhibit GLUT4 translocation in adipocytes treated with
endothelin (Lawrence and Birnbaum, 2001; Bose et al., 2001). In contrast to
insulin, which binds to and activates a receptor tyrosine kinase, endothelin
binds to a G-protein coupled receptor, linked to the heterotrimeric GTPases
Gαq or Gα11. In fact, the effects of endothelin on GLUT4 traffic could be
mimicked by expression of activated Gαq or Gα11 mutants. Several other
important differences between the two signaling pathways were also noted.
First, insulin-induced glucose transport was sensitive to inhibition of PI 3-
kinase by wortmannin, whereas endothelin-induced transport was not (Bose
et al., 2001). Second, while insulin induced translocation of both GLUT4
and a second glucose transporter, GLUT1, endothelin stimulated GLUT4,
but not GLUT1 transport (Lawrence and Birnbaum, 2001). Third, while
both insulin and endothelin induced reorganization of cortical actin, only
endothelin-induced actin assembly was inhibited by Arf6-N27 (Bose et al.,
2001).
These results, while intriguing, raise several important questions. First,
how can the same transport process be differentially sensitive to Arf6? One
 ARF6 2897

possibility, for which there is experimental evidence, is that GLUT4 exists in

two distinct endosomal pools (Millar et al., 1999). One of these pools is
thought to reside in a specialized storage compartment, analogous to
regulated secretory granules, which would presumably be sensitive to insulin
but not endothelin. The other pool may represent transporters residing in the
constitutive recycling pathway, and would respond to endothelin signaling
via Arf6 (Figure 1).

Figure 1. Alternative models for the role of Arf6 in GLUT4 transport. In model A, ligation of
endothelin receptors activates Arf6 through a PI3-kinase independent mechanism (possibly
EFA6) and this stimulates the recycling of GLUT4 transporter via a constitutive recycling
pathway. Insulin mediated signaling stimulates translocation of both GLUT4 and GLUT1 via
a distinct, insulin sensitive pathway that does not require Arf6 function. The same results
could also be explained by model B, in which expression of Arf6-N27 leads to selective
sequestration of endothelin receptors within the recycling endosomal compartment, reducing
the number of receptors at the cell surface.
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8 CASANOVA

A more puzzling question is the role of PI 3-kinase. Direct evidence

exists in adipocytes that insulin stimulates the PI 3-kinase-dependent
recruitment of two different exchange factors for Arf6, ARNO and GRP1,
to the plasma membrane via their PH domains (Venkateswarlu et al., 1998;
Langille et al., 1999). Presumably Arf6 is activated under these conditions,
yet GLUT4 translocation is not inhibited by Arf6-N27. Conversely,
endothelin triggers translocation independently of PI 3-kinase, yet Arf6
activation is necessary for this event. It is possible that endothelin and
insulin recruit distinct Arf GEFs (e.g., EFA6 vs. ARNO or GRP-1), however
this remains to be tested. Another possibility is that Arf6-N27 sequesters
endothelin, but not insulin receptors, within an endosomal compartment, so
that they are not available for activation at the cell surface, a prospect that
was not examined in either study. Although it is clear that more work is
necessary to clarify its precise role in glucose transporter traffic, these data
suggest that Arf6 regulates some, but not all forms of endosomal recycling.

4.3 Regulation of endocytosis in epithelial cells

An endocytic function for Arf6 has also been described in polarized

epithelial cells. As noted above, endogenous Arf6 has been localized by
electron microscopy to the apical brush border membrane and to apical
endocytic structures in kidney proximal tubule epithelia (Londono et al.,
1999; Maranda et al., 2001). Altschuler et al. (Altschuler et al., 1999) have
demonstrated a similar distribution for ectopically expressed Arf6 in two
renal tubule cell lines, MDCK and LLC-PK1. However, in contrast to its
inhibitory effects in non-polarized cells, expression of Arf6-L67 in MDCK
cells actually stimulated clathrin-mediated endocytosis (Altschuler et al.,
1999). Moreover, the observed increase in endocytic rate was restricted to
the apical pole, consistent with the predominantly apical distribution of both
the wild-type and mutant Arf6. The enhanced endocytic rate correlated with
an increased density of clathrin-coated pits at the apical membrane
(Altschuler et al., 1999), again in contrast with observations in non-polarized
cells (Peters et al., 1995). The ability of Arf6 to influence apical endocytosis
may be related to its capacity to modulate actin cytoskeleton assembly (see
below). Although the precise relationship between actin and clathrin
assembly at the apical membrane is unclear, apical endocytosis is acutely
sensitive to actin depolymerizing drugs such as cytochalasin D, whereas
basolateral endocytosis is not (Gottlieb et al., 1993). A more puzzling
observation, however, was the finding that the dominant inhibitory mutant,
Arf6-N27, also stimulated apical endocytosis (Altschuler et al., 1999). The
reasons for this are not readily apparent, and will clearly require further
study to resolve.
 ARF6 2919

Palacios et al. (Palacios et al., 2001) have reported a very different

distribution of Arf6 in MDCK cells. In their hands, the same epitope-tagged
Arf6 construct localized to lateral rather than apical membranes. Although
apical endocytosis was not measured, these authors found that expression of
Arf6-L67 led to disassembly of adherens junctions, accompanied by
internalization of the laterally localized cell adhesion molecule, E-cadherin.
Endocytosis of E-cadherin is not thought to be clathrin-mediated, and was
not assayed by Altshuler, et al. In contrast, the dominant negative mutant
(Arf6-N27) inhibited junction breakdown and E-cadherin internalization in
response to hepatocyte growth factor (HGF), which normally induces
separation and migration of epithelial cells. Again, this observation
disagrees with the results in non-polarized cells, where Arf6-N27 inhibits
recycling, not endocytosis. However, in this context the effect of Arf6 on E-
cadherin internalization may be indirect. Cell surface cadherin is anchored
to the actin cytoskeleton, which limits its diffusion in the plane of the
membrane, but may also prevent its constitutive internalization. If Arf6 is
activated by HGF, this could lead to reorganization of cortical actin,
dissociation of E-cadherin from the cytoskeleton, and its subsequent
endocytosis. The ability of Arf6-N27 to inhibit this process would be
consistent with such a hypothesis, but this remains to be tested directly.

4.4 Regulated Secretion

A role for Arf in regulated secretion has been described in a number of

experimental systems including HL-60 cells, neutrophils, RBL basophilic
leukemia cells (a mast cell model), adrenal chromaffin cells, and their tumor
derivative, PC12 cells. A consensus among workers in this field seems to be
that Arf6 promotes regulated secretion by stimulating the activity of the
well-characterized Arf effector phospholipase D (PLD) at the plasma
membrane. For instance, Bader and coworkers (Caumont et al., 1998) have
shown that in resting adrenal chromaffin cells Arf6 is localized to the
membranes of secretory granules, while PLD resides on the plasma
membrane. Stimulation of these cells resulted in translocation of Arf6 to the
plasma membrane, coincident with an increase in PLD activity and granule
content release. PLD activity in permeabilized cells could be stimulated by
recombinant, myristoylated Arf6, and inhibited by isoform-specific Arf6
antibodies (Caumont et al., 1998), demonstrating that Arf6 is not simply
carried with the granule membrane to the cell surface but plays an important
role in the exocytic event.
In contrast, Wakelam and coworkers (Brown et al., 1998) have shown
that PLD resides on secretory granules in RBL cells (rather than the plasma
membrane), and that it translocates to the plasma membrane in response to
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antigen stimulation. Little colocalization of Arf6 with PLD was observed

prior to stimulation, but significant overlap was demonstrated in stimulated
cells. Thus it appears that both Arf6 and PLD are required for regulated
secretion but the mechanisms for bringing the two proteins together at the
plasma membrane differ among cell types. Notably, it is PLD1b that
appears to be the target of Arf6 in most cells. In transfected PC12 cells, a
catalytically inactive PLD1b mutant inhibited the K+-induced release of
stored growth hormone, while a similar PLD2 mutant did not (Vitale et al.,
2001). Powner et al. demonstrated directly, using surface plasmon
resonance, that PLD1b can bind Arf6 (Powner et al., 2002). Furthermore,
PLD1b also binds Rac1 and PKCα simultaneously with Arf6, and maximal
PLD activity is achieved only when all three regulatory proteins are bound
(Powner et al., 2002).
Fractionation of membranes from resting chromaffin cells revealed that
ARNO colocalized with plasma membrane markers, suggesting that Arf6
encounters its GEF upon delivery to the plasma membrane. At least in
chromaffin cells, the GEF responsible for the agonist-induced activation of
Arf6 appears to be ARNO (Caumont et al., 2000). Incubation of
permeabilized cells with anti-ARNO antibodies inhibited both calcium-
evoked PLD activity and catecholamine secretion in a dose-dependent
manner. Similarly, expression of a catalytically inactive ARNO mutant in
PC12 cells also inhibited secretion induced by membrane depolarization.
Together, these data provide strong support for the notion that Arf6 plays an
important role in regulated secretion, primarily through its modulation of
PLD activity.

4.5 Regulation of G-protein-coupled receptor

desensitization

Accumulating evidence indicates that Arf6 can regulate the internalization

and/or desensitization of a subset of G-protein coupled receptors,
particularly those that are linked to arrestins (Mukherjee et al., 2000; Perry
and Lefkowitz, 2002). Because this topic is covered in other chapters (see
Chapters 8 and 15) it will not be described in detail here.

4.6 A role for Arf6 in actin cytoskeleton dynamics

A large body of evidence indicates that the actin cytoskeleton can be

dynamically assembled and disassembled in response to extracellular cues,
through signaling pathways that involve small GTPases of the Rho family
(Van Aelst and D'Souza-Schorey, 1997). In most cell types, activation of
 ARF6 293
11

RhoA leads to formation of stress fibers, while activation of Rac or Cdc42

results in the formation of lamellipodia or filopodia, respectively. Members
of the Arf family, particularly Arf6, can also modulate cytoskeleton
assembly, and may do so coordinately with Rho GTPases.
Endogenous Arf6 has been localized to areas of the cell undergoing
active cytoskeletal remodeling, including the leading edge of motile cells
(Santy and Casanova, 2001) and the periphery of spreading cells (Song et
al., 1998). Donaldson and colleagues first provided evidence that Arf6 plays
an active role in actin remodeling by showing that treatment of Arf6-
expressing HeLa cells with aluminum fluoride (AlFn) induced the formation
of actin rich membrane protrusions (Radhakrishna et al., 1996). AlFn
stabilizes the interaction between many GTPases and their associated GAPs,
thereby inhibiting GAP function and shifting the GTPase cycle toward the
GTP-bound state. (It should be noted that AlFn also activates heterotrimeric
G proteins directly by mimicking the gamma phosphate. An additional
complication of this reagent is that it can inhibit a wide variety of
phosphotransfer reactions in cells. Therefore, when added to intact cells,
AlFn presumably activates many GTPases.) However protrusions only
appeared in cells expressing exogenous Arf6 and similar structures were
observed in cells transiently expressing Arf6-L67, suggesting that their
formation in this context was Arf6-dependent (Radhakrishna et al., 1996).
Similar observations were made in CHO cells, where expression of Arf6-
L67 led to formation of structures more closely resembling classical
lamellipodia (D'Souza-Schorey et al., 1997).
Several lines of evidence suggest that Arf6 modulates actin assembly
through crosstalk with GTPases of the Rho family, probably by acting
upstream of the Rho protein. First, expression of Arf6-L67 (Boshans et al.,
2000) or of the Arf exchange factors ARNO (Frank et al., 1998) or EFA6
(Franco et al., 1999) interferes with assembly of actin stress fibers, a process
mediated by RhoA. Direct measurement of RhoA activation revealed a
robust increase in RhoA•GTP levels in control cells treated with
lysophosphatidic acid (a stimulator of Rho activation), and this was
paralleled by an increase in stress fiber formation (Boshans et al., 2000).
However, in cells expressing Arf6-L67, no such increase in RhoA activation
or stress fiber assembly was observed. In contrast, abundant stress fibers
were formed in cells co-expressing Arf6-L67 and an activated RhoA mutant
(RhoA-V14), indicating that Arf did not affect signaling downstream of Rho
leading to stress fiber assembly (Boshans et al., 2000). Presumably Arf6
inhibits Rho activation by decreasing Rho-GEF activity, increasing Rho-
GAP activity, or both. The recent discovery of several Arf GAPs that also
possess Rho GAP domains (Miura et al., 2002; Krugmann et al., 2002)
294
12 CASANOVA

suggests that recruitment of such Arf GAPs to sites of Arf6 activation could
lead directly to the down regulation of Rho.
As mentioned above, in many cell types activation of Arf6 also leads to
the formation of flat, actin-rich structures resembling lamellipodia. Motile
cells extend lamellipodia in the direction of movement, and inhibition of this
process dramatically slows cell motility (Ridley et al., 1999). Membrane
ruffling and lamellipodial extensions can be induced by a variety of
extracellular signals including growth factors (PDGF, EGF, HGF, CSF-1,
insulin), integrin ligation, and a number of G-protein coupled receptor
agonists (e.g., bombesin) through signaling pathways coupled to the
activation of Rac. Initial evidence of crosstalk between Arf and Rac came
from the discovery that the two GTPases share a common effector,
POR1/Arfaptin2 (D'Souza-Schorey et al., 1997; see Chapter 11). Although
the function of this protein is not well understood, D’Souza-Schorey et al.
showed that POR1 mutants interfered with actin remodeling induced by
either activated Rac1 or Arf6-L67, suggesting that these two proteins operate
on parallel pathways to modulate actin dynamics. However,
POR1/Arfaptin2 interacts with Arf1 as well as Arf6, and exhibits a striking
localization to the Golgi rather than the plasma membrane (Kanoh et al.,
1997). While it remains possible that a fraction of POR1/Arfaptin2
functions in the cell periphery to modulate cytoskeleton assembly, it seems
more likely that its primary function is at the Golgi, and the inhibitory
effects of mutant expression on lamellipodial extension are due to
mislocalization of the overexpressed constructs.
Further evidence of crosstalk between Rac and Arf6 is provided by the
observation that Arf6-N27 inhibits membrane ruffling in response to EGF
(Honda et al., 1999), bombesin (Boshans et al., 2000) or CSF-1 (Zhang et
al., 1999). In macrophages, Arf6-N27 dramatically inhibits Fcγ-mediated
phagocytosis (Zhang et al., 1998), a process also known to require activation
of both Rac and Cdc42. Similarly, activation of Arf6 by expression of the
exchange factor EFA6 induces membrane ruffling that is inhibited by co-
expression of dominant negative Rac1-N17 (Franco et al., 1999).
Furthermore, expression of the Arf GEF ARNO in MDCK cells is sufficient
to induce a dramatic morphological transition from a cuboidal, stationary
phenotype to a flattened, motile phenotype with pronounced lamellipodia
(Santy and Casanova, 2001). This transition was accompanied by the
activation of both endogenous Arf6 and Rac1. As in the case of EFA6-
induced ruffling (Franco et al., 1999), lamellipodia did not form in the
presence of dominant inhibitory Rac1-N17, suggesting that Rac lies
downstream of Arf6 in a GTPase signaling pathway.
How might Arf6 activation lead to activation of Rac? One model that has
gained favor in recent years is that Arf6 regulates the translocation of an
 ARF6 295
13

endosomal pool of Rac to the plasma membrane (Radhakrishna et al., 1999).

Several groups have reported the localization of either endogenous
(Radhakrishna et al., 1999) or exogenous (Boshans et al., 2000) Rac1 to
endosomal membranes and the colocalization of this endosomal pool with
Arf6. Moreover, stimulation of cells with either AlFn (Radhakrishna et al.,
1999) or bombesin (Boshans et al., 2000) resulted in an apparent shift of
both Rac and Arf6 from the endosomal pool to the plasma membrane.
Importantly, expression of Arf6-N27 both prevented this translocation and
inhibited membrane ruffling.
Together, these data form the basis of a model in which Arf6 regulates
the translocation of endosomal Rac to the plasma membrane where Rac-
specific GEFs would presumably activate it. This model is appealing
because it incorporates the role of Arf6 in endosomal membrane recycling
with its observed effects on the organization of the actin cytoskeleton.
However, there are several problems with this model that bear further
investigation. First, in most cells, a substantial pool of cytosolic Rac exists
bound to Rho-GDI (Del Pozo et al., 2002), which could be recruited to the
plasma membrane without invoking a vesicular transport mechanism.
Second, Arf6-N27 inhibits membrane ruffling, induced by an activated Rac
construct anchored to the plasma membrane by fusion to a heterologous
transmembrane domain (Zhang et al., 1999) (suggesting that translocation to
the plasma membrane is not sufficient to induce ruffling). Third, inhibition
of Arf6 function impairs some, but not all aspects of Rac signaling; Boshans
et al. have shown that while Arf6-N27 dramatically inhibits bombesin-
induced membrane ruffling, it has no effect on bombesin-induced MAP
kinase activation, which is also Rac-dependent (Boshans et al., 2000). This
last observation indicates that physiological agonists can activate Rac even if
Arf6 function is inhibited, and supports previous evidence that the effects of
Rac on membrane ruffling and on signaling to the nucleus can be uncoupled.
Direct measurement of Rac•GTP levels in the presence and absence of Arf6-
N27 should resolve this issue.
An alternative mechanism for Arf-induced Rac activation has been
proposed, in which Arf6 activation is coupled to the recruitment of a Rac
GEF. A subset of the Arf GAPs, the GITs (see Chapter 8) interact directly
with the Rac GEF, β-PIX, which is in turn coupled to a Rac effector, the
p21-activated kinase, PAK (Manser et al., 1998). The intrinsic nucleotide
exchange activity of β-PIX is quite low, however it is significantly enhanced
by its interaction with PAK. In this model, the GIT/PIX/PAK complex is
recruited to sites of local Arf6 activation through the interaction of the GIT
Arf GAP domain with Arf6•GTP. This would lead to the localized
activation of Rac, the subsequent activation of the bound PAK [if recruited
as a complex], phosphorylation of PAK substrates and consequent
296
14 CASANOVA

remodeling of the actin cytoskeleton (Figure 2). A variation on this theme is

that the GIT/PIX/PAK complex is constitutively localized to the plasma
membrane in an inactive conformation, and is then activated by its
interaction with Arf6•GTP. Testing of each model is currently underway.

Figure 2. Assembly of a GIT/PIX/PAK complex. Binding of GIT to Arf could stimulate

either membrane recruitment or enzymatic activity of PIX and PAK.

4.7 Arf6, phospholipid signaling and actin remodeling

Another mechanism by which Arf6 may regulate actin dynamics is through

its effects on membrane phospholipid composition. All Arfs are allosteric
activators of two lipid-modifying enzymes, phospholipase D (PLD, see
Chapter 11), and an isoform of phosphatidylinositol 4-phosphate, 5-kinase
(PI(4P) 5-kinase α or PI(4P)5K). These two enzymes are functionally
linked, in that PI(4P)5K is dependent on both phosphatidic acid, the product
of PLD, and interaction with Arf•GTP for its activity (Honda, et al., 1999).
The result of this linkage is lipid signaling in which the activation of PLD by
Arf leads, through PI(4P)5K, to enhanced local production of PI(4,5)P2 (see
Figure 3). Phosphatidic acid (PA) itself has a variety of biologically
important properties; its charged head group can attract proteins to the
membrane through electrostatic interactions, it can increase the fluidity of
membrane lipids, and it can be further metabolized to generate
diacylglycerol (an activator of PKC) as well as lysophosphatidic acid (a G-
protein coupled receptor agonist) (for review see Liscovitch et al., 2000).
PI(4,5)P2 also has a number of biological properties, including functions in
signaling, endocytosis, exocytosis, regulation of ion channels, and a well-
characterized ability to modulate the activity of a variety of actin-associated
proteins including vinculin, ezrin, profilin, α-actinin and WASp.
However, a confounding aspect of the PI(4,5)P2/PLD relationship is that
PI(4,5)P2 is also required for PLD activity. Both PLD1 and PLD2 possess
PH domains whose binding to PI(4,5)P2 mediate the interaction of these
proteins with cellular membranes (Liscovitch et al., 2000). Thus, in this
 ARF6 297
15

context, a basal level of PI(4,5)P2 is necessary to bring PLD together with its
substrate. Therefore, determining whether PI(4,5)P2 is serving solely to
recruit PLD or is truly required as a downstream effector necessitates careful
experimental design. Recently, Cockroft and coworkers used an Arf1
mutant that can activate PI(4P)5K but not PLD (Arf1-R52), to demonstrate
that 45% of the Arf-stimulated PI(4,5)P2 synthesis in HL-60 cells was
dependent on PLD activity, whereas the remaining 55% was PLD-
independent (Skippen et al., 2002). Thus, a significant fraction of PI(4,5)P2
can be generated by a pathway that does not require new PA synthesis.

Figure 3. Phospholipid signaling downstream of Arf6.

It has become possible in recent years to localize phosphoinositides in

cells using GFP fusions to PH domains of varying phosphoinositide
specificity. Using such a GFP construct fused to the PH domain of PLC-δ,
Brown et al. have shown that PI(4,5)P2 colocalizes extensively with Arf6,
particularly at the plasma membrane and in the distal portions of tubular
endosomes (Brown et al., 2001). Moreover, these authors found that
activation of Arf6 by expression of the exchange factor EFA6 induced the
formation of numerous large pinosomes whose membranes were enriched in
both EFA6 and PI(4,5)P2. While these pinosomes were dynamic, similar
large pinocytic structures were observed in cells expressing Arf6-L67,
except that these accumulated intracellularly (Brown et al., 2001). Schafer
et al. have also demonstrated increased numbers of pinosomes in cells
expressing activated Arf6, as well as actin-rich foci on the ventral surface of
the cells (Schafer et al., 2000). The pinosome-like structures often had
actin-rich “tails” associated with them, and were highly motile. This
motility was actin-dependent, required PLD activity and D-3
phosphoinositides, but was apparently not dependent on PI(4,5)P2 as
microinjection of anti-PI(4,5)P2 antibodies did not impair movement.
Activation of Arf6 in MDCK cells resulted in increased activation of
Rac, formation of broad lamellipodia and the onset of motility (Santy and
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16 CASANOVA

Casanova, 2001). In testing the role of PLD in these phenomena, we found,

to our surprise, that induction of lamellipodia was dependent on PA
production while the activation of Rac was not. In cells treated with low
concentrations of 1-butanol (which substitutes for water in the hydrolysis of
PC, leading to the production of biologically inert phosphatidylbutanol
rather than PA), levels of active Rac remained high, yet lamellipodia did not
form (Santy and Casanova, 2001). These findings suggest that PA is
required for the dynamic reorganization of the actin cytoskeleton that is
orchestrated by Rac; however, it is unclear whether it is PA itself, PI(4,5)P2,
or one of their metabolites that is the necessary lipid component. Given the
caveats described above, a combined approach using pharmacological
inhibitors, dominant negative proteins and/or anti-sense inhibition of PLD
and PIP5K isoforms will be necessary to resolve this question.
Taken together, these results indicate that Arf6 plays an important role in
the regulation of actin cytoskeleton dynamics. The mechanisms by which
Arf6 modulates actin assembly are complex, and probably include effects on
endosomal membrane traffic, crosstalk with Rho family GTPases and
changes in local membrane lipid composition. Deciphering the contribution
of each will be a difficult, but achievable goal.

5. SUMMARY

In attempting to summarize the data presented above, one thing is

abundantly clear: the function of Arf6 in eukaryotic cells is complex, highly
variable between cell types, and incompletely understood. However, it is
possible to extract some unifying principles. First, the ability of Arf6 to
regulate post-endocytic recycling appears to be a common feature in all cells
where it has been examined. The mechanisms by which this is
accomplished remain mysterious, but may involve the assembly of a novel
protein coat on endosomal membranes. Alternatively, or additionally,
recycling may utilize components of the actin cytoskeleton whose function is
modulated by Arf6. The ability of actin-depolymerizing drugs to inhibit
recycling in HeLa cells (Radhakrishna and Donaldson, 1997) and adipocytes
(Bose et al., 2001) suggests that this may be the case.
The capacity of Arf6 to affect actin cytoskeleton dynamics is related in
part to its interaction with GTPases of the Rho family. In the case of Rho
itself, this interaction is likely to occur through a subfamily of Arf GAPs that
also possess Rho GAP domains (Miura et al., 2002; Krugmann et al., 2002;
Santy and Casanova, 2002). Crosstalk between Arf6 and Rac also occurs in
all cells where it has been examined. Although the precise nature of the
 ARF6 299
17

interaction remains unclear, it may also involve the participation of Arf

GAPs or shared effectors, e.g., PLD or Arfaptin 2/POR1.
Finally, it is likely that the functions of Arf6 in both membrane traffic
and cytoskeleton dynamics can be attributed, at least in part, to its
modulation of membrane lipid composition. In most cases where it has been
examined, inhibition of PLD activity abrogates the downstream effects of
Arf6 activation. Unfortunately, due to the lack of specific pharmacological
inhibitors, the tool of choice for this purpose is often treatment of cells or
membrane preparations with primary alcohols. While effective, this
treatment does not distinguish between PLD isoforms and may have other
nonspecific effects. The emerging use of catalytically inactive PLD mutants
as dominant inhibitors should help to clarify the specific roles of PLD
isoforms in Arf-dependent processes.

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Chapter 15
A ROLE FOR ARF6 IN G PROTEIN-COUPLED
RECEPTOR DESENSITIZATION

MARY HUNZICKER-DUNN
Department of Cell and Molecular Biology, Northwestern University Medical Schoo1,
Chicago, USA

Abstract: In addition to its roles in changes in the actin cytoskeleton and lipid
metabolism at the plasma membrane, recent results suggest that Arf6 also
participates in desensitization and internalization of G-protein-coupled
receptors. The evidence to support this novel function for Arf6 is reviewed in
this chapter.

1. INTRODUCTION

The superfamily of seven membrane-spanning receptors that can couple to

heterotrimeric guanine nucleotide binding (G) proteins responds to a
multitude of extracellular signals. These G-protein-coupled receptors
(GPCRs) in turn regulate a variety of cellular responses that reflect their
diverse group of specific agonists, including muscle contractility, ovulation,
hormone secretion, gene expression, vision, taste, and neuronal excitation.
All GPCRs exhibit conserved amino acid residues that comprise seven
transmembrane (TM) alpha helices connected by variable extracellular and
intracellular loops. In the absence of agonist, GPCRs exist in an inactive
conformation, based on the interactions of key amino acid residues
contained in highly conserved domains. Upon binding agonist, these
interactions are altered and the receptors assume an activated conformation
in which they catalyze the rate-limiting release of GDP from heterotrimeric
G proteins. G protein activation then drives increased effector activity,
resulting, for example, in increased formation of second messengers such as
cAMP, inositol trisphosphate and diacylglycerol, and opening or closing of
ion channels.
305
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2 HUNZICKER-DUNN

Coupling of GPCRs to G protein(s) is regulated both by the rates of

association and dissociation of receptor ligands as well as by an apparently
common receptor desensitization mechanism. Desensitization functions to
uncouple agonist-bound receptor from its G protein and is mediated by a
family of proteins known as Arrestins. The two models of GPCR
desensitization that have been most intensely investigated are light
regulation of rhodopsin and isoproterenol regulation of the β2-adrenergic
receptor (β2-AR). It is well established that GPCR desensitization is
triggered by agonist-dependent receptor phosphorylation catalyzed by a
family of G protein receptor kinases (GRKs). Arrestin then binds with high
affinity to the active, phosphoreceptor. Arrestin binding to receptor
interrupts the ability of the receptor to activate G proteins.
Recent results reveal an unexpected function for Arf6 in this sequence of
events leading to homologous (agonist-specific) receptor desensitization.
Utilizing the luteinizing hormone receptor (LHR) as a prototypical GPCR in
a model in which only endogenous proteins are present in their native
environment, we have shown that Arf6 functions to sequester a pool of
Arrestin that is obligatory for LHR desensitization. Agonist activated
receptor not only activates its heterotrimeric G protein but also activates
Arf6, resulting in the undocking of Arrestin so that it is now available to
bind to this agonist-bound receptor. In this chapter we will review the
results that led to these conclusions and attempt to integrate our data
obtained using a plasma membrane model with the bulk of the literature in
which the Arrestins and GPCRs are overexpressed in intact cellular models.

2. CLASSIC MODEL FOR GPCR

DESENSITIZATION

With more than 1,000 GPCRs and less than 20 G proteins, there is a clear
need for regulation of the temporal aspects of G protein mediated signaling
or simply alternative mechanisms of signal termination. Homologous
desensitization is the phenomenon in which exposure to agonist produces a
state in which reduced responses are observed upon subsequent challenge by
agonist. This type of desensitization can result from covalent modifications
to the receptor (e.g., phosphorylation), changes in the binding partners of the
receptor (e.g., Arrestins), or changes in the location of the receptor
(internalization).
Most agonist-bound GPCRs require GRK-catalyzed phosphorylation of
the receptor in order to achieve high-affinity Arrestin binding (Krupnick and
Benovic, 1998). The family of Arrestins consists of the visual Arrestins
(Arrestin1 in rod cells and Arrestin4 in cone cells) and the ubiquitous
 ROLE OF ARF6 IN GPCR DESENSITIZATION 3073

Arrestin2 (β-Arrestin1) and Arrestin3 (β-Arrestin2) (Palczewski, 1994).

Arrestin is converted into an active conformation that binds GPCRs with
high affinity (in the pM range) upon engagement of two binding sites: one
site that recognizes the active conformation of the receptor and a second site
that interacts directly with GRK-phosphorylated residues on the receptor
(Gurevich and Benovic, 1993; Gurevich et al., 1995; Kovoor, 1999; Celver
et al., 2002). The basic requirements for GRK-catalyzed receptor
phosphorylation and the obligatory binding of Arrestin molecules in GPCR
desensitization were established in studies using a reconstituted model
consisting of phospholipid vesicles, receptor agonist, and recombinant β2-
AR, GRK and Arrestin (Lohse et al., 1990; Lohse et al., 1992). The validity
of this paradigm for GPCR desensitization has now been established for a
large number of GPCRs using intact cell models in which receptors, GRKs,
and (often tagged) Arrestins are co-transfected and expressed at artificially
high levels (Krupnick and Benovic, 1998; Ferguson, 2001; Claing et al.,
2002).
Because Arrestins also target the receptor for translocation to an
intracellular compartment (internalization), based on their ability to bind
both clathrin and β2-adaptin of the AP-2 protein complex (Goodman et al.,
1996; Laporte et al., 1999; Laporte et al., 2000), receptor internalization is
often assayed as an indicator of desensitization. However, desensitization
and internalization of GPCRs are not necessarily linked events. For
example, there is evidence for some GPCRs that desensitization and
internalization are mediated by the binding of distinct Arrestins to distinct
receptor sites (Cheng et al., 1998; Kohout et al., 2001; Paing et al., 2002;
Mukherjee et al., 2002). In such cases desensitization and internalization
cannot be sequential events in a linear pathway. There is also evidence that
internalization of some GPCRs is Arrestin-independent (Pals-Rylaarsdam et
al., 1995; Bhatnagar et al., 2001) while, to date, GPCR desensitization is
Arrestin-dependent. However, despite these caveats in data interpretation,
using either receptor desensitization or internalization as the endpoint,
overexpression of GRKs and/or Arrestins drives desensitization or
internalization of nearly all GPCRs that have been investigated (Krupnick
and Benovic, 1998). Indeed, the ability of GRKs and Arrestins to promote
desensitization or internalization has often been used as evidence that a
specific GPCR requires receptor phosphorylation and Arrestin binding to
achieve desensitization or internalization (Carmen and Benovic, 1999).
Upon receptor activation, overexpressed, tagged Arrestins are uniformly
recruited from a cytosolic compartment to the plasma membrane by an
unidentified mechanism (Ferguson et al., 1996). The recruitment of
Arrestins to the membrane has even been developed as an assay to detect
receptor activation (Barak et al., 1997) as well as to estimate the affinity of
308
4 HUNZICKER-DUNN

Arrestin’s interaction with agonist-bound receptor (Oakley et al., 2000).

Consistent with the fundamental role of Arrestin binding for GPCR
desensitization, Arrestin2 knockout mice show impaired β2-AR
desensitization (Conner et al., 1997) and Arrestin3 knockout mice show
prolonged responsiveness to analgesics like morphine (Bohn et al., 2000).
Mouse embryonic fibroblasts from Arrestin2/Arrestin3 knockout mice also
show impaired desensitization of the angiotensin II type 1A receptor
(Kohout et al., 2001). Thus, the levels of Arrestins and their regulated
interactions with GPCRs clearly play a role in GPCR signaling.

3. DEVELOPMENT OF A NEW MODEL FOR LHR

DESENSITIZATION

3.1 Arrestin2 is stably bound to plasma membranes

Desensitization of some GPCRs, including the LHR, has been demonstrated

under cell-free conditions utilizing a receptor/effector-enriched plasma
membrane model (Bockaert et al., 1976; Roy et al., 1976; Anderson and
Jaworski, 1979; Ezra and Salomon, 1980; Ezra and Salomon, 1981). This
assay involves a two-stage incubation: in stage 1, membranes are
preincubated without or with agonist to promote receptor desensitization; in
stage 2, the ability of receptor in the absence or presence of agonist to couple
to the stimulatory G protein (Gs) is evaluated using adenylyl cyclase
activity. Diminished adenylyl cyclase activity upon restimulation with
agonist in stage 2 results from receptor desensitization. Under the
appropriate in vitro incubation conditions, 80-100% desensitization of LHR
is achieved with a time-course equivalent to that seen in the intact animal
(Marsh et al., 1973; Hunzicker-Dunn, 1981; Ekstrom and Hunzicker-Dunn,
1990). These results challenge the conclusion that Arrestins must be
recruited from a cytosolic compartment to effect receptor desensitization in
all cases.
For a GPCR to be desensitized in a phosphorylation- and Arrestin-
dependent manner in this cell-free model, both the Arrestin and the GRK
must be present in the plasma membrane-enriched fraction. As the high
affinity binding of Arrestin to a GPCR is the crucial step that uncouples
receptor from its cognate G protein, this model leads to the prediction of a
pool of Arrestin, localized to the membrane fraction used in the two step
assay. Arrestin2 is readily detected by western blotting in the membrane
fraction isolated from ovarian follicles and available to the LHR to mediate
desensitization (Mukherjee et al., 1999a). Similarly, subcellular
 ROLE OF ARF6 IN GPCR DESENSITIZATION 3095

fractionation of other cells has revealed detectable levels of immunoreactive,

endogenous Arrestins in membrane fractions (Bennett et al., 2000; Santini et
al., 2000). The inclusion of Arrestin antibodies that block the ability of
Arrestin to interact with GPCRs (Knospe et al., 1988; Palczewski, 1994)
completely abrogated agonist-dependent LHR desensitization in follicular
membranes (Mukherjee et al., 1999a). This result shows that the
endogenous Arrestin2 present in this LHR-enriched membrane fraction is
necessary and sufficient for LHR desensitization. Addition to the membrane
fraction of either visual Arrestin1 or Arrestin2 promoted desensitization of
active LHR in a dose-dependent manner, while Arrestin3 did not (Mukherjee
et al., 1999a; Mukherjee et al., 2002). Moreover, the concentration of
Arrestin2 that promoted 50% desensitization of LHR activity was in the sub-
nM range, consistent with the probable high affinity binding of Arrestin2 to
the LHR.
In order to identify the Arrestin2 binding sites on the receptor that
promoted LHR desensitization, we first determined whether synthetic
peptides made to various regions of the receptor could compete with
receptor for Arrestin2 and block LHR desensitization. Only peptides
corresponding to the third intracellular (3i) loop, and not those
corresponding to a scrambled 3i peptide, the 2i loop, or an N-terminal
portion of the cytoplasmic tail, abrogated LHR desensitization (Mukherjee et
al., 1999b). The blockade by the 3i loop peptide was rescued by addition of
recombinant Arrestin2, indicating that the 3i peptide blocked LHR
desensitization by competing with receptor for binding of Arrestin2.
Arrestin2 bound directly to the 3i peptide with pM affinity while Arrestin3
bound with only mM affinity (Mukherjee et al., 2002). Asp-564 in the 3i
loop, which interacts with Arg-464 at the base of TM 3 to maintain the
receptor in an inactive conformation (Ballesteros et al., 2001; Shenker,
2002), is required for Arrestin2 to bind to the agonist-bound receptor
(Mukherjee et al., 2002). Even substitution with a negatively charged amino
acid (Glu) did not support Arrestin2 binding to the receptor, suggesting that
the specific geometry of Asp-564 at this site in the 3i loop is required for
Arrestin2 binding to the LHR.
In contrast to many GPCRs, Arrestin2 binds to the LHR without the
requirement for receptor phosphorylation (Ekstrom and Hunzicker-Dunn,
1989; Ekstrom et al., 1992; Zhu et al., 1993; Lamm and Hunzicker-Dunn,
1994; Lamm et al., 1994; Wang et al., 1997). This high affinity binding
appears to be mediated, in part, by Asp-564 in the 3i loop of the receptor,
although the negative charge of this residue is not simply mimicking the
negatively charged phosphate group of phosphorylated GPCRs (Mukherjee
et al., 2002). The homologous Asp residue is conserved in the glycoprotein
hormone and cannabinoid receptors but is replaced with a negatively
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6 HUNZICKER-DUNN

charged Glu in most other receptors (Schulz et al., 2000). Although the
other glycoprotein hormone receptors are believed to require GRK-catalyzed
phosphorylation to bind Arrestins for desensitization (Iacovelli et al., 1996;
Troispoux et al., 2000), it is possible that the presence of this Asp residue in
their 3i loops, as with the LHR, may allow them to bind Arrestin for
desensitization in a phosphorylation-independent manner.
For Arrestin2 to promote LHR desensitization it should bind only to
active and not to inactive receptor. Moreover, the Arrestin2 binding site at
Asp-564 in the 3i loop is not expected to be accessible in the inactive
receptor conformation. Under conditions in which neither receptor nor
Arrestin is overexpressed, Arrestin2 preferentially co-immunoprecipitated
with agonist-bound LHR (Mukherjee et al., 1999a; Mukherjee et al., 2000).
Because the presence of Arrestin2 in LHR-enriched membranes is
independent of agonist and interacts only with agonist-bound LHR,
Arrestin2 must be bound at a site distinct from the LHR, when the receptor
is not active (as shown in Figure 1, panel A). Moreover, there must be a
mechanism that stimulates the “undocking” of Arrestin2 so it is available to
bind to agonist-bound receptor. A clue to the regulation of the Arrestin2
undocking mechanism was revealed by the demonstration that the pool of
Arrestin2 present in the plasma membrane-enriched fraction requires GTP to
be released and effect the desensitization of LHRs.

3.2 LHR desensitization is GTP-dependent

That GPCR desensitization might be more complex and require proteins in

addition to the GRKs and Arrestins was suggested by studies in the early
1980’s by Ezra and Salomon. They used a cell-free model to demonstrate a
GTP requirement for desensitization of the LHR (Ezra and Salomon, 1980;
Ezra and Salomon, 1981). Similar reports soon followed for the β2-AR and
vasopressin receptor in kidney cells (Roy et al., 1976; Anderson and
Jaworski, 1979). Due to the relatively high concentrations of GTP in intact
cells, the GTP requirement for GPCR desensitization could not be
investigated in an intact cell model. This GTP requirement for GPCR
desensitization could theoretically be fulfilled by the activation of a
heterotrimeric G protein, possibly to recruit GRKs via released βγ subunits
(Krupnick and Benovic, 1998). However, as LHR desensitization is
independent of receptor phosphorylation (as reviewed above), βγ-dependent
GRK recruitment cannot be a prerequisite for LHR desensitization. A
requirement for heterotrimeric G protein activation in LHR desensitization
 ROLE OF ARF6 IN GPCR DESENSITIZATION 3117

Figure 1. Proposed model depicting LHR desensitization: the LHR promotes the activation
of ARF6, leading to cessation of LHR coupling to Gs and adenylyl cyclase. (A) The inactive
receptor and key proteins detected by immunoblot or activity assays in ovarian follicle plasma
membrane are shown. (B) Agonist binding promotes a conformational change in the LHR,
resulting in activation of Gs and adenylyl cyclase (AC). Dashed arrows represent the directed
movements of ARNO to bind the NPXXY motif in TM7 of the LHR with consequent
activation of ARF6. This, in turn, releases Arrestin2, allowing its binding to the 3i loop of the
LHR and effecting desensitization. The large dot in the 3i peptide represents Asp-564. (C)
The desensitized LHR is shown with Arrestin2 bound to the 3i loop, ARNO bound to TM 7,
and Gs uncoupled and returned to its basal state.
312
8 HUNZICKER-DUNN

was ruled out by the inability of C-terminal Gα peptides, Gα antibodies,

or βγ internalization to affect LHR desensitization (Rajagopalan-Gupta et al.,
1999). Interestingly, LHR desensitization could be completely reversed in
the membrane model by incubating desensitized LHR with GDPβS,
resulting in re-coupling of the receptor to Gs and adenylyl cyclase (Ekstrom
et al., 1992). More recent studies have led to the identification of the site of
action of GTP that is required for desensitization in in vitro systems.

3.3 Arf6 sequesters Arrestin2

Recombinant Arrestin2 promoted desensitization of the active LHR both in

the presence and absence of GTP (Mukherjee et al., 2000), so the GTP-
dependent step of LHR desensitization could not be the binding of Arrestin2
to the receptor. To determine whether the GTP-dependent step consisted of
the undocking of Arrestin2 from its membrane binding site, membranes
were preincubated with water or 100 µM GTP, diluted ~25-fold, then
repelleted and analyzed. Preincubation of membranes with 100 µM GTP (in
the absence of receptor agonist) resulted in release from the membranes of
more than 80% of the bound Arrestin2 (Mukherjee et al., 2000), which was
detectable in the wash buffer (S. Mukherjee, unpublished observation).
Insufficient Arrestin2 was retained in the membranes to promote LHR
desensitization (Mukherjee et al., 2000). These results demonstrate that the
GTP-dependent component of LHR desensitization is the release of
Arrestin2 from its docking site in the membrane. The identity of the GTP
binding protein responsible for release of Arrestin2 from membranes was
sought.
We turned to the large family of monomeric GTPases as candidates for
the regulator of the availability of Arrestin2 for LHR desensitization.
Members of the Rho, Ras, Rac and Rap families of GTPases, were
discounted based on evidence that the active fragments of the large
Clostridial toxins, C. difficile toxin B, and C. sordelli lethal toxin, did not
affect LHR desensitization (Mukherjee et al., 2000). Arfs 1-5 were also
ruled out based on the inability of brefeldin A, which inhibits many Arf
guanine nucleotide exchange factors (GEFs), to block LHR desensitization
(Mukherjee et al., 2000). However, the Arf GEFs which have been identified
as Arf6 activators are resistant to brefeldin A (Jackson and Casanova, 2000),
and Arf6 is often localized to plasma membrane-enriched cellular fractions
(Cavenagh et al., 1996). To investigate a possible role for Arf6 in LHR
desensitization, we utilized a synthetic peptide corresponding to the N-
terminus of Arf6, which has been shown to block Arf6 activation (Kahn et
al., 1992; Lenhard et al., 1992; Galas et al., 1997; Caumont et al., 1998).
The corresponding N-terminal Arf1 peptide was used as a control. The N-
 ROLE OF ARF6 IN GPCR DESENSITIZATION 3139

terminal Arf6 peptide abrogated LHR desensitization while the

corresponding Arf1 peptide was ineffective (Mukherjee et al., 2000).
Moreover, the Arf6 peptide blocked the GTP-dependent release of Arrestin2
(Mukherjee et al., 2000). These results suggest that Arf6•GDP sequesters
Arrestin2, either directly or indirectly, and that upon activation, Arrestin2 is
released from its membrane docking site, as shown in Figure 1, panel B.

3.4 ARNO promotes Arrestin2 undocking and LHR

desensitization

Consistent with an obligatory role for Arf6 activation in liberating Arrestin2

for receptor desensitization, addition of the Arf1/6 activator ARNO (see
Chapters 2, 4-6) to plasma membranes decreased the concentration of GTP
required for release of Arrestin 2 or for receptor desensitization from 100
µM to 1 µM. Neither catalytically inactive ARNO-K156 (Frank et al., 1998)
nor a catalytically active ARNO containing a mutation in its pleckstrin
homology domain (which interferes with its binding of phosphoinositides
(Nagel et al., 1998)) promoted LHR desensitization (Mukherjee et al., 2000).
Moreover, LHR agonist-dependent Arrestin2 undocking and consequent
LHR desensitization were blocked when membranes were incubated with
catalytically inactive ARNO-K156 (Mukherjee et al., 2001). These results
suggest that the exogenous, catalytically inactive ARNO mutant competes
with endogenous ARNO, or a similar Arf6 activator, to block Arf6
activation Arrestin2 release from its membrane docking site.
In support of a primary role for Arf6 in regulating the availability of
Arrestin2 for LHR desensitization, Arf6 is readily detected in a Triton X-
100-insoluble fraction in plasma membrane preparations from ovarian
follicles by western blotting (Mukherjee et al., 2000; Salvador et al., 2001)
and is activated upon LHR activation (Salvador et al., 2001). Moreover,
Arf6 activation by LHR is significantly reduced in the presence of ARNO-
K156 (Salvador et al., 2001).
Additional evidence in support of a linear pathway from the LHR to
regulate the activation of Arf6 is provided by recent evidence that ARNO
binds directly to the conserved NPXXY motif located at the base of TM 7 of
the LHR (personal observation). This conclusion is based on results
showing that ARNO binds to a synthetic peptide containing this conserved
amino acid sequence and not to one in which the amino acid residues are
scrambled. ARNO also preferentially co-immunoprecipitates with the
agonist-activated form of the receptor. Moreover, this TM 7 peptide
promotes Arrestin2 release from its membrane docking site in the absence of
LHR agonist, and supports LHR desensitization in the presence of agonist.
LHR desensitization stimulated by this TM 7 peptide is blocked by
314
10 HUNZICKER-DUNN

catalytically inactive ARNO-K156 and by the N-terminal Arf6 peptide,

indicating that the TM 7 peptide directs activation of ARNO, or a similar
GEF, and consequently Arf6.

3.5 Model for LHR desensitization

Taken together, these results indicate that Arf6•GDP, present in the

receptor/effector-enriched follicular membrane fraction, sequesters Arrestin2
either directly or indirectly (see Figure 1, panel A). Agonist-dependent LHR
activation results in movement of TM 3 and TM 6 and the consequent
shifting of the 3i loop into a more exposed position (Ballesteros et al., 2001),
resulting in activation of the heterotrimeric Gs and consequent activation of
adenylyl cyclase (see Figure 1, panel B). Gαs appears to interact with the 3i
loop of the LHR (Abell and Segaloff, 1997). LHR activation may also be
associated with the movement of the cytoplasmic end of TM 7 (Abdulaev
and Ridge, 1998; Altenbach et al., 1999; Shenker, 2002), resulting in
exposure of the highly conserved NPXXY motif. ARNO, also present in the
membrane preparation (Mukherjee et al., 2001), binds to the region of the
LHR at the base of TM 7 containing the NPXXY motif and promotes
activation of Arf6, resulting in the undocking of Arrestin2 (panel B).
Arrestin2 then binds to the agonist-bound LHR at the 3i loop, resulting in
uncoupling of the receptor from Gs and reduced production of cAMP (panel
C). As indicated in panel B, there is limited time period (~8-10 min in our
membrane model) after agonist addition when the LHR is actively signaling
to adenylyl cyclase (i.e., prior to desensitization). Addition of exogenous,
recombinant Arrestin2 to active receptor (see panel B, when endogenous
Arrestin2 is still docked with Arf6) likely abbreviates this time course and
promotes desensitization. Similarly, addition of exogenous recombinant
ARNO to active receptor likely stimulates desensitization by accelerating
Arf6 activation, resulting in the undocking of endogenous Arrestin2. The
apparent rate-limiting step in LHR desensitization is the relatively slow
binding of ARNO to the LHR. Neither the events involved in ARNO
binding to the LHR nor the mechanism by which activated Arf6 promotes
the release of Arrestin2 is understood.

4. ARRESTIN SEQUESTRATION: A WIDESPREAD

FUNCTION OF ARF6?

The principal unique feature of LHR desensitization that we have uncovered

is the finding that Arrestin2 is sequestered by Arf6•GDP and liberated with
 ROLE OF ARF6 IN GPCR DESENSITIZATION 315
11

Arf6 activation. Arrestin2 is released from its membrane docking site only
in response to (a) Arf6 activation by ARNO, in the presence of 1 µM GTP,
(b) high concentrations of GTP that override the GEF requirement and
activate all GTPases, or (c) LHR activation by agonist. Thus, the release of
Arrestin2 is regulated and specific for reagents that activate Arf6.
Moreover, sufficient Arrestin2 is retained in a membrane fraction to achieve
complete LHR desensitization under cell-free conditions. These results
suggest that the Arrestin available for receptor desensitization in cells may
be limiting under some circumstances. Indeed, there is evidence to support
this hypothesis in intact cells. In COS-7 cells in which a constitutively
active parathyroid hormone receptor was expressed and coupled to Gs, the
overexpression of Arrestin3 was required to reduce cAMP production by this
receptor (Ferrari and Bisello, 2001). Even more compelling is the report that
internalization of the β2-AR in stably transfected HEK293 cells was reduced
>65% by activation of the V2 vasopressin receptor coexpressed in the same
cells, an effect that was reversed by overexpression of Arrestin2 and
Arrestin3 (Klein et al., 2001).
Does the Arrestin2 that is not localized at the plasma membrane by Arf6
participate in LHR desensitization in intact cells? We do not know.
Subcellular fractionation of ovarian granulosa cells revealed that the
majority of immunoreactive Arrestin2, in contrast to Arf6 (Mukherjee et al.,
2000), was detected in a soluble fraction (S. Mukherjee, unpublished
observation). Perhaps this soluble Arrestin has additional functions. For
example, Arrestin2 binds to cytosolic Disheveled proteins, which lie
downstream of the Frizzled receptor in the Wnt pathway (Chen et al., 2001).
It is also not known if the Arrestin present in the soluble fraction of
untransfected cells is docked at specific cellular locations, perhaps bound
(directly or indirectly) to other Arfs, or if it is diffusely distributed
throughout the cell. In one of the few studies in which the distribution of
endogenous Arrestins was evaluated by immunofluorescence, both non-
visual Arrestins were found distributed in a punctate pattern throughout the
interior of RBL-2H3 cells (a rat mast cell line) (Santini et al., 2000),
consistent with the existence of specific Arrestin binding sites.
Does Arf6 sequester Arrestin in cells other than ovarian follicular cells?
And does GPCR activation universally promote the undocking of Arrestin
from an Arf6-dependent binding site, or is this pathway unique to the ovary
and the LHR? We do not yet know the complete answer to these questions.
However, the findings that LHR artificially expressed in kidney cells can
activate a pathway that leads to Arf6 activation and Arrestin2-dependent
LHR desensitization, shows that the GPCR-dependent Arf6 activation
pathway to undock Arrestin is not restricted to ovarian cells (Mukherjee et
al., 2002). Additional studies are required to ascertain whether or not other
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GPCRs use this GTP-dependent pathway to undock Arrestins and if so,

whether Arf6 universally fulfills this role. However, in cells expressing
recombinant receptors, GRKs, and Arrestins, the hallmark of GPCR action is
the recruitment of Arrestin from a cytosolic fraction to the receptor
(Ferguson et al., 1996; Barak et al., 1999; Evans et al., 2001). Does this
result simply reflect the exposure of high affinity binding sites for Arrestins
on activated receptors and resulting movement of Arrestin to these localized
high-affinity binding sites, and does it represent the normal cellular response
to receptor activation? Additional studies are required to resolve these
important questions. Perhaps in the native cellular environment, the two
non-visual Arrestins can be recruited to receptors by distinct mechanisms.
While Arrestin2 is recruited from its plasma membrane-localized docking
site in response to LHR-stimulated Arf6 activation for desensitization,
Arrestin3 is not detected in follicular membranes (Mukherjee et al., 1999a)
and therefore is not sequestered by Arf6 at this location. Rather, Arrestin3
appears to promote LHR internalization (Min et al., 2001) and must be
recruited from another cellular location to perform this function. Using a
cell line that expresses relatively high and equivalent levels of endogenous
Arrestin2 and Arrestin3 and in which β2-AR was stably expressed (at
~80,000 receptors/cell), Arrestin3 was selectively translocated from a
soluble compartment to clathrin-coated pits in response to agonist
stimulation of the β2-ARs (Santini et al., 2000). This result indicates that
under conditions in which Arrestins are not overexpressed, Arrestin3 is still
recruited from the cytosol to the receptor. In a number of cell types, Arf6 is
localized not only to the plasma membrane but also to a tubulovesicular
endosomal membrane compartment (Peters et al., 1995; Radhakrishna and
Donaldson, 1997; D'Souza-Schorey et al., 1998; Frank et al., 1998; Peters et
al., 2001; Brown et al., 2001) which could co-fractionate with the plasma
membrane even on sucrose gradient centrifugation (Radhakrishna and
Donaldson, 1997). We therefore cannot rule out the possibility that in intact
granulosa cells, Arrestin2 along with Arf6 and possibly ARNO, are recruited
to the plasma membrane from this tubulovesicular compartment. However,
immunofluorescence results with rat granulosa cells indicate that the
majority of Arf6 is localized at the plasma membrane both in the absence
and presence of receptor agonist (Mukherjee et al., 2000).
Finally, there is recent evidence for the β2-AR and possibly other GPCRs
that ARNO and Arf6 are involved in receptor internalization. This
conclusion was based on studies by Premont and collaborators, as discussed
in Chapter 8, in which they demonstrated that overexpression of the GRK
interacting protein GIT1, which possesses Arf GTPase activating protein
(GAP) activity, reduced β2-AR internalization (Premont et al., 1998). This
effect of GIT1 was dependent on its GAP activity (Premont et al., 1998).
 ROLE OF ARF6 IN GPCR DESENSITIZATION 317
13

GIT1 overexpression reduced the internalization only of those GPCRs that

were internalized in an Arrestin-dependent manner and not those
internalized by an Arrestin-independent pathway (Bhatnagar et al., 2001).
That overexpression of GIT1 inhibits Arrestin-dependent GPCR
internalization suggests that Arf•GTP is required for GPCR internalization.
Subsequent studies by this group showed that transient overexpression of
ARNO, in cells overexpressing β2-AR, accelerated receptor internalization;
although inexplicably, overexpression of either constitutively active Arf6-
L67 or dominant negative Arf6-N27 reduced β2-AR internalization (Claing
et al., 2001). Interestingly, these authors also showed that Arrestin2 and
Arrestin3 interacted directly with ARNO and Arf6•GDP but not with
Arf6•GTP in GST pull-down assays. When Arrestins, Arf6, ARNO, and β2-
AR were overexpressed in HEK293 cells and proteins were cross-linked,
Arf6 and Arrestins co-immunoprecipitated in an agonist-dependent manner
while Arrestins and ARNO were constitutively associated. These authors
concluded that with Arrestin/ARNO recruitment to the agonist-activated and
phosphorylated β2-AR, Arrestin functions both to recruit Arf6•GDP and as a
cofactor to enhance the activity of ARNO to activate Arf6. Arf6•GTP was
predicted to dissociate from the Arrestin/receptor complex and to participate
in recruitment of vesicle coat proteins leading to β2-AR endocytosis (Claing
et al., 2001) in a manner analogous to that of Arf1 and Arf5 in Golgi
membranes where they stimulate coat assembly by binding AP-1 (Zhu et al.,
1999; Drake et al., 2000). However, Arf6 is not generally associated with
clathrin-coated vesicles (Peters et al., 1995; Cavenagh et al., 1996;
Radhakrishna et al., 1996; Radhakrishna and Donaldson, 1997; Brown et al.,
2001) and recent studies do not support an association of Arf6 and AP-2
(Austin et al., 2002). Arf6 could stimulate clathrin coated vesicle
endocytosis at the plasma membrane indirectly, e.g., as a result of changes in
lipid metabolism (Honda et al., 1999; Godi et al., 1999). Alternatively, the
stimulatory effects of ARNO and inhibitory effects of GIT1 on β2-AR
internalization could be interpreted to support our model (see Figure 1) in
which ARNO stimulates Arf6 activation to liberate Arrestin to promote
receptor desensitization and, for some GPCRs, receptor internalization,
while GIT1-driven Arf6•GDP sequesters Arrestin, thereby inhibiting
receptor internalization.

5. SUMMARY

Taken together these data support a novel function for Arf6 in Arrestin-
dependent GPCR desensitization and internalization. The key features of the
role of Arf6 in receptor desensitization are as follows. In the resting state
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there exists a pool of Arrestin that is bound to Arf6•GDP. Upon ligand

binding, receptors assume an activated conformation, couple to G proteins,
and recruit ARNO. The ARNO then activates Arf6, resulting in release of
Arrestins, which in turn bind the receptor and promote uncoupling and
internalization. This model is based largely on results from follicular cells
and LRH but Arf6•GDP appears to fulfill a similar function in human
embryonic kidney cells (Mukherjee et al., 2002). As a result, signaling of
the LHR to Gs is halted, and perhaps signaling to other pathways is then
initiated (Pierce et al., 2001). However, because the domain to which
ARNO binds on the LHR is highly conserved among the superfamily of
GPCRs, it is tempting to speculate that ARNO binds to the agonist-bound
conformation of other GPCRs to activate Arf6 and regulate the availability
of Arrestins to GPCRs. Roles for lipid signaling (e.g., ARNO-activated Arf
activating phospholipase D) in these processes cannot be ruled out as
playing a role in receptor desensitization or internalization. The β2-AR may
use a similar mechanism to localize and regulate the release of Arrestin from
an Arf6-bound site (Claing et al., 2001). For Arrestin to bind to the β2-AR,
both agonist binding and GRK-dependent receptor phosphorylation are
required to convert Arrestin to a conformation that binds with high affinity
(Pierce et al., 2001). While ARNO binds to the LHR and promotes Arf6
activation and LHR desensitization, the participation of another Arf6 GEF,
like EFA6 (Franco et al., 1999; Dulin et al., 2001), in these events has not
been ruled out.
These studies thus reveal a previously unappreciated level of complexity
in the signaling pathway regulating LHR coupling to heterotrimeric G
proteins, which places Arf6 at a pivotal position to direct the availability of
Arrestin2. As our understanding of how the Arrestins are delivered to
GPCRs unfolds, new avenues for basic research and drug development for
the treatment of a variety of diseases will likely emerge.

6. ACKNOWLEDGMENTS

I would like to thank the members of my laboratory, especially Sutapa

Mukherjee, Evelyn Maizels, Regina Horvat, and Lisa Salvador, as well as
Andrew Shenker, Vsevolod Gurevich, and James Casanova for helpful
discussions. I would also like to thank Andrew Shenker for providing me
with the receptor bundles used in the figure.
 ROLE OF ARF6 IN GPCR DESENSITIZATION 319
15

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Mukherjee, S., Palczewski, K., Gurevich, V. V., Benovic, J. L., Banga, J. P., and Hunzicker-
Dunn, M. (1999a). A direct role for arrestins in desensitization of the luteinizing
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19

hormone/choriogonadotropin receptor in porcine ovarian follicular membranes. Proc.

Natl. Acad. Sci., USA , 96, 493-498.
Mukherjee, S., Palczewski, K., Gurevich, V. V., and Hunzicker-Dunn, M. (1999b). βarrestin-
dependent desensitization of luteinizing hormone/choriogonadotropin receptor is
prevented by a synthetic peptide corresponding to the third intracellular loop of the
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Nagel, W., Zeitlmann, L., Schilcher, P., Geiger, C., Kolanus, J., and Kolanus, W. (1998).
Phosphoinositide 3-OH kinase activates the beta2 integrin adhesion pathway and induces
membrane recruitment of Cytohesin-1. J. Biol. Chem., 273, 14853-14861.
Oakley, R. H., Laporte, S. A., Holt, J. A., Caron, M. G., and Barak, L. S. (2000). Differential
affinities of visual arrestin, β arrestin1, and β arrestin2 for G protein-coupled receptors
delineate two major classes of receptors. J. Biol. Chem., 275, 17201-17210.
Paing, M. M., Stutts, A. B., Kohout, T. A., Lefkowitz, R. J., and Trejo, J. (2002). β-Arrestins
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distinct perturbations of nonoverlapping membrane compartments. J. Cell. Biol., 128,
1003-1017.
Pierce, K. L., Lefkowitz, R. J., and Lefkowitz, R. J. (2001). Classical and new roles of β-
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Birnbaumer, L., and Hunzicker-Dunn, M. (1999). Roles of Gi and Gq/11 in mediating
desensitization of the luteinizing hormone/choriogonadotropin receptor in porcine ovarian
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Santini, F., Penn, R. B., Gagnon, A. W., Benovic, J. L., and Keen, J. H. (2000). Selective
recruitment of arrestin-3 to clathrin coated pits upon stimulation of G protein-coupled
receptors. J. Cell. Sci., 113, 2463-2470.
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and Reiter, E. (2000). Involvement of G protein-coupled receptor kinases and arrrestins in
desensitization to follicle sitmulating hromone action. Mol. Endo., 13, 1599-1614.
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receptor facilitates uncoupling of the receptor from adenylyl cyclase and endocytosis of
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receptor with a severely truncated cytoplasmic tail (LHR-ct628) desensitizes to the same
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coat assembly on synthetic liposomes. Proc. Natl. Acad. Sci., USA, 96, 5013-5018.
Chapter 16
ARF-LIKE PROTEINS

ANNETTE SCHÜRMANN AND HANS-GEORG JOOST

German Institute of Human Nutrition, Potsdam-Rehbrücke, Germany

Abstract: At least 10 different proteins have homology to members of the Arf family but
each lacks activities that define “true” Arfs. In addition, the percent identity
between these 10 proteins, when aligned with Arfs or to each other, is lower
(typically 40-60%) than that between Arfs (greater than 65% identity). A
summary of current information on each is presented below. Despite
extensive conservation of structure between Arfs and Arls, the Arls represent a
much more functionally divergent collection of GTPases, with apparent roles
in vesicle traffic (Arl1, Sar1), microtubule organization (Arl2, Arl3),
spermatogenesis (Arl4), and differentiation (Arfrp1). However, the overlap in
binding partners between functionally distinct Arls suggests that Arfs and Arls
are likely to have a complex web of overlapping and distinct functions.

1. INTRODUCTION

ADP-ribosylation factor-like (Arl) proteins are GTPases that were identified

on the basis of their sequence similarity with Arfs (33-60% identical amino
acids). At present, 10 mammalian isotopes (Arl1-7, with two different
proteins designated Arl6, Arfrp1 and Sar1) have been described (see Figure
1). GTP-binding has been shown for most Arl proteins, but all Arls are
essentially devoid of GTPase activity and activities described for Arf
isotypes, i.e., activity as cofactor in the ADP-ribosylation of GαS by
bacterial toxins (Kahn and Gilman, 1984; Kahn and Gilman, 1986),
activation of phospholipase D (Brown et al., 1993; Cockcroft et al., 1994), or
activation of phosphatidylinositol 4-phosphate 5-kinase (Honda et al., 1999).
No Arl has ever been purified or cloned on the basis of an activity.
Furthermore, no Arl can rescue the synthetic lethality of arf1-/arf2- mutants
in Saccharomyces cerevisiae, an effect that is characteristic for mammalian
(Kahn et al., 1991; Lee et al., 1992) and other Arfs (Drosophila
melanogaster, Murtagh et al., 1993; Giardia lamblia, Lee et al., 1992).
325
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These data lead to the conclusion that Arfs and Arls lack a functional
overlap, except for their role as GTPase-operated molecular switches. In
addition, there appears to be little functional overlap between Arls, as
reflected by a considerable heterogeneity of their expression in tissues and of
their subcellular distribution. Some Arls appear to be involved in the
regulation of protein and/or vesicle transport between cell organelles (Arl1,
Arl4, Sar1) or in the regulation of enzymatic activities controlling these
processes (Arfrp1). Furthermore, the physiological functions of the different
family members, in as far as they are known, appear to be quite diverse (e.g.,
Arl4, spermatogenesis; Arfrp1, differentiation of mesoderm during
embryogenesis).

2. ARL1

Arl1 is the closest relative to the Arfs, appears to have some functional
similarities to the Arfs, and should rather be considered an atypical Arl
protein. Although the subcellular localization and several interaction
partners of Arl1 were identified in the past, the exact role of Arl1 is still
unknown.

2.1 Similarities and differences of Arl1 to the Golgi-

related function of Arf

Arl1 was originally cloned from Drosophila melanogaster and is essential

for normal development because arl1- mutants exhibited a recessive lethal
phenotype (Tamkun et al., 1991). The mammalian isoform that was
subsequently identified (Schürmann et al., 1994) exhibits 79% amino acid
identity with Drosophila Arl1, and 57% with human Arf1. The identity with
other Arl isotypes is lower (40% with Arl2, 45% with Arl3, and 41% with
Arl4; Figure 1). Because mammalian Arls are so highly conserved (97-99%
identity between human, mouse, rat and bovine) the human proteins are used
in alignments and sequence analyses (see Figure 1) but results will hold true
for Arls from at least these other mammals. Northern blots revealed that
Arl1 is ubiquitously expressed in rat tissues (brain, heart, spleen, lung, liver,
skeletal muscle, kidney, testis and fat (Schürmann et al., 1994; Lowe et al.,
1996). Binding of GTPγS to recombinant Arl1 appears to be partially
dependent on the presence of lipids and detergent (Tamkun et al., 1991), a
feature prominent for the Arfs.
 ARF-LIKE PROTEINS 3273

Figure 1. Dendrogram of the Arf family. The human proteins were aligned with the
CLUSTAL program. Bars denote the classes I-III of Arfs and the Arl4 subgroup. All
mammalian Arl proteins are highly conserved (97% to 99% identity between human, mouse,
rat and bovine).

2.2 Subcellular localization and conditions of

redistribution between Golgi and cytosol

Like most Arf proteins, Arl1 is associated with the Golgi complex. Lowe et
al. (1996) detected endogenous Arl1 in the perinuclear region of normal rat
kidney (NRK) cells by indirect immunofluorescence, co-localized with the
Golgi markers p28 (a cis-Golgi protein) and mannosidase II. The same
subcellular localization was found in other cell lines. Furthermore, treatment
of cells with nocodazole, a microtubule depolymerizing drug that causes
fragmentation of the Golgi complex, dispersed Arl1 labeling into many
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4 SCHÜRMANN AND JOOST

punctate structures throughout the cell. A similar pattern was observed for
both Golgi markers p28 and mannosidase II (Lowe et al., 1996).
A different pattern of cellular redistribution was observed for Arl1 and
mannosidase II when brefeldin A was used to disrupt the Golgi structures.
After short treatment (2 min) with brefeldin A, the labeling pattern of Arl1
and mannosidase II was unaffected. After 5 min of brefeldin A-treatment,
mannosidase II was detected in tubulovesicular extensions. Arl1 labeling
did not colocalize with these structures, but was detected in the cytosol.
After 30 min of brefeldin A treatment, Arl1 was completely redistributed to
the cytosol, whereas mannosidase II was transferred into an extensive
reticular network.
The observed response of Arl1 to brefeldin A differs from those of Arf
and COPI (β-COP), components of non-clathrin-coated transport vesicles.
Arf and COPI redistributed into the cytosol faster (within 2 min) than Arl1.
This suggests that the effect of brefeldin A on Arl1 may be indirect or
secondary, as compared to Arfs. As is the case for Arfs, brefeldin A could
block guanine nucleotide exchange via interaction with a brefeldin A-
sensitive, Arl1-specific exchange factor (GEF). If the normal turnover rate
of Arl1 cycling on and off Golgi membranes is slower than that of Arfs, then
the inhibition of this GEF could cause the delayed release of Arl1 from the
Golgi (Lowe et al., 1996). Alternatively, brefeldin A may affect Arl1
indirectly. For example, inhibition of an Arf-specific GEF by brefeldin A
leads to the fast redistribution of Arf1 into the cytosol. Subsequently, the
lack of Arf1 within Golgi membranes could result (via an unknown
mechanism) in the release of Arl1 into the cytosol, suggesting that Arf1 is
acting upstream of Arl1. Somewhat different results were obtained by van
Valkenburgh et al. (2001). They found that epitope tagged Arl1 (Arl1-myc)
was released from the Golgi with similar kinetics to endogenous Arfs. This
discrepancy may result from the use of epitope tagging or overexpression of
the GTPase. There is now extensive evidence that either approach can
produce data that differ from the location or behavior of endogenous
GTPases, particularly for members of the Arf family. Unfortunately, the
levels of expression of Arl1 are typically low and make studies of
endogenous protein more difficult than for Arfs. We should probably
consider tentative any conclusions based solely on results obtained with
overexpressed and or epitope tagged proteins.
Additional evidence that the subcellular distribution of Arf1 and Arl1 is
regulated independently was obtained with aluminum fluoride. AlF
activates heterotrimeric G-proteins and reverses the effects of brefeldin A on
COPI, mannosidase II, and Rab6, but not that on TGN38. When NRK cells
were preincubated with AlF for 20 min prior to addition of brefeldin A, both
Arl1 and COPI were more tightly associated with the Golgi complex (Lowe
 ARF-LIKE PROTEINS 3295

et al., 1996). In contrast, AlF failed to reverse the effect of brefeldin A on

the redistribution of Arfs into the cytosol (Donaldson et al., 1991).
Association of Arl1 with the Golgi appears saturable (Lu et al., 2001).
Moderate overexpression of Arl1 gave rise to enrichment of the protein at
the trans side of the Golgi apparatus. In contrast, when expression was high,
the majority of Arl1 was detected in the cytosol. This saturable Golgi
association was explained by a rate-limiting activation step that depends on
nucleotide-exchange factors. As expected, the Golgi association depends on
N-terminal myristoylation. A mutant of Arl1 lacking the myristoylation
motif (Arl1-A2) was found in the cytosol and the nucleus but was absent
from the Golgi.
Arl1 seems to be necessary for maintaining normal Golgi structure, as
overexpression of Arl1-N31, a nucleotide exchange defective mutant, causes
a disintegration of the Golgi apparatus (Lu et al., 2001). This phenotype was
similar to that observed in cells treated with brefeldin A (Klausner et al.,
1992) or cells overexpressing Arf1-N31 (Dascher and Balch, 1994). In
addition, the perinuclear, Golgi-associated localization of AP-1 (γ-adaptin)
was also disrupted in Arl1-N31 cells, suggesting that Arl1 is involved in
Golgi recruitment of AP-1 (Lu et al., 2001).
Overexpression of Arl1-L71, the activated, GTPase-defective mutant,
resulted in an expanded perinuclear Golgi-like structure (Lu et al., 2001).
Van Valkenburgh et al. (2001) also described a more diffuse Golgi staining
in normal rat kidney (NRK) cells stably expressing Arl1-L71-myc. The
Golgi of cells expressing Arl1-L71 exhibited an engorged lumen,
particularly at the ends of the stacks. This phenotype was comparable to
early time points in the expression of Arf1-L71, suggesting similarities in
function of these proteins at the Golgi. However, the change in Golgi
morphology induced by Arl1-L71 was subtle and was not accompanied by
the progressive development of large perinuclear vesicles which often
surrounded the nucleus, as seen for Arf1-L71 (van Valkenburgh et al.,
2001). The expression of Arl1-L71 caused a massive recruitment of both
COPI (β-COP) and AP-1 (γ-adaptin) into the expanded Golgi (Lu et al.,
2001). Treatment with brefeldin A normally inhibits Golgi-association of
COPI and AP-1, and the formation of their respective vesicles. In cells
expressing Arl1-L71, in contrast, COPI and AP-1 could not be dissociated
by brefeldin A. Moreover, extensive association of Arfs with the expanded
Golgi-like structure was observed in cells overexpressing Arl1-L71,
suggesting that active Arl1 could stimulate Golgi recruitment of Arfs. Arfs
remain Golgi-associated even after brefeldin A treatment when Arl1-L71
was expressed in these cells (Lu et al., 2001). These results indicate that Arl1
has the potential to regulate COPI, AP-1, and Arf-mediated vesicle
formation in the Golgi.
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6 SCHÜRMANN AND JOOST

The effects of Arl1 on membrane traffic in the Golgi apparatus appear to

be specific (Lu et al., 2001). Overexpression of Arl1-L71 selectively
stimulated recruitment of COPI and AP-1, which mediate export from the
Golgi. In contrast, the localization of COPII (which mediates ER export),
and of α-adaptin (which mediates endocytosis from the plasma membrane),
was not affected by the Arl1 mutant.

2.3 Arl1 binding proteins

In order to test whether Arl1 and Arf1 have overlapping functions, van
Valkenburgh et al. (2001) analyzed the interaction of known Arf-binding
proteins with Arl1. Of these, Arfaptin2 (also termed POR1, see Chapter 11
for details) and mitotic kinesin-like protein-1 (MKLP1) exhibited GTP-
dependent binding with Arl1. Furthermore, Arfaptin2 increased the
stoichiometry of GTP binding to Arl1 (van Valkenburgh et al., 2001).
Arfaptin2 was previously identified as an effector of Rac (D’Souza-Schorey
et al., 1997; Kanoh et al., 1997), and has been proposed to be a mediator of
cross talk between Arf proteins and Rac. Arfaptin2 binds specifically to
GTP-bound Arf1 and Arf6, and binds to Rac•GTP and Rac•GDP with
similar affinities (Tarricone et al., 2001). The C-terminal domain of MKLP1
binds to all activated Arf isoforms (Boman et al., 1999) and also to Arl1, but
not to Arl2 or Arl3 (van Valkenburgh et al., 2001). MKLP1 is present in
central spindles and midbodies of mammalian cells, and is thought to be
involved in the re-organization of membranes during the terminal phase of
cytokinesis (Kuriyama et al., 2002).
In a two-hybrid screen performed with the GTPase-defective mutants of
Arl1-3, van Valkenburgh et al. (2001) identified PDEδ and HRG4 as GTP-
dependent binding proteins. In addition, the authors identified Golgin-245 to
bind to Arl1-L71 and Arl3-L71 (see Figure 2). Golgins are Golgi-associated
proteins with antigenic potency in autoimmune diseases. The 245 kDa
protein Golgin-245 contains a coiled-coil domain and is peripherally
associated with the cis-Golgi. Golgin-245 comprises a C-terminal GRIP-
domain that is believed to direct associated proteins to the Golgi apparatus.
Arl1 and the GRIP domain of Golgin-245 are co-localized when expressed
in NRK cells. In contrast to Arl1, binding of the GRIP-domain to the Golgi
was not disrupted by brefeldin A, suggesting that this domain can bind the
Golgi independently of Arl1.
SCOCO (Short Coiled-Coil; see Figure 2) is another binding protein of
Arl1-L71 that colocalizes with Arl1 in the Golgi. The function of SCOCO is
still unknown (van Valkenburgh et al., 2001). Its 82 amino acid residues are
predicted to fold almost completely into a coiled-coil structure. SCOCO
message was widely distributed in human tissues with the highest levels
 ARF-LIKE PROTEINS 3317

detected in brain, heart, and skeletal muscle. The protein was found
associated with the Golgi apparatus and the plasma membrane. Like Arl1,
the binding of SCOCO to the Golgi was rapidly reversed by brefeldin A.
Purified SCOCO had no detectable GTPase activating protein (GAP)
activity for Arl1.

Figure 2. Specific and shared binding partners of Arfs, Arls and Arfrp1 are shown. Arf
family members are shown in boxes (center), with binding partners that are shared by
multiple members shown above them and those specific to the Arfs or an Arl, shown below.

3. ARL2

Arl2 is widely expressed in mammals, Drosophila melanogaster,

Caenorhabditis elegans and yeast. Arl2 is a regulator of microtubule
dynamics and therefore plays an essential role in cytokinesis. A
subpopulation of Arl2 is located in the mitochondria and may be involved in
adenine nucleotide transport. Arl2 and Arl3 are closely related, exhibit a
similar structure, and share some features. Both proteins interact with PDEδ
and are most likely involved in PDEδ-mediated transport of prenylated
proteins.
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8 SCHÜRMANN AND JOOST

3.1 Potential involvement of Arl2 in the biogenesis of

microtubules and interaction with the mitochondrial
adenine nucleotide transporter (ANT)

Arl2 was identified in a PCR-based cloning approach on the basis of its

sequence similarity with Arf (Clark et al., 1993). It is expressed in all
mammalian tissues with highest levels in neural cells. Arl2 binds GDP or
GTP much faster than Arf, even in the absence of lipids or detergents (Clark
et al., 1993). The presumed budding yeast ortholog of Arl2, CIN4, was
identified in a genetic screen for mutants defective in microtubule function
(Hoyt et al., 1990; Stearns et al., 1990). In addition, Radcliffe et al. (2000)
cloned the Arl2/CIN4 ortholog, alp41, from fission yeast, and demonstrated
that it is essential for the cofactor-dependent tubulin pathway. Disruption of
the alp41 gene resulted in microtubule dysfunction and growth polarity
defects. The phenotype observed for the alp41- mutant was very similar to
that of cells lacking the gene encoding cofactor B or E (alp11 and alp21,
respectively), suggesting that Alp41 is involved in the microtubule
biogenesis pathway. Interestingly, expression of Alp1 (ortholog of cofactor
D) or expression of Alp21 (cofactor E) rescued the phenotype of alp41-
mutants. These genetic analyses indicate that Alp41/Arl2 plays an essential
role in the cofactor-dependent tubulin pathway (Radcliffe et al., 2000).

Figure 3. Model depicting the diversity in localization and actions of Arl proteins in (1) the
biogenesis of microtubules (Arl2; Bhamidipati et al., 2000; R. A. Kahn, unpublished
observations), (2) mitochondrial adenine nucleotide transport (Arl2; Sharer et al., 2002), (3)
nuclear transport (Arl4; Jacobs et al., 1999), (4) vesicle budding from the endoplasmic
reticulum (Sar1; Springer et al., 1999), membrane traffic through the Golgi (Arl1), and
nuclear localization of Arl4, Arl6/Arf4L and Arl7. (C, D, E = cofactors C, cofactor D and
cofactor E).
 ARF-LIKE PROTEINS 3339

Microtubules are polarized polymers of α/β tubulin heterodimers that

participate in several cellular functions. The dynamic behavior of
microtubules is controlled by polymerization-dependent GTP hydrolysis
catalyzed by the β-subunit and associated proteins. Tubulin heterodimers
are generated in a multi-step process that involves several chaperone
proteins designated cofactor A-E. They assemble the α/β-tubulin
heterodimer, interact with native tubulin, and act as a β-tubulin GTPase
activating protein (GAP; see Figure 3; Levis et al., 1997; Tian et al., 1997).
Regulation of microtubule dynamics by Arl2 was also shown in
mammalian cells (Bhamidipati et al., 2000). The authors demonstrated that
Arl2 interacts with cofactor D and modulates the interaction of this tubulin-
folding protein with native tubulin (Figure 3). In addition, Arl2 down
regulates the tubulin GAP activity of cofactors C, D and E, and inhibits the
binding of cofactor D to native tubulin (Figure 3). Overexpression of
cofactor D or E in cultured cells resulted in the destruction of the tubulin
heterodimer and of microtubules. Arl2 specifically prevented the destruction
of tubulin and microtubules by cofactor D, but not by cofactor E. Using
different mutants of Arl2, Bhamidipati et al. (2000) demonstrated that Arl2
preferentially interacts with cofactor D in its GDP-bound form. As binding
of the effector loop mutant Arl2-A47 to cofactor D was indistinguishable
from that of wild-type Arl2, the authors concluded that the switch from the
GDP-bound to the GTP-bound conformation is not essential for the
interaction between Arl2 and cofactor D. However, a second effector loop
mutant, Arl2-A50, failed to bind cofactor D, suggesting that the cofactor is
an effector of Arl2•GDP. While Bhamidipati et al. (2000) suggested that
Arl2 is involved in a tubulin destruction pathway others (Y. Li, W. Kelly,
and R. A. Kahn, unpublished observations) described an essential role for
Arl2 in tubulin biosynthesis during embryogenesis and in the germline. The
authors used RNA-mediated interference to target Arl2 in C. elegans.
Injection of Arl2 double-stranded RNA resulted in embryonic lethality or
sterility in early offspring. Germ cells often exhibited defects in
cellularization, forming regions with numerous nuclei sharing a complete
cytoplasm. As embryos lacking Arl2 exhibited decreased tubulin levels, Li,
et al. (unpublished observations) concluded that Arl2 is required for the
biosynthesis of tubulin. Interestingly, the phenotype observed after injection
of double stranded Arl2 RNA was similar to that seen with injection of the
334
10 SCHÜRMANN AND JOOST

C. elegans ortholog of cofactor D RNA, leading to the conclusion that Arl2

and cofactor D co-participate in cell-cycle related events in C. elegans.
A second important function of Arl2 has been established by Sharer and
Kahn (1999). Arl2•GTP interacts with a 19 kDa protein, designated Bart
(Binder of Arl Two), with an apparent KD of, 20 nM. Like Arl2, Bart is
expressed ubiquitously and is most abundant in brain. Binding of Arl2 to
Bart was detected in solution (Sharer and Kahn, 1999), and no detectable
membrane association was found for either protein. This observation is of
particular interest, because binding of all other interacting proteins to Arf
isotypes requires the presence of membranes or lipids. Thus, binding of Arfs
to membranes had been considered a critical part of Arf action.
In contrast to every other member of the Arf family, no incorporation of
[³H]myristate into Arl2 could be detected either when coexpressed in
bacteria with N-myristoyltransferases, or in cultured mammalian cells
(Sharer et al., 2002). Lack of covalent N-myristoylation preserves the N-
terminal amphipathic α-helix as a potential mitochondrial import sequence.
Recently, Sharer et al. (2002) indeed demonstrated that the Arl2-Bart
complex is located in the mitochondria within the intermembrane space
(Figure 3). By overlay assays with mitochondria isolated from five different
tissues, the mitochondrial adenine nucleotide transporter (Ant) was
identified as a binding partner of the Arl2-Bart complex (Sharer et al., 2002).
Ant1 appeared to be a specific binding partner of the Arl2•GTP-Bart
complex, as the Ant2 isotype did not bind the complex. Ant proteins are
abundant, integral proteins of the mitochondrial inner membrane, where they
homodimerize to form channels used for exchange of ATP and ADP,
thereby controlling the levels of ATP in the cytoplasm (Fiore et al., 1998).
In rodents, Ant1 is the major isotype present in skeletal muscle, cardiac
muscle, and brain. In contrast, expression of Ant2 is ubiquitous with lower
levels than those of Ant1 in heart and skeletal muscle (Stepien et al., 1992;
Graham et al., 1997; Levy et al., 2000).

4. ARL3: A POTENTIAL REGULATOR OF THE

PDEδ-MEDIATED PROTEIN TRANSPORT

Arl3 (Cavenagh et al., 1994) exhibits 43% identity with Arf1. The GTPase
is expressed in most tissues, with elevated mRNA levels detected in several
human tumor cell lines. Recombinant Arl3 binds guanine nucleotides but
lacks intrinsic GTPase activity (Cavenagh et al., 1994). However, Arl3
exhibits an unusually low affinity for guanine nucleotides, with a KD of 24
nM for mant-GDP and 48 µM for mant-GTP (Linari et al., 1999a). The
Arl3•GTP, but not the Arl3•GDP binds the δ subunit of the cGMP
 ARF-LIKE PROTEINS 335
11

phosphodiesterase (PDEδ) (Figure 2). The binding decreased the

dissociation rate of GTP, thereby stabilizing Arl3•GTP (Linari et al., 1999a).
PDEδ is also an effector of Arl2 (Hanzal-Bayer et al., 2002).
PDEδ was isolated as the fourth subunit of bovine rod PDE (PDE6;
Gillespie et al., 1989). PDE6 is the effector for the trimeric G protein
transducin after its activation by light-sensitive rhodopsin. PDEδ binds to
the prenylated C-terminus of PDEα,β, resulting in a translocation of the
PDE6 holoenzyme from the membranes to the cytoplasmic fraction (Florio
et al., 1996). PDEδ also transfers membrane-bound Rab13 to the cytoplasm
(Marzesco et al., 1998), suggesting that the function of PDEδ is similar that
of GDP-dissociation inhibitors (GDIs), which extract prenylated proteins
from cellular membranes. This conclusion is strongly supported by
structural similarities between PDEį and Rho GDI (Hanzal-Bayer et al.,
2002). PDEδ also interacts with the retinitis pigmentosa guanine receptor
(RPGR; Linari et al., 1999b) and a number of prenylated GTPases (Hanzal-
Bayer et al., 2002). RPGR contains a domain homologous to the GEF for
the GTPase Ran (RCC1, regulator of chromosome condensation; Meindl et
al., 1996; Renault et al., 1998). The finding that PDEδ binds to RPGR
(Linari et al., 1999b) and Arl3 (Linari et al., 1999a) in the absence of any
post-translational modifications indicates that PDEδ binding is not
prenylation-dependent, despite the presence of a hydrophobic pocket similar
to the one in Rho GDI that binds prenyl groups.
Because PDEδ not only binds the subunits of PDE6 but also several other
proteins (Arl2, Arl3, Rab13, RPGH, H-Ras, Rheb, Rho6 and Gαi1; Hanzal-
Bayer et al., 2002), it was suggested that PDEδ is not a genuine subunit of
the PDE holoenzyme, but merely an associated protein involved in protein
transport. In addition, the structure of PDEδ in a complex with Arl2•GTP
(see below) led to the hypothesis that PDEδ is a transport factor for
prenylated proteins, and that Arl2 and Arl3 regulate the PDEδ-mediated
transport (Hanzal-Bayer et al., 2002).

5. ARL4

Arl4, Arl6/Arf4-like and Arl7 represent a subgroup of the Arl proteins that
share sequence similarities and the presence of C-terminal nuclear
localization signals (NLSs). Arl4 is highly expressed in testis and is
involved in the differentiation of spermatocytes. It has been suggested that
Arl4 is involved in the regulation of nuclear protein transport during cell
division or differentiation.
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12 SCHÜRMANN AND JOOST

5.1 Arl4 is involved in male germ cell development

Arl4 was cloned as a GTPase that is abundantly expressed in differentiated

cells of the pre-adipocyte cell line 3T3-L1, but not in undifferentiated cells
(Schürmann et al., 1994). The GTPase was found predominantly in testis;
lower levels of Arl4 mRNA were detected in spleen and intestine, brain,
heart, fat, liver, lung and thymus (Jacobs et al., 1998). The subcellular
distribution of Arl4 differs from other Arf and most Arl proteins because it is
in the nucleus and nucleoli (Lin et al., 2000).
On the basis of sequence similarity, Arl4, Arl6 and Arl7, form a
subgroup within the ARF family (Figure 1). In addition, a striking
characteristic of these three GTPases is their C-terminal NLS and unusually
high guanine nucleotide exchange rate. Jacobs et al. (1999) demonstrated
that the C-termini of Arl4, Arl6 and Arl7 are capable of targeting green
fluorescent protein (GFP) to the nucleus of COS-7 cells, and suggested that
these GTPases are involved in the regulation of protein transport to or from
the nucleus. Transport through the nuclear pore complex is GTP-dependent
and is regulated by Ran (Görlich and Mattaj, 1996; Nigg, 1997). Cargo
proteins bind to importin-α by their NLS, and Ran regulates the
translocation and subsequent dissociation of the complex inside the nucleus.
Data from yeast two-hybrid and in vitro protein interaction assays indicated
that Arl4 does not bind importin-α in a GTP-dependent manner (Lin et al.,
2000). Thus, Arl4 appears to be a cargo protein rather that a Ran-like
regulator of nuclear import.
Several lines of evidence indicate that Arl4 is involved in male germ cell
development. In situ hybridization showed that Arl4 is expressed in testis of
pubertal and adult rats, but not in testis of prepubertal animals (Jacobs et al.,
1998). In the mouse, Arl4 expression starts at day 16 when spermatogenesis
proceeds to the late pachytene. In adult testis, Arl4 was detected
immunohistochemically in pre- and post-meiotic cells, spermatocytes, and
spermatids, but not in spermatogonia and mature spermatozoa (Schürmann
et al., 2002). Deletion of the Arl4 gene produced a significant reduction in
testis weight and number of spermatozoa without affecting their mobility or
reproductive function. Thus, Arl4 is required for progression through
meiosis, but not for the division of spermatogonia or the function of mature
spermatozoa (Schürmann et al., 2002).
Because of the differential expression of Arl4 in 3T3-L1 adipocytes
(Schürmann et al., 1994), a defect in the development of adipose tissues was
expected in the Arl4-/- mice. Therefore, the weight of epididymal fat pads
and several metabolic parameters were analyzed in Arl4+/+ and Arl4-/- mice.
However, no differences in epididymal fat pad weight, blood glucose levels,
serum cholesterol, serum triglycerides, or blood cell count were detected.
 ARF-LIKE PROTEINS 337
13

Lin et al. (2000) have shown that Arl4 is developmentally expressed

during mouse embryogenesis. Arl4 mRNA appears transiently at day 8.5 to
10.5 in a rostro-caudal direction, suggesting that Arl4 is involved in somite
formation and central nervous system differentiation. However, because
mice lacking the Arl4 gene were viable with normal growth and no apparent
abnormality it was concluded that Arl4 is dispensable for embryonic
development (Schürmann et al., 2002).
The organization of the Arl4 gene appears unique among the Arf family
in that its coding region, its 3’ UTR, and a considerable portion of its 5’
UTR are located on a single exon. This observation suggested that the Arl4
gene has evolved by retrotransposition, and that its evolution is a relatively
recent event (Jacobs et al., 1998). Interestingly, two separate promoters
direct the expression of Arl4 in different tissues. The downstream promoter
appeared to be active in most tissues, whereas the upstream promoter
specifically drives the large expression in testicular germ cells (Jacobs et al.,
1998).

6. ARL5

Arl5 was identified by PCR cloning on the basis of its sequence similarity
with members of the Arf family (Breiner et al., 1996). Arl5 exhibits 48.2%
and 49.2% identity with Arf1 and Arl1, respectively, and is mainly
expressed in brain, thymus and intestine. No other information (e.g.,
subcellular localization, nucleotide binding properties, function) is yet
available.

7. ARL6/ARF4L

Arl6, also named Arf4-like protein (Arf4L; Smith et al., 1995; accession
number L38490), exhibits the highest similarity to Arl4 (61%) and Arl7
(59%) (Jacobs et al., 1999). Arl6/Arf4L binds guanine nucleotides rapidly
and in a Mg2+-dependent manner. The exchange rate of Arl6/Arf4-like
protein alone was considerably higher than that of Arf1 in the presence of its
exchange factor Sec7-2/ARNO. Like its close relatives Arl4 and Arl7,
Arl6/Arf4L contains an NLS in its C-terminus, suggesting that it too is
located within the nucleus (Jacobs et al., 1999).
Unfortunately, a different Arl has also been named Arl6 (accession
number AF031903). The amino acid sequence of this GTPase is 41%
identical with that of Arf1 (and only 34% identical to Arl6/Arf4L). Its
cDNA was identified by differential display (Ingley et al., 1999). The gene
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14 SCHÜRMANN AND JOOST

exhibits an expression pattern that is unique within the Arf and Arl family,
with highest mRNA levels present in brain and kidney. In addition, this
Arl6 transcript is up regulated during erythropoietin-induced differentiation
of erythroid cells and down regulated during interleukin 6-induced
macrophage differentiation, suggesting that it plays a role in hematopoietic
development. The protein was found mainly in the cytoplasm (Ingley et al.,
1999). Stimulation of cells with GTPγS produced an increase in membrane
association, indicating that the protein may cycle between the cytoplasm and
membranes in a GTP-dependent manner. A yeast two-hybrid screen and co-
immunoprecipitation revealed that Arl6 interacts with Sec61β, a subunit of
the core component of the ER translocon (Ingley et al., 1999).

8. ARL7 IS PRESUMABLY INVOLVED IN PROTEIN

TRANSPORT INTO OR OUT OF THE NUCLEUS

Arl7 was cloned by Jacobs et al. (1999), and shown to be a close relative of
Arl4 (71% identical amino acids) and Arl6/Arf4L (59% identical amino
acids). The C-terminal NLS of Arl7 targets a GFP fusion protein to the
nucleus of transfected COS-7 cells. Like Arl4 and Arl6/Arf4L, Arl7 binds
GTPγS rapidly and in a Mg2+-dependent manner. Arl7 mRNA expression
was highest in brain, with lower levels seen in spleen, thymus, esophagus,
stomach, intestine and uterus (Jacobs et al., 1999).

9. ARF-RELATED PROTEIN 1 (ARFP1)

Arfrp1 exhibits the lowest similarity to Arf proteins and several differences
to Arf and Arl proteins. It inhibits activation of phospholipase D (PLD) as a
result of its interaction with an Arf-specific activator (cytohesin 1, an Arf
GEF). Because deletion of the Arfrp1 gene resulted in marked defects in
early gastrulation, it was speculated that Arfrp1 is involved in adhesion-
dependent morphogenesis, cytoskeletal reorganization, or development of
cell polarity, occurring during this stage of embryogenesis.

9.1 ARFRP1 is essential in embryogenesis

Arfrp1 is a 25 kDa GTPase that exhibits somewhat lower similarity to Arf

and Arf-like proteins (33 and 39% identical amino acids to Arf1 and Arl3,
respectively) than do the Arls. Guanine nucleotide exchange on
recombinant Arfrp1 is slower than most GTPases, requiring an hour to reach
steady state in vitro. Arfrp1 shares two features with most Ras and GĮ
 ARF-LIKE PROTEINS 339
15

proteins that distinguish it from other members of the Arf family; Arfrp1 can
hydrolyze GTP in the absence of a GAP (0.093 min-1), and it lacks the site
for N-myristoylation (glycine 2). Arfrp1 was detected in the plasma
membrane and was absent from the cytosol (Schürmann et al., 1995) of 3T3-
L1 cells. The mechanism of this tight attachment to membranes is unknown.
Arfrp1 appears to be involved in a pathway that can inhibit Arf activities,
e.g., activation of PLD (Schürmann et al., 1999). In a two-hybrid screen
designed to identity proteins interacting with Arfrp1, a partial sequence
encoding the N-terminus and most of the Sec7 domain of the Arf-specific
guanine nucleotide exchange factor cytohesin 1 was isolated. The
interaction between Arfrp1 and the Sec7 domain was GTP-dependent and
resulted in the inhibition of the Arf/Sec7-dependent activation of PLD in a
system of isolated membranes. In addition, transfection of HEK-293 cells
with a constitutively active mutant of Arfrp1 inhibited the stimulation of
PLD by the muscarinic acetylcholine receptor-3, and the translocation of Arf
from the cytosol to membranes.
The closest relative of Arfrp1 in yeast is a protein with 43% identity that
has been identified by Huang et al. (1999) and designated ScArl3 (see
Chapter 1). Like ScArl1 and ScArf3, ScArl3 is not essential for cell
viability. However, disruption of ScArl3 resulted in cold-sensitive growth.
At the permissive temperature (37°C), maturation of vacuolar proteins was
retarded and, at the non-permissive temperature (15°C), impairment in fluid
phase endocytosis was observed. Unlike mammalian Arfrp1, ScArl3 was
detected in the cytosol and in part associated with the ER-nuclear envelope
structure, suggesting that the GTPase is involved in the regulation of
vesicular transport pathways (Huang et al., 1999).
The murine Arfrp1 gene spans approximately 7 kb and contains 8 exons.
The proximal 5’-flanking regions of mouse and human Arfrp1 lack a TATA
box and a CAAT box, are highly GC rich and contain potential transcription
factor binding sites. Sequence analysis of human Arfrp1 showed that its 5’-
flanking region contains the first exon of another gene (DJ583P15.3,
ensemble data base) on the opposite strand. Promoter analysis revealed that
the intergenic region between both genes (54 bp) exhibits bi-directional
promoter activity. However, deletion analysis demonstrated that
transcription of both genes is regulated by different cis-elements.
Mutational analysis and electrophoretic mobility shift assays indicated that
two short cRel- and cEts1-like elements in the 5’-flanking region of Arfrp1
are critical for the regulation of Arfrp1 expression (Mueller et al., 2002a).
Arfrp1 null-mutant mice, generated by gene targeting in embryonic stem
cells, were the first knockout mice of an Arf or Arl protein (Mueller et al.,
2002b). Heterozygous Arfrp1 mutants developed normally, whereas
homozygosity for the mutant allele resulted in embryonic lethality. Cultured
340
16 SCHÜRMANN AND JOOST

homozygous Arfrp1-null mutant blastocysts were indistinguishable from

wild-type blastocysts, suggesting that disruption of Arfrp1 did not alter the
growth of stem cells or the development of the blastocysts. Furthermore,
blastocysts implanted in vivo and formed egg-cylinder-stage embryos that
appeared normal until day 5. At day 6-6.5, the time of early gastrulation,
Arfrp1-/- embryos exhibited profound alterations of the distal part of the egg
cylinder. Rounded pyknotic cells were found within this area of the egg
cylinder in the proamniotic cavity, and some of these were only loosely
attached to the ectodermal cell layer. In contrast, the proximal
extraembryonic part of the embryo appeared normal (Figure 4). In order to
determine whether the morphologically abnormal cells within the
proamniotic cavity were apoptotic, TUNEL (terminal deoxy-nucleotidyl-
transferase-mediated UTP end labeling) assays were performed with sections

Figure 4. Schematic overview of the egg cylinder stage (E6.5) and the primitive streak stage
(E7.5) in control and Arfrp1-/- mouse embryos. (PS = primitive streak).

adjacent to those stained for histological examination. This analysis

demonstrated that the pyknotic epiblast cells in the area of the primitive
 ARF-LIKE PROTEINS 341
17

streak region were TUNEL-positive and therefore apoptotic (Figure 4;

Mueller et al., 2002b).
Early gastrulation is a stage during embryogenesis in which a set of
morphogenetic movements coupled with cell proliferation and
differentiation is necessary to convert the two germ layers into three; the
ectoderm, the mesoderm and the endoderm (Figure 4). A likely explanation
for the embryonic lethality is that deletion of Arfrp1 prevents the proper
integration of dividing cells into the ectodermal cell layer. We speculate that
Arfrp1 plays a critical role in processes of adhesion-dependent
morphogenesis, cytoskeletal reorganization, or development of cell polarity,
which are known to occur in early gastrulation (Mueller et al., 2002b).

10. SAR1 REGULATES VESICLE BUDDING FROM

THE ENDOPLASMIC RETICULUM

Sar1 exhibits weak similarity with the Arf family (30% identity with Arf1
and Arf5). The GTPase controls protein-protein and protein-lipid
interactions that direct budding from the endoplasmic reticulum (Springer et
al., 1999). In S. cerevisiae, only a single isotype was identified (Nakano and
Muramatsu, 1989), whereas two isotypes, Sar1a and Sar1b (91% identity)
were found in mammalian cells (Kuge et al., 1994). Unlike Arf proteins,
Sar1 lacks the myristoylated glycine residue at position two, and does not
undergo any other lipid modification. In NRK and CHO (Chinese hamster
ovary) cells, Sar1 was detected in the juxtanuclear Golgi region; in rat
pancreatic insulin cells, the protein was abundant at smooth transitional
elements of the endoplasmic reticulum (ER), and at vesicular profiles in the
proximal Golgi cisternae facing the transitional region (Kuge et al., 1994).
Sar1 is an essential component of COPII vesicle coats, and regulates the
selective export of membrane-bound and soluble proteins from the ER. The
COPII coat forms by the sequential recruitment of Sar1 and two large
complexes, Sec23/24 and Sec13/31 (Barlowe et al., 1993; Matsuoka et al.,
1998), and is necessary for the deformation of ER membranes into vesicles
as well as for the selection of cargo molecules (Schekman and Orci, 1996;
Rothman and Wieland, 1996). Sar1•GTP exhibits the unique ability to
interact directly with both lipid membranes and Sec23/24; therefore, the
switch between the GDP and GTP-bound forms controls assembly and
disassembly of COPII coats. Assembly is driven by the exchange of GDP
for GTP, which is catalyzed by Sec12. Sec12 is a transmembrane ER
glycoprotein containing a 40 kDa cytoplasmic domain and a ∼10 kDa
lumenal domain. The cytosolic domain operates independently of
membranes as a Sar1-specific GEF (Barlow and Schekman, 1993;
342
18 SCHÜRMANN AND JOOST

Weissmann et al., 2001). Activation of Sar1 by Sec12 leads to the formation

of ER transitional elements (Aridor et al., 2001), recruitment of cargo to pre-
budding complexes, and assembly of the Sec23/24 and Sec 13/31
complexes, leading to vesicle budding from the ER (Aridor et al., 1998;
Kuehn et al., 1998; Balch et al., 1994). The disassembly of COPII coats
requires GTP-hydrolysis on Sar1, which is stimulated by the COPII coat
subunit Sec23/24 (a Sar1 GAP), to promote vesicle uncoating and the
subsequent delivery of cargo to the Golgi complex through Rab1-dependent
tethering and fusion factors (Allan et al., 2000; Moyer et al., 2001).

11. THE STRUCTURE OF ARF-LIKE PROTEINS

The conformational changes that accompany the binding of GDP and GTP
can alter the affinity of the GTPases for proteins, lipids, and membranes.
These conformational changes are centered in two critical regions, referred
to as switch I and switch II (Lacal and McCormick, 1993). Residues in these
switches make direct contact with effectors and GAPs in a variety of
GTPases, including Arfs (Goldberg, 1998; Goldberg, 1999). The
interactions and the mechanism of regulation of Arf proteins differ from
those of other GTPases of the Ras superfamily. Arf proteins have an
additional nucleotide-sensitive region, the “myristoyl switch”, an extension
in the N-terminus and a covalently attached myristate which cooperate to
coordinate GTP binding (activation) and membrane association (Goldberg,
1999). In addition, there is evidence that the N-terminus may modify
effector binding or activity (Kahn et al. 1992; Zhu et al., 2000). The N-
terminal region (residues 1-17) of Arf is sufficient to confer Arf activity and
increase GTPase activity to an Arl protein, despite the distance between the
N terminus and the switch or nucleotide binding region (Kahn et al., 1992).
Amor, et al. (2001) analyzed the structure of yeast Arf2 and Arl1 and
reported that the secondary structure of GDP-bound yeast Arf2, yeast Arl1
and human Arf1 were essentially identical. Their structures include seven β-
strands, six α-helices, and 12 connecting loops. The core of the protein is an
invariant six-stranded β-sheet surrounded by six α-helices. The seventh
strand forms an edge to the core and corresponds to the switch I region
(Lacal and McCormick, 1993). The N-terminal α-helix (αNT) is wedged
between the C-terminal helix α-E and loop L-2/3 which connects strands β2
and β3 (also referred to as the interswitch region, ISR). The presence of
αNT is in contrast to the rather undefined secondary fold seen for the N-
terminal sequence in the Arl3 model (see Figure 5; Hillig et al., 2000). Like
Arl3•GDP (Hillig et al., 2000), yeast Arl1•GDP appears to form a covalent
 ARF-LIKE PROTEINS 343
19

homodimer, linked by a disulfide bond between the two Cys81 residues

present in switch II. However, dimerization of Arl1 does not

Figure 5. Ribbon diagram of Arl3•GDP and Arl2•GTP (Hanzal-Bayer et al., 2002), with
switches in blue, interswitch region in green, N-terminus in yellow, C-terminal helix in
magenta, nucleotides in orange, and Mg2+ as red sphere (see arrow).
344
20 SCHÜRMANN AND JOOST

appear to be essential for its function, since knock-in of the Arl1-S31 mutant
produced no effect in S. cerevisiae.
In contrast to Arf1, Arf2 and Arf6, the electrostatic surface of yeast Arl1
is characterized by an overall negative electrostatic potential. Arl1 is N-
myristoylated but, in contrast to Arf proteins, no GTP-dependent binding to
membranes or lipid vesicles has been detected. The difference in the
electrostatic potential led to the hypothesis that the binding of Arl1 to
membranes is dependent on protein-protein interactions at the membrane
(Amor et al., 2001).
The comparison of the structures of Arfs with that of Arl1 revealed
differences in the N-terminal region. The 17 N-terminal amino acids of
Arfs, which form the αNT and loop (L-NT), are involved in membrane
binding, nucleotide affinity, phospholipid dependence of GTP-binding, and
nucleotide hydrolysis (Kahn et al., 1992; Kahn et al., 1996). The N-terminal
helix and the length of the loop L-NT can affect the structure of switch I and
the interswitch region and therefore alter the nucleotide binding (Kahn et al.,
1992; Zhu et al., 2000). In comparison to Arf1 and Arf6, the loop L-NT in
yeast Arl1 is much longer, and this difference was proposed to be
responsible for different biophysical and biochemical properties of Arf and
Arl proteins (Amor et al., 2001).
Like Arl1, the structure of Arl3 predicts that the N-terminal extension
folds into an elongated loop region, suggesting that Arl3 releases its N-
terminus and undergoes a β-sheet register shift upon the binding of GTP.
Structure as well as kinetic experiments demonstrated high affinity GDP (but
not GTP) binding in the absence of Mg2+. The disturbed magnesium binding
site and the lack of Mg2+-dependence of GDP binding distinguish Arl2 and
Arl3 from Arf proteins. In contrast to Arf proteins, the conformational
change of Arl2/3•GTP did not require the presence of membranes and might
thus be energetically unfavored (Hillig et al., 2000).
Recently, Hanzal-Bayer, et al. (2002) presented the crystal structure of
full-length Arl2•GTP in a complex with its effector PDEδ. In this complex,
Arl2•GTP shows a typical G domain fold with six-stranded β sheet
surrounded by five α helices, with an additional α helix at the N terminus.
Because Arl3 is the closest relative of Arl2 (53% identity, Figure 1) and also
a binding partner of PDEδ (Figure 2), the authors compared the structure of
Arl2•GTP with Arl3•GDP (Figure 5). During nucleotide exchange, the
interswitch region of Arl2 has to undergo a β sheet register shift, with β
strands β2 and β3 sliding by two residues relative to the remaining β sheet.
This shift results in displacement and conformational change of the N-
terminus, which appears to interact with a membrane, as observed with Arf
proteins (Hanzal-Bayer et al., 2002).
 ARF-LIKE PROTEINS 345
21

The interaction of Arl2•GTP and PDEδ is strikingly GTP-dependent and

involves the switch region of the molecule (Hanzal-Bayer et al., 2002). It
was concluded that PDEδ is an effector of Arl2. Interestingly, the authors
concluded that the structure of PDEδ is closely related to Rho-GDI and
features a hydrophobic pocket similar to that found in Rho-GDIs. This
conclusion provides a structural explanation for the ability of PDEδ to
extract the catalytic subunit of PDE from the membrane, and to generate a
cytosolic pool of the enzyme (Cook et al., 2001). A GDI-like function was
also supported by the identification of four new putative targets that bind to
PDEδ: H-Ras, Rheb, Rho6 and Gαi1. The C-terminus of H-Ras is
absolutely required for the binding to PDEδ (Hanzal-Bayer et al., 2002).
Thus, PDEδ may function as a transport factor for H-Ras and other
prenylated proteins, and Arl2 and Arl3 might mediate their release and/or
uptake.

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