BB

ELSEVIER B i o c h i m i c a et B i o p h y s i c a A c t a 1286 (1996) 7 5 - 9 3

Biochi~ic~a
et Biophysica A~.ta

The secretory pathway: mechanisms of protein sorting and transport

Cordula Harter, Felix Wieland *
lnstitut fiir Biochemie L UniL'ersit~t Heidelberg, lm Neuenheimer Feld 328, D-69120 Heidelberg, Germany
R e c e i v e d 8 D e c e m b e r 1995; revised 18 J a n u a r y 1996; accepted 18 J a n u a r y 1996

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

2. Organelles o f the secretory p a t h w a y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

2.1. E n d o p l a s m i c reticulum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
2.2. The G o l g i c o m p l e x . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

3. Characterization o f vesicular transport intermediates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

3.1. Exit f r o m the E R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
3.2. C O P I coat in E R transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
3.3. C O P I I coat in ER transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
3.4. E R m e m b r a n e proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
3.5. Intra-Golgi transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
3.6. C O P I coat in intra-Golgi transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
3.7. M e m b r a n e proteins o f G o l g i - d e r i v e d C O P I vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
3.8. R e t r o g r a d e transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

4. Protein sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
4.1. Protein sorting in the ER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
4.2. Protein sorting in the G o l g i c o m p l e x . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

5. M a c h i n e r y o f vesicular transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
5.1. F o r m a t i o n of a coated vesicle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
5.2. T a r g e t i n g a n d fusion of a vesicle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

6. C o n c l u s i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

* C o r r e s p o n d i n g author. Fax: + 49 6221 544366; e-mail: bc I @ novsrv 1 .pio 1.uni-heidelberg.de.

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76 C. Harter, F. Wieland/Biochimica et Biophysica Acre 1286 (1996) 75-93

1. Introduction of experimental approaches has allowed to define these

The secretory pathway was originally defined by Palade
for the pancreatic exocrine cell [1]. Along this pathway
secretory proteins are transported from the endoplasmic
reticulum (ER) to the plasma membrane. The stations
include 'transitional elements' of the ER, the cis side of
the Golgi apparatus (cis Golgi network, CGN), the en-
trance port of the Golgi, the Golgi stack and finally the
trans side of the Golgi ( t r a n s - G o l g i network, TGN) as the
last station before the plasma membrane. It was suggested
that proteins are transported by distinct vesicles rather than
via continuous tubular connections between the ER and the
Golgi complex. Already in 1975, it was proposed that not
only highly specialized but rather all eucaryotic cells use
the secretory pathway to deliver proteins from the ER to
intracellular organelles or to the plasma membrane. Fur-
thermore, a relationship between membrane traffic and the
maintenance of cellular organization has been postulated.
Meanwhile, it is well established that transport from the
ER and through the Golgi apparatus occurs in vesicles that
travel vectorially in a cis to trans direction [2,3]. A variety
transport intermediates of the secretoy pathway [4,5].
Components involved in the formation and fusion of trans-
port vesicles have been isolated and a picture emerges how
intracellular protein transport is performed and how the
organization of the endomembrane system is maintained
[6].
The principles that are employed by cells for most
intracellular transport and sorting events most likely work
in all cell types with some variations. For example, in
polarized cells the sorting problem is more complex due to
the presence of functionally distinct membranes [7,8]. In
cells capable o f regulated secretion, fusion of a secretory
vesicle is mediated by a stimulus, a feature that adds
another layer of complexity to the fusion event [9]. In the
last few years, it has also become clear that not only
proteins but also lipids are selectively sorted along the
secretory pathway [10]. Research to unravel the biosyn-
thesis of lipids and lipid traffic in a cell is still in an early
phase [ 11 ].
In this review we will focus on the molecular machin-
ery underlying protein sorting and transport within the ER

::i:i:i:::::i

CGr,
ER GOLGI STACK PM

O COP I coatedvesicle ER endoplasmicreticulum

O COP II coatedvesicle LY lysosome

CGN cis Golgi network PM plasmamembrane
EN endosome SV secretoryvesicle
TGN trans Golgi network
Fig. 1. Transport of proteins along the secretory pathway. Proteins can exit from the ER at two distinct exit sites which are characterized by COPI and
COPII coats. Subsequent transport to the cis-side of the Golgi complex (CGN) may occur in COPI- or COPII-coated vesicles. The type of coated vesicle
which is chosen might be determined by the vesicle cargo and the cell type. Intra-Golgi transport is mediated by COPI-coated vesicles. At the TGN
proteins are sorted to their final destinations: lysosomes, secretory vesicles or the plasma membrane. TGN resident proteins as well as cell surface proteins
may recycle between the TGN and the plasma membrane. ER resident proteins that escaped the ER as well as lipids and membrane proteins of anterograde
transport vesicles which are involved in vesicle targeting and fusion may be retrieved by retrograde moving COPI-coated vesicles. For detailed discussions
see text.
 c. Harter, F. Wieland/ Biochimica et Biophysica Acta 1286 (1996) 75-93
and the Golgi apparatus, and we will discuss the mecha-
nism of targeted membrane fusion, best studies for the
neuronal synapse. Special aspects of the secretory path-
way, like sorting in polarized cells [ 12,13] and intracellular
trafficking of lipids [14-16], as well as biosynthesis of
secretory graules [17] will not be covered. The interested
reader is referred to the reviews cited above.
2. Organelles of the secretory pathway
2. I. Endoplasmic reticulum
The entry station for all proteins of the secretory path-
way (this includes secretory proteins, plasma membrane
proteins and proteins destined for various compartments of
the endomembrane system) is the endoplasmic reticulum.
Besides its role in detoxification of substrates by UDP-
glucuronyl transferases, the preparation of antigens to be
presented on the cell surface by major histocompatibility
complex and its capacity to synthesize lipids, the ER is
responsible for the synthesis and translocation of proteins,
and the release of properly processed, folded and assem-
bled proteins to allow transport to their destinations. These
functions of the ER are mainly performed by three sets of
proteins:
(i) the translocation machinery that transports the
nascent chain of a growing polypeptide through the ER
membrane (reviewed in [18-21]). In mammalian cells
translocation occurs predominantly co-translationally
[22,23]- although post-translational import has also been
described [24-26]. The translocation site is composed of
membrane proteins that target the ribosome-bound nascent
chain to the cytosolic face of the ER membrane [27-30],
and of proteins that form an ER-membrane channel through
which the polypeptide is translocated [31-34]. On the
lumenal site of the ER membrane, an oligosaccharyl trans-
ferase catalyzes co-translational N-glycosylation of most
polypeptides [35,36];
(ii) molecular chaperones that are present as soluble
proteins in the lumen of the ER or in a membrane-bound
form facing the lumen of the ER (reviewed in [37]).
Chaperones associate with nascent chains or with the
polypeptide after its release from the ribosome in order to
control and facilitate proper folding and assembly of the
newly synthesized protein (reviewed in [38-40]). An abun-
dant soluble ER-chaperone is BiP/grp74 [41] that not only
modulates protein folding in the ER lumen but also seems
to play a role in translocation of precursors across the ER
membrane [42]. Calnexin, a transmembrane protein, and its
soluble homologue calreticulin are lectins that have been
suggested to interact transiently with mono-glucosylated
N-linked oligosaccharides during folding of glycoproteins
[43-451;
(iii) a third major group of ER resident proteins com-
prises folding enzymes that assist in one way or the other
77
the proper folding of newly synthesized proteins, e.g.,
protein disulfide isomerase and peptidyl-prolyl cis-trans
isomerase (reviewed in [37,46]). Only properly folded and
assembled proteins pass the quality control of the ER and
are allowed to exit. Mal-folded proteins are not capable to
escape but are rapidly degradated in the ER [47,48].
2.2. The Golgi complex
The Golgi complex serves on its entry side as an
acceptor compartment of newly synthesized material from
the ER and on its exit as a distribution center for post-
translationally modified proteins to their destinations [49]
(Fig. 1). During their passage through the Golgi apparatus
proteins are subject to various kinds of post-translational
processing, most remarkably the remodeling of N-linked
oligosaccharides side chains of glycoproteins and the step-
wise biosynthesis of O-linked glycans. Processing of
carbohydrates occurs in an ordered sequence of enzyme-
catalyzed reactions [50,51 ]. Accordingly, transport through
the Golgi complex is directional. The functional complex-
ity of the Golgi apparatus calls for an organelle with
distinct polarities that is devided into subcompartments
equipped with distinct sets of enzymes.
The most characteristic features of the Golgi complex
have originally been revealed by electron microscopic
studies (reviewed in [49,52]): (i) its typical stack of 3-8
flattened cisternae; (ii) its polarity: the cis side is usually
oriented towards the ER and the trans side, where
clathrin-coated vesicles and tubular endosomes are found,
faces secretion granules or centrioles; and (iii) the compo-
sitional heterogeneity of distinct cisternae, e.g., cistemae
of the cis-Golgi contain resident enzymes distinct from
cisternae of the trans Golgi. In its simplest version, the
Golgi complex is composed of three subcompartments: the
cis-Golgi network (CGN) receives newly synthesized pro-
teins from the ER, the Golgi stack processes glycoproteins,
and the trans-Golgi network (TGN) sorts proteins to their
final destination (Fig. 1).
Recent data suggest that the CGN comprises vesicular
and tubular extensions that may be connected with the first
cisternae of the Golgi stack [52,53]. The CGN is not only
involved in receiving material from the ER but also in the
sorting and recycling of proteins and lipids to the ER with
only a limited role in glycosylation [54-56]. The opposite
side, the TGN, appears as a highly variable structure
depending on the cell type [57-59]. In secretory cells the
TGN is rather small, while in cells with an extensive
lysosomal system (e.g., Sertoli cells) the TGN forms an
sacculotubular network extending from the trans cisternae
[59]. It has been suggested that the TGN itself is composed
of functionally distinct subdomains ([60]; T. Nilsson, per-
sonal communication). Subdomains might house glyco-
sylation enzymes, e.g., sialyl transferase [61], as well as
enzymes that perform late Golgi modifications, such as
tyrosine sulfation [62], or might be involved in the segrega-
78 C. Harter, F. Wieland / Biochimica et Biophysica Acta 1286 (1996) 75-93
tion of membrane and lumenal proteins into different types
of transport vesicles destined for endosomes, lysosomes
[63], secretory granules [64] and the plasma membrane
[65-68]. Another type of subdomain might receive and
sort endocytosed material from the plasma membrane
[69,70]. Thus, the TGN is not a mere exit station of the
Golgi apparatus but a rather complex structure where
exocytic and endocytic pathways intersect.
Another view of the Golgi apparatus derives from the
drawing of biochemical rather than morphological bound-
aries [71]. This view is based on studies that examined the
distribution of glycosylation enzymes involved in the syn-
thesis of complex N-linked oligosaccharides [61]. For
example, o~1,3-1,6-mannosidase II and /31,2-N-acetylg-
lucosaminyltransferase I reside in both the medial and the
trans-cisternae with a more or less equal distribution over
both cisternae. Thus, a biochemical compartment would be
defined by an overlapping distribution of these two Golgi
resident enzymes. Co-localization of Golgi residents within
the same subcompartment also plays a role in the forma-
tion of domains as a means of retention of Golgi enzymes.
This topic will be discussed in chapter 4.2.
3. Characterization of vesicular transport intermediates
A combination of morphological studies, biochemical
assays and yeast genetics led to the current picture we
have from the secretory pathway. Morphological data pro-
vided the concept of the topographical and structural orga-
nization of organelles of the secretory pathway [1,52].
Intercompartmental transport in mammalian cells has been
characterized mainly by biochemical assays [72] and is
best understood for transport within the Golgi complex
[73]. These in vitro studies have allowed to characterize a
set of proteins involved in vesicular transport along the
Golgi stack, and subsequent work has revealed the general-
ity of the principles established (reviewed in [6]). This
includes genetic studies in S. cere~'isiae that have led to
the identification of distinct sets of genes that are involved
in intercompartmental transport [74] and that have allowed
to confirm some functions of the gene products in distinct
transport steps in vivo (reviewed in [5]).
3.1. Exit from the ER
Transport of newly synthesized proteins from the ER
has been characterized by various approaches both in vitro
and in vivo [75-78]. Based on morpholigical studies in the
60's, so-called transitional elements have been defined
within the rough ER (RER) [79]. In the original view of
the secretory pathway these transitional elements have
been suggested to represent the exit sites of proteins from
the ER [1] and during the last decade detailed immuno-
cytochemical and biochemical studies have led to a de-
tailed characterization of exit sites within the ER mem-
brane [80-82].
An issue that it still debated is whether proteins are
transported directly from the ER to the CGN or whether a
separated post-ER-pre-Golgi compartment, designated in-
termediate compartment (IC) [83], exists. A putative IC
has been defined biochemically by marker proteins, most
notably ERGIC 53, and morphologically by a thermal
block at reduced temperature [84,85]. At 15°C proteins are
segregated into tubulo-vesicular structures that are distinct
from the typical ER [86]. These structures do not seem to
belong to the Golgi complex since proteins do not receive
typical Golgi-modifications at 15°C. The IC, also referred
to as the 'salvage compartment' [87] or '15°C compart-
ment', has been suggested to be structurally and function-
ally continuous with the RER rather than representing a
physically distinct entity [88].
Two distinct exit sites of the transitional ER of mam-
malian cells have been recently characterized by immuno-
cytochemistry [82], one defined by a coat protein complex,
coatomer [89], and the other by a protein complex that
includes Sec23p complex [90,91] (Fig. 1). Coatomer is
referred to as COPI coat and the Sec23p containing com-
plex as COPII (reviewed in [92,93]). Both proteins com-
plexes have been shown to play key roles in vesicular
transport. The function of coatomer is not restricted to
export from the ER but extends to various sites of the
secretory pathway (section 3.2., 3.6. and 3.8.). Indeed,
coatomer was the first protein coat complex shown to be
involved in vesicular biosynthetic transport and charac-
terized as the major envelope constituent of Golgi-derived
transport vesicles [94,95]. The function of Sec23p complex
seems to be restricted to ER-Golgi transport both in mam-
mals and in yeast (section 3.3.).
3.2. COPI coat in ER transport
Evidence that COPI is involved in ER to Golgi trans-
port in mammals is based on several biochemical and
immunocytochemical assays:
(i) microinjection of antibodies against a subunit of the
COPI complex (/3-COP) efficiently blocks transport of
proteins en route to the Golgi [78];
(ii) at 15°C, a condition that allows exit from the ER
but prevents entry to the Golgi, COPI co-localizes together
with cargo proteins (e.g., the glycoprotein of vesicular
stomatitis virus in infected cells) in specialized tubular
structures of the ER, also designated the IC (section 3.1)
[96];
(iii) immunocytochemical studies under certain condi-
tions (reduced temperature, energy-depletion, or treatment
of cells with the drug brefeldin A (BFA) that induces a
redistribution of the Golgi apparatus to the ER) led to the
identification of special domains of the ER that are en-
riched in COPI complexes and consequently were desig-
 C. Harter, F. Wieland/ Biochimica et Biophysica Acta 1286 (1996) 75-93 79

Table 1 tion of this motif is unknown. It might represent an

Coat proteins of COPI- and COPII-coated vesicles oligomerization motif or it might be involved in the bind-
Protein complex Subunits (M.W.) Homologues/features ing of coatomer to membranes. /3-, 6- and ~'-COP show
(M.W.) weak homolgies to subunits of the adaptin complexes of
COPI clathrin-coated vesicles [110], while 3/- and e-COP are not
cofltomer a-COP (140 kDa) Ret I p/WD-40 repeats related to any known protein. So far, no enzymatic activity
(700 kDa) could be attributed to any of the coatomer subunits.
/3-COP (107 kDa) Sec26p//3-adaptin of AP-2
/T-COP (102 kDa) Sec27p/WD-40 repeats
T-COP (97 kDa) Sec21p; KKXX binding
3.3. COPII coat in ER transport
6-COP (57 kDa) AP47 of AP- 1
e-COP (35 kDa)
~'-COP (20 kDa) AP17/19 of AP-2/AP-I A role of Sec23p in ER to Golgi transport was first
ARF (20 kDa) Sarlp/Yptlp; GTP-binding suggested in S. cerevisiae [90] based on the observation
COPII
Sec 13 complex Secl 3p (34 kDa) WD-40 repeats that mutations of the corresponding gene lead to a defect
(700 kDa) in vesicle formation and accumulation of core-glycosylated
Sec31p (150 kDa) WD-40 repeats proteins that are not transported out of the ER. Cross-reac-
Sec23 complex Sec23p (85 kDa) GAP for Sarlp tion of an anti-Sec23p antibody with a protein on transi-
(400 kDa) tional elements of the ER from mammalian cells then
Sec24p (105 kDa) indicated that this protein is likely to play a role in
Sarlp (21 kDa) ARF family; GTP-binding
ER-to-Golgi transport in various cell types [91].
For references, see text. Further mutant genes that show phenotypes similar to
sec23 include sarl[l 11], sec 12 [112,113] and secl3 [114].
Sarlp is a 21 kDa GTPase that belongs to the class of
nated coatomer-rich ER (CRER) [82]. Although at present small GTP-binding proteins and shares significant homol-
the exact function of these special sites is not clear, it is ogy to the ARF family of GTPases (reviewed in [115]).
likely that these domains are actively involved in transport; Nucleotide exchange on Sarlp is catalyzed by Secl2p, an
(iv) a role of COPI in export from the ER has also been integral membrane glycoprotein on the cytosolic face of
suggested for S. cerevisiae. Mutations in some coatomer the ER [116]. Sec23p (85 kDa) is a Sarlp-specific
subunits result in defects in ER-to-Golgi transport as judged GTPase-activating enzyme (GAP) [117] and is associated
by the accumulation of ER-membranes and ER-derived with Sec24p (105 kDa) in a cytosolic complex with an
transport vesicles, and by the processing of carboxypepti- apparent molecular weight of 400 kDa [118]. Another
daseY, a protein that is normally synthesized in the ER as soluble complex involved in ER-to-Golgi transport con-
an immature precursor and subsequently processed in the sists of Secl3p (34 kDa) [114] and Sec 31p (150 kDa)
Golgi [97,98]. [119]. Both Secl3p and Sec31p contain WD-40 motifs that
Coatomer complex was first isolated from cytosol of have also been found in a- and /T-COP of COPI (section
bovine brain [89]. It is present in all eukaryotic cells 3.2.). The set of soluble proteins consisting of Sarlp,
examined, including Drosophila [99] and yeast [97,100]. Sec23p complex and Secl3p complex, designated COPII,
The discovery of a membrane-bound form of coatomer on is sufficient for the formation of functional ER-derived
Golgi-derived transport vesicles was the first hint for a role vesicles in yeast [120] (Table 1).
of this complex in intracellular trafficking [95] (section Recently, mammalian homologues of Sarlp [121] and
3.6.). Secl3p [122] have been identified. Like mammalian
Coatomer (700 kDa) consists of seven subunits (Table Sec23p, these proteins reside in a region of the transitional
1): a-COP [101,102], /3-COP [95,103], /T-COP [104,105], ER. Although COPII seems conserved from yeast to mam-
y-COP [106], 6-COP (Faulstich et al., submitted), e-COP mals, no discrete function has been attributed to COPII-
[107] and ~'-COP [108]. cDNA-derived primary structures coated transport vesicles in mammals nor have those vesi-
are known for all mammalian COPs. In S. cerevisiae, cles been isolated, so far.
homologues of a-, /3-, /3'- and y-COP are the products of Since COPII-coated vesicles can perform ER-to-Golgi
the genes RET1 [101], SEC26, SEC27 [98] and SEC21 transport in yeast, the question arises as to the function of
[97], respectively. COPI in ER transport in this species. Recent studies have
The most remarkable feature of the primary structure of proven that indeed both COPI and COPII are capable to
ce- and /T-COP is the presence of four and five WD-40 drive the formation of distinct types of coated vesicles
repeated motifs (a conserved ~ 40 amino acid stretch from isolated ER membranes [276]. Both types of vesicles
terminating in the residues Trp, Asp), respectively (re- share some integral membrane proteins (section 3.4.), and
viewed in [109]). WD-40 repeats are typically found in for COPII coats a selectivity for some specific cargo
/3-subunits of trimeric G proteins but also in other subunits proteins has been demonstrated [276]. The role of ER-de-
of various hetero-oligomeric protein complexes. The func- rived COPI-vesicles in yeast remains to be established.
80 C. Harter, F. Wieland/Biochimica et Biophysica Acta 1286 (1996) 75-93
3.4. ER membrane proteins
The recruitment of cytosolic coat proteins to mem-
branes during the budding reaction calls for specific inte-
gral membrane proteins as binding partners. A candidate
for a functional role in recruiting COPII-components from
the cytosol is Emp24p, a type I membrane protein present
in ER-derived COPII vesicles of S. cereL~isiae [123]. Muta-
tions in the EMP24 gene have been shown to affect export
from the ER of a set of proteins.
In addition, mutational analysis in S. cereuisiae has
identified several genes that code for integral components
of the ER membrane required for the transit of proteins
from the ER to the Golgi [124]. Unlike Emp24p, these
proteins seem to be required for the correct docking of
transport vesicles rather than for recruiting coat compo-
nents. Betlp, Sec22p and Boslp are cytoplasmically ori-
ented hydrophilic small integral membrane proteins with a
stretch of hydrophobic residues at their C-termini [[125],
[126], [127]]. Bosl was identified by its ability to suppress
the betl mutant (bet stands for blocked early in transport)
[128]. Boslp and Sec22p are constituents of ER-derived
transport vesicles, whereas it is less clear whether Betlp
also resides in those vesicles [120,129,276]. These compo-
nents will be discussed in the context of vesicle targeting
and fusion (section 5.2.).
3.5. Intra-Golgi transport
The breakthrough towards the elucidation of the molec-
ular mechanisms of vesicular transport was the reconstitu-
tion in a cell free system of vesicular flow (reviewed in
[72,130]). This was achieved by incubation of two differ-
ent populations of Golgi membranes with cytosol and ATP
as an energy source at 37°C [73,131]. A 'donor' population
containing the cargo to be transported, like for example
VSV G protein, is incubated with an 'acceptor' population
of Golgi that contains a glycosylating enzyme, GlcNAc
transferase, whose activity is absent in the donor popula-
tion. If, after addition of UDP-3H-GIcNAc, the radioactive
aminosugar is transferred to the cargo protein, this label
serves as a measure for the amount of protein transported
from the donor to the acceptor compartment [132]. The
transporting vesicles were visualized by immunoelectron
microscopy and were found to bud preferentially from the
rims of the Golgi cisternae [94,133]. The vesicles are
uniform in size, with a diameter of about 75 nm. They
have an electron-dense, fuzzy protein envelope, distinct
from the clathrin coat of endocytic vesicles. In fact, this
envelope was the first protein coat defined on biosynthetic
transport vesicles, consisting of coatomer and most re-
cently named COPI-coat. Coated vesicles were predomi-
nantly found in budding regions and this was taken as a
first indication that the coat drives the budding reaction by
deforming the membrane into a spherical shape. Omission
of either cytosol or an ATP regenerating system as well as
incubation at 0°C completely blocked coated vesicle for-
mation [134]. Addition of GTPTS, a nonhydrolyzable ana-
logue of GTP, during the generation of Golgi-derived
transport vesicles in vitro results in inhibition of transport
with a concommitant accumulation of coated vesicles [135],
and has allowed their isolation in amounts sufficient for a
biochemical characterization of their major protein compo-
nents [95]. Final purification was achieved by isopycnic
sucrose gradient centrifugation. Fractions at about 40%
sucrose ( w / v ) contained the putative transport vesicles as
has been shown by the following criteria: (i) electron
microscopy, that revealed the presence of a homogenous
population of coated vesicles of about 75 nm in diameter
and (ii) immunological analysis that demonstrated these
fractions to contain typical cargo proteins: VSV G protein,
if VSV infected CHO cells were used as the donor Golgi
source, or albumin, if liver cells were used. On SDS-poly-
acrylamide gels these vesicles gave rise to a characteristic
protein pattern. The major part of these proteins turned out
to be membrane-associated rather than integral membrane
proteins, and biochemical and immunocytochemical stud-
ies have shown that they represent the fuzzy coat observed
on these vesicles by electron microscopy [95] (reviewed in
[92]).
3.6. COPI coat in intra-Golgi transport
The coat of Golgi-derived transport vesicles - - termed
COPI - consists of two types of proteins: the coatomer
-
complex with seven subunits, the COP proteins [89,95]
(section 3.2) and ADP ribosylation factor (ARF), a small
GTP-binding protein [136] (Table 1).
Each individual COP is a constituent of both the cytoso-
lic coatomer and of Golgi-derived transport vesicles as
demonstrated by immunolocalization and peptide sequence
comparisons [95,102,104,106-108]. (-COP is the only
coatomer subunit that, in addition to its coatomer-associ-
ated form, is also present as a soluble monomer in the
cytosol [ 108].
Similar to coatomer, ARF is mainly cytosolic with only
minor amounts present on membranes [137]. ARF was
originally discovered as co-factor in Cholera toxin-cata-
lyzed ADP-ribosylation of stimulatory G protein ce-sub-
units [138]. It belongs to the Ras family of GTPases, but
differs from other members of this family by the absence
of a carboxyl-terminal prenylation motif and by the pres-
ence of a myristyl residue on its amino-terminal glycine
residue (m-ARF), a modification essential for membrane
association (reviewed in [139]). In vitro experiments with
purified components as well as immunocytochemistry have
proven that m-ARF and coatomer are the only cytosolic
proteins required for the formation of coated buds and
vesicles from Golgi membranes [140,141]. When Golgi
membranes are incubated with m-ARF and coatomer in the
presence of GTF'TS coated buds and vesicles are formed.
 c. Harter. F. Wieland/ Biochimica et Biophysica Acta 1286 (1996) 75-93
3.7. Membrane proteins o f Golgi-deri~'ed COPI uesicles
Recently, two major integral membrane proteins of
Goigi-derived COPI-coated vesicles from mammalian cells
have been characterized, p24 [142] and p23 (Sohn et ah, in
preparation). They belong to a family (p24 family) with
homologues present in yeast (Emp24p of COPII-coated
ER-derived vesicles [123]; section 3.3) and mammals [142].
All p24 family members contain heptad repeats of hy-
drophobic residues in their lumenal domain and short
cytoplasmic tails. Heptad repeats were predicted to form
coiled coils that might be involved in protein-protein
interactions to form homo- or hetero-oligomers [143].
Likewise, these motifs may trigger periplasmic fusion at
the base of a coated bud followed by the release of a
coated vesicle. A role of p24 in vesicle budding is sup-
ported by studies with yeast strains that were deleted in the
gene of p24 [142]. Production of transport vesicles was
reduced in these strains, while viability and growth was
unaffected. Some of the p24 family members contain
dibasic motifs at their C-termini which are related to the
KKXX motif believed to serve as a signal for the retrieval
of membrane proteins from the Golgi to the ER [144]
(section 4.1.2.). Recently, this KKXX motif has been
shown to bind specifically to coatomer [145]. p24 of COPI
vesicles contains an RRXX sequence at its C-terminus
[142], p23 contains a C-terminal KKXXX motif (Sohn et
al., in preparation). It is not known at present whether
these motifs bind to coatomer under physiological condi-
tions.
3.8. Retrograde transport
The continous flow of membrane from the ER to the
Golgi apparatus - - also referred to as anterograde trans-
port - - would ultimately lead to a depletion of ER mem-
branes and to a steady extension of the Golgi complex.
Consequently, in order to maintain the functional and
structural identity of the endomembrane system, lipids and
proteins must be recycled. Retrograde transport from the
Golgi back to the ER in membrane-bound compartments
would serve several purposes:
(i) to ensure the recycling of lipids that are needed for
the formation of anterograde moving transport vesicles
simultaneously maintaining the surface area of the ER;
(ii) to provide a means to retrieve ER resident proteins
that had escaped from this organelle;
(iii) to recycle membrane proteins that are involved in
the targeting and fusion of transport vesicles from the
donor compartment to the appropriate acceptor compart-
ment, e.g., from the ER to the cis-Golgi (Fig. 1).
Interestingly, the effects of the fungal antibiotic brefeldin
A (BFA) have been taken as evidence for the existence of
a physiologically relevant retrograde pathway from the
Golgi to the ER [146]. BFA reversibly blocks transport of
secretory proteins to the Golgi apparatus [147], and in-
81
duces a dramatic fusion of most of the Golgi membrane
with the ER [146]. Treatment of many mammalian cells
with BFA leads to an immediate dissociation into the
cytosol of Golgi-bound COPI-coats [148,149].
However, the BFA-induced fusion of Golgi and ER-
membranes might have nothing to do with retrograde
transport, because a fundamental function of coatomer
seems to be a protective one: to prevent uncontrolled
fusion, thereby coupling fusion to vesicle budding [150].
Thus, BFA-induced dissociation of coatomer would allow
uncontrolled fusion of Golgi with ER membranes rather
than retrograde transport.
Recently, direct biochemical as well as yeast genetic
data have provided evidence for a functional role of
coatomer also in retrograde transport [101,145]. Coatomer
binds in vitro to protein constructs that contain a di-lysine
motif (KKXX) present in membrane proteins that reside in
the ER [151 ]. Furthermore, yeast strains that are mutated in
the genes coding for a-COP (retlp), /3'-COP (Sec27p) or
y-COP (Sec21p) are defective in the retrieval of a con-
structed KKXX-bearing protein [101]. In wild-type cells
the KKXX-containing construct is retrieved to the ER,
whereas in the coatomer mutants this protein is transported
to the cell surface. This was taken as a strong indication
that - - in addition to their well-established role in antero-
grade transport from the ER to the Golgi and across the
Golgi stack - - COPl-coated vesicles are mediating retro-
grade transport from the Golgi to the ER [152,153] (Fig.
1). In a set of experiments using photolabile peptides the
coatomer subunit y-COP was shown to be the direct
binding partner of the cytosolic KKXX tail motif [154].
This is surprising because mainly a-COP has been sug-
gested to be involved in this binding according to the
genetic studies in yeast [101].
The mechanisms to regulate whether a COPI-coated
transport vesicle moves forward or backward are presently
unknown. The following scenarios may be considered:
(i) the existence of distinct but related populations of
coatomers: one for anterograde, the other for retrograde
transport;
(ii) coatomer binds to the various cytoplasmic tail mo-
tifs found in Golgi-membrane proteins with different
affinities. Thus, binding and movement of a COPI-coated
vesicle would be regulated by the concentration of these
cytoplasmic tails. If, for example, many KKXX-containing
proteins escaped from the ER, their local concentration in
a particular Golgi cisterna would be high, and this would
result in coatomer binding and subsequent retrieval to the
ER, A prerequisite for the binding of coatomer is the
accessibility of KKXX motifs on a membrane. As cytoso-
lic KKXX-motifs are typical of ER-resident membrane
proteins one would predict that most of the coatomer is
bound to the surface of the ER. However, under physio-
logical conditions coatomer is found predominantly at the
Golgi apparatus. Thus, the KKXX motif protruding from
the ER must somehow be masked. A possible candidate to
82 C. Harter, F. Wieland / Biochimica et Biophysica Acta 1286 (1996) 75-93
form a matrix on the ER membrane that masks cytosolic
KKXX tails are microtubules [144,155] (discussed in sec-
tion 4.1.2.). If coatomer is involved in both anterograde
and retrograde transport from the Golgi, and binds via
KKXX motifs, the motifs must be accessible on the Golgi
surface. Anterograde COP-coated vesicles form, as de-
scribed in section 3.6., in an ARF-GTP-dependent manner.
Nothing is known about the physiological conditions that
allow KKXX-binding of coatomer so far, but the in vitro
studies have shown that binding to this motif is indepen-
dent of ARF and GTP. Thus, dependence on ARF-GTP
may discriminate whether an anterograde or retrograde
COPI-coated vesicle forms depending on the concentration
of KKXX-tails that are accessible in any case, and binding
partners for the formation of anterograde vesicles may be
made accessible only by a mechanism that involves ARF-
GTP;
(iii) a completely different view is that coatomer is
involved in vesicle-mediated transport only in the retro-
grade direction [152,156]. Two alternative mechanisms
have been proposed for anterograde transport: first, the
existence of a set of still unknown coat proteins [152] and
second, the formation of tubuluar extensions that emanate
from one Golgi cisterna and fuse with an adjacent cisterna
[ 156]. In this concept anterograde transport would be medi-
ated by tubules rather than by vesicles.
Binding studies with various cytoplasmic tail motifs and
the isolation and molecular characterization of retrograde
transport vesicles will help to resolve this problem.
4. Protein sorting
A critical characteristic of protein traffic within the
secretory pathway is that each component chooses its
correct destination. Failure in doing so would profoundly
affect the identiy of the individual organelles. What does,
for example, cause a protein to stay in a particular or-
ganelle or to depart to the next organelle, a basic feature of
sorting? Protein sorting can principally occur by two dif-
ferent mechanisms:
(i) it can result from a signal that causes the protein to
be retained within a specific location, i.e., the protein is
prevented to enter a departing transport vesicle. Retention
signals may consist of specific peptide domains of a few
amino acids that are recognized by a receptor-like molecule.
Alternatively, retention might also be induced through
signals that induce the formation of oligomers or aggre-
gates. Such protein aggregates might be too large to enter a
transport vesicle;
(ii) a protein can be tagged with a signal that specifies it
as cargo for export. The presence of a transport signal
would result in selective packaging of a given protein into
a budding transport vesicle. As for a retention signal, some
type of receptor would be required that specifically recog-
nizes a transport signal for a particular organelle. Lumenal
proteins would be recognized by a transmembrane protein,
and integral membrane proteins by specific coat compo-
nents. The best known example for a targeting signal that
is recognized by a receptor is the mannose 6-phosphate tag
of lysosomal hydrolases which is bound to the mannose
6-phosphate receptor (reviewed in [157,158]). A well-
established transport signal of plasma membrane receptors
is defined by a tyrosine-containing cytoplasmic tail motif
(NPXY (or related), also referred to as coated-pit localiza-
tion signal; reviewed in [159,160]). This motif interacts
with a component of the adaptin II complex of clathrin-
coated vesicles I110].
All proteins that neither contain a retention signal nor a
transport signal would be consequently exported by de-
fault. This also implies that proteins are transported from
the donor to the acceptor compartment without concentra-
tion, a mechanism termed bulk flow [3,161 ].
4.1. Protein sorting in the ER
A vast subset of proteins that are synthesized into the
ER resides in this organelle at steady state. As outlined
above, residency of a protein in a given organelle may
result from a retention signal that prevents the protein to
enter a budding transport vesicle. However, a resident
protein of a certain organelle might escape and must be
carried back by use of a retrieval signal. Resident proteins
of the ER, both lumenal and integral membrane proteins,
have been shown to be capable to reach post-ER compart-
ments from which they can be retrieved [162]. Therefore,
ER residency may be achieved by retrieval mechanisms in
those cases where retention is rather weak [144].
4.1.1. Sorting of lumenal ER resident proteins
The first retrieval signal identified was the C-terminal
tetrapeptide Lys-Asp-Glu-Leu (K-D-E-L) in mammalian
cells and H-D-E-L or a related sequence in S. cerevisiae
[163,164]. These sequences are found in many soluble ER
proteins, like protein disulfide isomerase, BiP/grp78,
grp94 and calreticulin (reviewed in [162,165]) (Table 2). If
transplanted to various reporter proteins this peptide causes
localization of the constructs in the ER demonstrating that
the KDEL-sequence is both necessary and sufficient for
ER-localization [163]. However, such reporter proteins
were found to acquire Golgi modifications, an indication
that they must have been transported out of and returned to
the ER [164,166]. For example, the lysosomal enzyme
cathepsin D tagged with a C-terminal KDEL sequence is
efficiently retained in the ER, but carries a GlcNAc-1-
phosphate residue, a modification that is thought to occur
in a post-ER compartment [164]. Furthermore, in tempera-
ture-sensitive mutant yeast cells that are defective in ER-
to-Golgi transport, HDEL-bearing fusion proteins undergo
Golgi modifications at permissive temperature (at this
temperature mutant cells behave similar to wild-type cells).
At non-permissive temperature, however, these proteins do
 C. Harter, F. Wieland / Biochimica et Biophysica Acta 1286 (1996) 75-93
not receive Golgi modifications [166]. The capacity to
retrieve KDEL-tagged molecules is not restricted to the
Golgi but extends throughout the secretory pathway as
recently shown by an elegant approach: a KDEL-tagged
probe was introduced into the TGN and its retrieval from
this compartment to the ER was monitored [167].
It has been suggested that ER proteins are carried along
the secretory pathway with other proteins, and are recov-
ered by a receptor-mediated process in a post-ER compart-
ment [165,166]. The receptor-ligand complexes are then
incorporated into a special class of transport vesicles that
return to the ER where the ER proteins are released. A
putative sorting receptor was first identified in yeast by a
genetic approach [168,169]. Yeast mutants unable to retain
protein constructs bearing the HDEL signal were isolated.
This led to the identification of two genes, termed ERD1
and ERD2 (for ER retention defective). Mutations in ERD1
affect Golgi function and seem to block ER retrieval
indirectly [170]. The ERD2 gene encodes a transmembrane
protein with properties that are expected for a receptor
[169]. The S. cerevisiae receptor is a 26 kDa protein with
7 putative transmembrane domains, a short cytoplasmic
C-terminal tail and an amino-terminus that is oriented
towards the lumen. Mutation or underexpression of the
gene causes secretion, while overexpression in wild-type
cells improves retention of HDEL-containing proteins. The
ability of the ERD2 gene product to increase the capacity
of a cell to retrieve HDEL-tagged proteins strongly sup-
ported that this gene indeed codes for the HDEL receptor
[169]. Based on mutational analysis it has been suggested
that ligand binding is dependent on charged residues in the
transmembrane domains, while retrograde movement of
the receptor-ligand complexes seems to involve a critical
aspartic acid residue in the seventh transmembrane domain
[171]. Interestingly, cells depleted in the ERD2 gene are
not only defective in retrieval but also in growth and, more
intriguingly, in protein secretion [ 169]. How can this find-
ing be explained? If the HDEL receptor is indeed involved
Table 2
Mechanisms of protein residency
Protein Localization Motif
BiP ER-lumen C-terminal K/H-D-E-L
PDI
Grp94
calreticulin
E3/ 19 ER-membrane KKXX-COO-
UDP-gluc.T KXKXX-COO-
Iip33 +H 3N-XXRR
NAGT I medial-Golgi transmembranedomain/lumenal domain
Man II medial-Golgi
GalT trans-Golgi/TGN
SialT trans-Golgi/TGN transmembranedomain
transmembranedomain/cytoplasmictail (Y-Q-R-L)
TGN38/41 TGN
For references, see text.
83
in transport from Golgi compartments to the ER, i.e.,
retrograde transport, then a block in the retrieval of Golgi
membranes to the ER might result in a loss of membrane
proteins that are needed for anterograde transport. Thus,
the results obtained with HDEL receptor deficient cells
strongly support a function of this receptor in the forma-
tion of retrograde moving transport vesicles [ 169]. A role
of the erd2-protein/HDEL-receptor in ER-Golgi mem-
brane traffic is further supported by the observation that its
overexpression results in a BFA-like phenotype: (i) redis-
tribution of Golgi enzymes into the ER, (ii) release of
/3-COP from Golgi membranes into the cytosol, and (iii)
block of anterograde transport from the ER [172]. This
phenotype may be due to uncontrolled fusion of Golgi
with ER membranes as discussed in section 3.8.
Homologues of the S. cere~'isiae receptor have been
identified in mammals, and are termed KDEL receptors
[173-175]. The human KDEL-receptor normally localizes
to the Golgi apparatus but moves towards the ER at high
levels of expression of KDEL-tagged proteins [176]. Opti-
mal binding to the KDEL-sequence occurs at acidic pH,
suggesting that the selective binding and release of the
ligand in the Golgi and the ER may be regulated by a pH
difference in these organelles [177]. In fact, the pH along
the secretory pathway becomes increasingly acidic from
the ER towards the TGN [178]. Thus, it seems consistent
that the KDEL receptor binds its ligand with higher affin-
ity at more acidic pH. KDEL binding in a post-ER com-
partment would induce a conformational change of the
receptor that triggers retrograde transport of the receptor-
ligand complexes to the ER, and the higher pH in the ER
would then promote a dissociation of the complex. Empty
receptors would be returned to their original localization
and await another round of ligand binding [ 179]. Retention
in the Golgi of the receptor has been proposed to be
mediated by interactions within the lipid bilayer that might
lead to a Golgi-specific conformational state of the recep-
tor [171 ]. The shift of the KDEL-receptor from the Golgi
Mechanism
KDEL-receptor(retrieval)
KDEL-r.,/Ca2+ ? (retention)
Coatomer/Microtubuli?
oligomerization (retention)
lipid bilayer(retention)
oligomerization/retrieval
84 C. Harter. F. Wieland / Biochimica et Biophysica Acta 1286 (1996) 75 93
to the ER induced by overexpression of KDEL-tagged
proteins [176] would indicate that dissociation at higher pH
(in the ER) is the rate-limiting step in the retrieval process.
4.1.2. Sorting of ER membrane proteins
As mentioned in section 3.8., several ER-resident mem-
brane proteins have been shown to contain short discrete
signals in their cytoplasmic tails that are responsible for
residency in the ER of these proteins [ 15 l, 180,181 ]. For
type I transmembrane proteins the motif contains two
lysine residues in position - 3 / - 4 or - 3 / - 5 relative
to the C-terminus (KKXX or KXKXX, where X is any
amino acid) [151,180]. ER residents with type II topology
are characterized by a double arginine motif (RR) which
has to be within the first five amino acid residues of the
protein [181] (Table 2). Similar to the studies performed
with KDEL-tagged fusion proteins, transplanting an KK or
RR motif to a protein that is normally transported to the
plasma membrane results in its localization to the ER
membrane [144,181]. Both KK- and RR-chimerae acquire
Golgi modifications and co-localize in post-ER vesicular
structures indicative of a common retrieval pathway for
type I and type II ER-resident proteins that have escaped
from the ER. However, it is not known whether both
membrane proteins (KKXX, XXRR) and lumenal proteins
(KDEL) of the ER are retrieved by the same retrograde
moving transport vesicles. Nor is it known whether
KK/RR-motifs are recognized in a receptor-dependent
manner similar to KDEL-proteins. It is also possible that
the cytosolic tails of these membrane proteins directly
interact with cytosolic proteins that drive the formation of
retrograde moving transport intermediates. As discussed
above (section 3.8.), two possible candidates that might
serve such a function are microtubules [144,155] and the
coatomer complex [101,145]. An involvement of micro-
tubules in retrieval/retention of KKXX-tagged proteins
has been studied in cells treated with nocodazole, a drug
that disrupts microtubules [144]. Intriguingly, in nocoda-
zole-treated cells the rate of carbohydrate modification
(measured by the addition of GIcNAc-residues) to
KKXX-bearing reporter proteins was increased, an indica-
tion that an intact microtubular network normally limits
the exposure of KKXX-proteins to the Golgi-localized
GIcNAc transferase [144]. Further support that micro-
tubules might be involved in retention of KKXX-proteins
comes from binding studies with a synthetic peptide corre-
sponding to the KKXX tail of E 3 / 1 9 K of adenovirus, a
well-studied resident protein of the ER membrane
[151,155]. This peptide has been shown to bind to /3-tubu-
lin and to promote tubulin polymerization in vitro [155].
However, it is not known whether binding of microtubuli
to the dibasic motifs of ER membrane proteins plays a role
in ER-transport in vivo. One might also envisage that
microtubules serve as a retention matrix for ER membrane
proteins and thus mask the KKXX motif and prevent
coatomer binding (see also section 3.8). A possible role of
coatomer in the sorting of ER membrane proteins has been
discussed in section 3.8.
4.1.3. Retriet,al t,ersus retention
Although it has been clearly demonstrated that the
KDEL sequence of soluble proteins as well as the dibasic
K K / R R motifs of membrane proteins are capable to re-
trieve reporter molecules to the ER that escaped from this
organelle, it is also clear that these motifs are not the sole
determinants for ER localization. Many endogenous ER
proteins do not receive detectable post-ER modifications
like, for example, calreticulin, a KDEL-tagged lumenal
ER-resident [43] or UDP-glucuronyl transferase, a
KXKXX-carrying resident of the ER membrane [182].
ER-residency of calreticulin has been suggested to occur
by two independently operating mechanisms: (i) by a
KDEL-based retrieval system, and (ii) by a Ca2+-depen -
dent direct retention mechanism [183]. In the case of
UDP-glucuronyl transferase removal of the KXKXX motif
does not result in a significant loss of the truncated protein
from the ER [144]. Another resident of the ER membrane,
the CD3E chain of the T-cell receptor, does not contain a
K(X)KXX motif [184]. In this case tyrosine and serine
residues in the cytoplasmic tail seem to be imported for
retention. Thus, in endogenous ER proteins retention sig-
nals must operate independently and in addition to the
retrieval signals discussed above. It seems likely that a
combination of retention and retrieval exists to optimize
retention of a protein in the ER membrane, and that for
endogenous proteins the extent of retrieval is kept at a
minimum.
4.2. Protein sorting in the Golgi complex
4.2.1. Retention of Golgi enzymes
Interestingly, no soluble Golgi protein is known to date.
All Golgi glycosylation enzymes characterized share the
same type II membrane topology and are anchored in the
membrane by an uncleared signal-anchor sequence near
the N-terminus. Transmembrane domains and in some
cases also the lumenal domain have been shown to be
critical for Golgi retention [185-188] (Table 2).
Transplanting the membrane-spanning domain and part
of its flanking regions of various Golgi resident proteins to
reporter molecules is sufficient to retain these constructs in
the Golgi [189-191]. Interestingly, the chimerae were even
retained in the 'correct' cisternae [190,192,193]. In con-
trast to maintaining a protein in the ER, retention of Golgi
enzymes does not seem to be saturable since overexpres-
sion does not lead to mis-localization to the plasma mem-
brane [189,190]. This was taken as an indication that the
membrane-spanning domain of Golgi proteins serves as a
'true' retention signal rather than as a retrieval signal.
Overexpression of two Golgi enzymes which normally
localize to the trans cisternae, /31,4-galactosyltransferase
(GalT) and o~2,6-sialyltransferase (SialylT), led to their
 C. Harter, F. Wieland / Biochimica et Biophysica Acre 1286 (1996) 75-93
retention in the ER [189,190]. This observation can be
explained by a premature oligomerization caused by high
expression levels: since the transmembrane domains and
part of the flanking regions were sufficient to cause this
effect, it has been postulated that these domains are in-
volved in the formation of aggregates that are too large to
enter antergrade transport vesicles [ 190,194,195]. A forma-
tion of hetero-oligomers between Golgi enzymes that lo-
calize to the same cisterna, termed kin recognition, has
been suggested to serve as a means to retain Golgi en-
zymes at their location [186,196]. Kin recognition would
also maintain the structural organization of the Golgi
complex [197]. That hetero-oligomers are indeed involved
in the formation of some kind of Golgi matrix has been
demonstrated by the expression of hybrid proteins. The
cytoplasmic domain of N-acetylglucosaminyl transferase I
(NAGT I), a Golgi enzyme that resides in medial and trans
cisternae, has been replaced with that of the invariant
chain, p33, a protein that contains an ER-retrieval signal,
XXRR [181]. If this hybrid protein is expressed in mam-
malian cells it localizes in the ER and at the same time
keeps back in the ER another medial/trans Golgi enzyme,
mannosidase II. This effect is so strong that overexpression
of the chimeria leads to a disappearance of the Golgi stack,
and this indicates that other medial Golgi enzymes might
also be involved in the formation of extensive oligomers
which are relocated to the ER [196]. Oligomerization can
be explained by kin recognition of Golgi enzymes that
reside within the same biochemical compartment [186]
(section 2.2.). Recently, it has been proposed that the
formation of multimeric structures involves the lumenal
domain of medial Golgi enzymes, and that retention by kin
recognition is restricted to this population of Golgi resi-
dents [ 188].
In the case of trans Golgi residents, such as SialytT, a
different retention mechanism has been suggested: lipid-
based sorting [188]. The observation that transmembrane
domains of various Golgi enzymes do not share any
homologies in their primary structure led to investigations
whether the length of this domain is important for reten-
tion rather than its amino acid sequence. In fact, the
membrane-spanning domain of SialylT could be replaced
with polyleucines without loss of Golgi retention [189].
However, increasing the length of the membrane spanning
domain of SialylT results in appearance of this enzyme at
the cell surface [189]. Investigation of the length of hy-
drophobic parts of transmembrane domains has revealed
that Golgi residents in average contain shorter membrane-
spanning domains than plasma membrane proteins do [198].
The authors suggest that residency of a Golgi enzyme
might be determined by the appropriate thickness of a
membrane. One factor that causes the diameter of a mem-
brane is an increase in its cholesterol content [199]. It has
been shown that a gradient of cholesterol exists increasing
from the ER, across the Golgi stack, towards the plasma
membrane, [200], and this cholesterol gradient correlates
85
with the thickness of the lipid bilayers from the ER to the
plasma membrane. Residency of a protein with a single
transmembrane domain along the secretory pathway would
then be determined by the length of its membrane-span-
ning domain and by the diameter of the recipient mem-
brane. Thus, a Golgi protein would be excluded because
the lipid bilayer of post-Golgi membranes were too thick
[1981.
4.2.2. Retriet~al of Golgi resident proteins
As outlined above, a variety of experimental approaches
points to a role of the membrane-spanning domains of
Golgi resident proteins in their retention in a particular
Golgi cisterna [194]. However, the exact mechanism by
which residency in the Golgi complex is achieved is not
known. It is probably a combination of several events that
leads to the correct localization of all Golgi residents.
Besides retention through the formation of oligomeric
structures one might also envisage that lipids play a role in
the sorting event, e.g., by the formation of microdomains
that maintain some proteins within a certain matrix, while
other proteins are able to move towards the budding sites
at the rim of a cisternae [16,188]. In this scenario it is
possible that some Golgi proteins can escape and are
subsequently retrieved by a similar mechanism as has been
suggested for ER residents.
A mechanism for retrieval from the plasma membrane
or pre-vacuole has been suggested to operate for proteins
that reside in the TGN or late Golgi compartments both in
mammals [201] and S. cererisiae [202] (Table 2). The
principial signal responsible for residency in the TGN has
been proposed to localize within the cytosolic tails of TGN
resident proteins. In Kex2p and diamino peptidyltrans-
ferase A (DPAP), both residents of a late Golgi compart-
ment in yeast, the signal consists of short stretches of
sequences that contain aromatic residues [202]. Both pro-
teins seem to take a common retrieval pathway, since
overexpression of Kex2p diminishes retention of a protein
that carries the DPAP tail [203]. In analogy to the interac-
tion between adaptin complexes and tyrosine-based
coated-pit localization signals of some plasma membrane
receptors, one might assume that similar interactions be-
tween coat proteins and cytoplasmic tails of TGN-residents
exist [68,201]. Retrieval of TGN residents in S. ceret,isiae
has been proposed to occur from an endosomal/pre-
vacuolar compartment and to require an intact clathrin
gene [204] as well as an intact vpsl gene [205]. The VPSI
mutant was isolated based on its phenotype of mis-sorting
vacuolar proteases [206]. In cells that carry a defective
clathrin gene transport to the endosome/pre-vacuole is
blocked and the protein is mis-localized to the plasma
membrane [207]. If vpslp, a member of the dynamin
GTPase family, is defective, Kex2p is mis-sorted to the
vacuole [205]. The mammalian homologue of Kex2p, fu-
rin, behaves similarly in many respects [208]: a TGN-lo-
calization signal is present within the cytosolic tail, and
86 C. Harter, F. Wieland / Biochimica et Biophysica Acta 1286 (1996) 75-93
deletion of the cytosolic tail results in loss of TGN-locali-
zation and transport to the lysosome for degradation. The
finding that a truncated form of furin is transported to the
lysosome rather than to the plasma membrane, as expected
if the default pathway were taken, points to the presence of
an additional determinant located in the transmembrane or
lumenal domain. Indeed, in the case of TGN 38/41, a
hetero-dimeric integral membrane protein that recycles
between the TGN and the plasma membrane [209], two
distinct signals have been identified that confer TGN
residency: one localized in the cytoplasmic tail
(SKYQRL)[210-212] and another localized within the
transmembrane domain. [213]. Thus, TGN38 can be con-
sidered both as a resident and as a recycling protein
[201,214]. It remains to be seen whether retention and
retrieval signals also co-exist in other Golgi residents.
5. Machinery of vesicular transport
Vesicular membrane traffic can be divided into three
consecutive steps: (i) vesicle budding, (ii) targeting and
(iii) fusion. The machinery required for these reactions
consists of coat proteins that drive the formation of a
vesicle, membrane proteins that specify a certain vesicle
with respect to the donor compartment from which it
derives, and membrane proteins that specify a target mem-
brane to fuse with, as well as proteins that initiate the
fusion between the vesicular membrane and the target
membrane.
5.1. Formation of a coated vesicle
The coat that surrounds a transport vesicle serves as a
mechanical device to force a flat membrane into a spheri-
cal shape. Besides this, a coat structure also prevents a
vesicle from a premature or random fusion with the accep-
tor membrane. A third possible function of coat proteins is
to sort cargo proteins present in the lumen or in the
membrane of a transport vesicle as discussed in section 4.
The molecular mechanisms underlying the steps in bud-
ding have first been elucidated for Golgi-derived COPI-
coated vesicles (Fig. 2). The initial step is the recruitment
to the Golgi membrane of the myristylated small GTP-bi-
nding protein ARF (m-ARF) [141,215,216] (reviewed in
[139]). Membrane-bound m-ARF contains GTP, while cy-
tosolic m-ARF is in its GDP-bound state [217].
Membrane-binding is triggered by a Golgi-localized en-
zyme that catalyzes exchange of GDP for GTP, and is
prevented by treatment with BFA [218,219] (see also
section 3.8). Thus, the nucleotide exchange factor is likely
to be the target for BFA. It has been suggested that the
nucleotide exchange results in the exposure of the amino-
terminal myristic acid residue. This hydrophobic tail is
believed to serve as a membrane anchor of m-ARF-GTP.
A certain population of m-ARF-GTP has been shown to
- O
Fatty_Ac~
ARF-GDP ~ ARF-GTP ~ COATOMER
Fig. 2. Steps in the budding of a COPI-coated Golgi-derived transport
vesicle. Membrane-binding of ARF is triggered by the exchange of GDP
for GTP. The myristic acid tail is exposed and serves as a membrane
anchor for ARF-GTP. Specific ARF-binding primes the membrane to
recruit coatomer. Coatomer binding initiates coat assembly, thus driving
the formation of buds. A coated vesicle is released by a process that
requires fatty acyl coenzyme A. The coat disassembles following hydrol-
ysis of GTP. Coat subunits can be re-used for another cycle of coated
vesicle formation. Model based on [140].
bind to Golgi membranes in a saturable fashion indicative
of the existence of an ARF-receptor [220]. The molecular
identity of this receptor is unknown. Saturable, specific
binding of m-ARF primes the Golgi membrane to recruit
coatomer. Coatomer binding initiates coat assembly thereby
triggering the formation of coated buds [216,221]. To
release functional active transport vesicles the presence of
palmitoyl-CoA is required [140,222]. This led to the as-
sumption that fission of vesicle membranes at the base of a
coated bud involves palmitoylation of a fusion protein
[223]. Indeed, palmitoylation of a 62-kDa protein that
localizes to the cis-Golgi has been demonstrated to affect
vesicular transport [224].
Subsequent to the release of a coated vesicle, hydrolysis
of GTP bound to m-ARF triggers destabilization of the
coat structure that leads to its disassembly [225]. Coat
subunits (m-ARF-GDP and coatomer) are shed into the
cytosol and can be re-used for another cycle of coated
vesicle formation (Fig. 2).
In principle the formation of ER-derived COPII-coated
vesicles follows a similar mechanism [120]. Among the
Ras-like GTPases, Sarlp is most closely related to the
ARF family (reviewed in [115]). In contrast to ARF, Sarlp
is not myristylated nor does it contain a C-terminal farne-
syl- or geranyl residue like many other members of the
family of small GTP-binding proteins. The major mass of
the COPII coat is formed by cytosolic proteins that exist as
pre-formed complexes similar to coatomer in the COPI
coat (section 3.3.) (Table 1). One major difference between
the budding of COPI and COPII coated vesicles is that the
former requires a long chain fatty acyl CoA, such as
palmitoyl-CoA in order to be released from the membrane
[222], whereas release of the latter doesn't show this
 C. Harter, F. Wieland / Biochimica et Biophysica Acta 1286 (1996) 75-93
dependence [120]. Another difference between a COPl-
and a COPII-coated vesicle concerns GTP-hydrolysis. Hy-
drolysis of GTP bound to Sarlp is catalyzed by Sec23p
[117], a component of the COPII coat. No such activity
could yet be attributed to any of the coatomer subunits.
However, a 49 kDa GTPase-activating protein (GAP) spe-
cific for ARF-GTP has been purified from rat liver cytosol
where it exists in a complex of 200 kDa (probably a
homo-tetramer) [226]. This finding suggests that the GAP-
activity required for dissociation of COPI coats from coated
vesicles may not be intrinsic to a coat component.
Most likely the formation of various types of coated
vesicles, for example vesicles involved in late steps of the
secretory pathway [66,227], underlies a similar general
principle as established for COPI- and COPII-coated vesi-
cles.
5.2. Targeting and fusion of a vesicle
Targeting requires receptors on the vesicle membrane
that specifically interact with receptors on the target mere-
m--
~]~[SNAP
q [
| in [241]);
v-SNARE ~] t-SNARE
Fig. 3. Targeting and fusion of a transport vesicle. Specific pairing of a
v-SNARE (vesicle-associated SNAP receptor) with a t-SNARE (target
membrane-associated SNAP receptor) targets a transport vesicle to the
appropriate membrane and forms the scaffold for the binding of the
general membrane trafficking factors NSF and SNAP. ATP hydrolysis
leads to the disassembly of the SNARE complex and to fusion of the
vesicle membrane with the target membrane.
87
brane. For example, a transport vesicle budding from the
ER is equipped with an ER-specific receptor that engages
with a receptor at the cis-Golgi compartment. A vesicle
derived from the cis-Golgi only pairs with a receptor at the
medial-Golgi, etc. The specific pairing of vesicle-associ-
ated receptors (v-SNAREs; SNARE stands for soluble
NSF-attachment protein receptor) with target-associated
receptors (t-SNAREs), referred to as the SNARE hypothe-
sis [228], forms the scaffold for the recruitment of the
general membrane trafficking factors NSF (N-ethylmalei-
mide sensitive fusion protein) [229,230] and SNAP (solu-
ble NSF-attachment protein) [231] from the cytosol. Bind-
ing of NSF and SNAP leads - - upon ATP-hydrolysis - -
to the dissociation of the SNARE complex followed by the
fusion of the vesicle membrane with the target membrane
(reviewed in [232]; Fig. 3).
5.2.1. SNARE proteins
SNARE proteins were functionally discovered from
detergent solubilized brain extracts [233]. These extracts
contain a stoichiometric complex of three membrane pro-
teins, designated core complex, that had been characterized
as constituents of synaptic membranes: the vesicle-associ-
ated membrane protein (VAMP, also known as synapto-
brevin) [234-236], the sy naptosome-associated protein of
25 kDa (SNAP-25; the acronym is coincidentally identical
to that of the soluble NSF attachment proteins) [237] and
syntaxinl [238]. Both of the latter proteins are localized to
the presynaptic plasma membrane. Due to their distinct
locations, VAMP was defined as a v-SNARE (vesicle-as-
sociated) and SNAP-25/syntaxinl as t-SNAREs (target
membrane-associated) [228,233] (Table 3). A role of these
proteins in neuronal exocytosis in particular and in intra-
cellular trafficking in general is supported by three find-
ings:
(i) all three proteins are targets of the clostridial neuro-
toxins: botulinium and tetanus toxins (reviewed in
[239,240]. These toxins are metallo-endoproteases with
high specificities for their respective substrates. Proteolytic
cleavage affects the proper assembly of the core complex
of v- and t-SNAREs and subsequent exocytosis (reviewed
(ii) related protein families have been identified in yeast
and mammals that are required for distinct intracelluar
membrane trafficking steps (reviewed in [242] (Table 3);
(iii) these proteins function as receptors for soluble
proteins that are highly conserved in evolution and in-
volved in several membrane fusion reactions including
fusion of ER- or Golgi-derived transport vesicles with
Golgi membranes [233].
A recently identified homologue of VAMP, cellubrevin,
has been shown to be ubiquitously present in coated
vesicles in a large variety of cells and tissue [243]. From
this observation it was concluded that cellubrevin acts as a
v-SNARE in the constitutive vesicular pathway. Similarly,
the family of syntaxins (syntaxin 1-5) shows a wide tissue
88 C. Harter, F. Wieland/Biochimica et Biophysica Acta 1286 (1996) 75-93
Table 3
Components and modulators of SNARE complexes
Organism Transport v-SNARE t-SNARE
step
mammals synapse VAMP syntaxin I
SNAP-25
mammals ? cellubrevin syntaxin 2 - 5
yeast ER-Golgi Bos I Sed5
Sec22
p26
Bet l ?
yeast post-Golgi Snc 1 / 2 Sso I / 2
Sec9
For references, see text.
distribution [244]. While syntaxin 1 is restricted to the
presynaptic plasma membrane of neuronal tissue, syntaxin
2 - 5 are ubiquitously distributed. When expressed in COS
cells, syntaxin 2 and 4 localize to the plasma membrane,
while syntaxin 5 is found on the CGN, where it co-local-
izes with /3-COP [245,246] (Table 3).
Recently, a SNARE complex involved in ER-to-Golgi
transport in yeast has been isolated [247]; Table 3). This
SNARE complex contains four known proteins: Sed5p
[248], Boslp, Betlp, and Sec22p [128], and 3 unknown
proteins: p28, p26 and p14. Boslp and Sec22p are concen-
trated in ER-derived transport vesicles [129], and p26
shows significant homology to Sec22p [247]. Therefore,
these proteins have been classified as v-SNAREs. In the
case of Betlp, contradictory observations exist as to its
localization: some authors detect Betlp on ER membranes
but not in vesicles derived from them [129], others find
this protein in both localizations [120]. Sed5p [248] and its
mammalian homologue syntaxin5 [245,246] have been lo-
calized to the cis-Golgi and are therefore putative t-
SNAREs (Table 3). The presence of at least three v-
SNAREs but only one t-SNARE in an isolated SNARE
complex raises the question whether the set of v-SNAREs
acts in the same vesicle or whether distinct populations of
v-SNAREs exist, may be moving in the anterograde and
retrograde directions [242,247]. In mammalian cells the
complete set of SNAREs that might act in ER-to-Golgi
transport awaits to be isolated.
Most of the SNAREs are type II membrane proteins
[249]. They lack a cleavable signal sequence and are
anchored to the membrane by a single hydrophobic se-
quence close to the C-terminus. Thus, the bulk of their
mass is exposed to the cytosol with a short lumenal or
extracellular C-terminal tail (tail-anchored). VAMP (and
probably other SNAREs) are inserted into the ER mem-
brane by a novel post-translational mechanism following
its transport, via the Golgi complex, to the synaptic vesicle
membrane [26]. The machinery that promotes translocation
of VAMP seems to be different from the classical co-trans-
lational translocation apparatus, but is at present unidenti-
Modulator fied. SNAP-25 and p26 do not have a membrane-spanning
domain. They are anchored in the membrane via lipid
n-See I moieties [250]. SNAP-25 contains a cluster of cysteine
rab3 residues that have been suggested to be a target for palmi-
complexin I
toylation [250]. p26 contains a C-terminal CAAX box
Sec l-family?
rab-family? (C-I-I-M), a consensus sequence for farnesylation [251].
complexin 117 Besides a domain that confers membrane anchorage,
Sly 1 SNAREs require a variety of further specific structural
Yptl elements:
(i) one domain that mediates the pairing with the match-
Sec 1 ing SNARE. The cytosolic domains of SNAREs have been
Sec4 proposed to adopt a coiled-coil structure that might be
crucial for v-SNARE-t-SNARE interactions [252,253];
(ii) SNAREs need to contain information for their own
targeting to the correct intracellular destination. Mutational
analysis of various SNAREs has shown that localization
signals are present in both the cytosolic and the transmem-
brane domains [245];
(iii) some kind of modulation is required that keeps the
protein in an inactive state until it reaches the correct
membrane. For example, a SNARE that acts at the plasma
membrane has to be transported along the secretory path-
way in an inactive state. Only upon its arrival at the correct
location is it allowed to be activated. If v-SNAREs and
t-SNAREs destined to operate late in the secretory path-
way were capable to partner already before they reach
their final destination, the functional and structural organi-
zation of the entire endomembrane system would be de-
stroyed;
(iv) upon targeting and fusion of a vesicle with the
target membrane, the v-SNARE has to be recycled to the
original organelle. Presently, it is not known what deter-
mines the direction in which a vesicle moves (section 3.8.).
5.2.2. Modulators of SNARE interactions
In order to ensure the fidelity of vectorial membrane
flow, the formation and function of SNARE complexes is
likely to be a regulated event. Various classes of proteins
have been implicated in regulation and control of the
pairing of matching SNAREs:
(i) a family of proteins related to the SECI gene
product has been shown to interact with t-SNAREs both in
mammalian and yeast cells [254,255]. For example, n-
Seclp (a neuronal Seclp homolgue, also known as muncl8)
prevents in vitro formation of the core complex of SNAREs
by binding to syntaxin [256-260]. This was taken as an
indication for a negative regulatory effect of Seclp in the
formation of a SNARE complex;
(ii) rab proteins, a class of small GTP-binding proteins
have been proposed to play a role in the specification of a
certain transport step (reviewed in [261]). Genetic interac-
tions between rabs and SNAREs have been established and
biochemical studies demonstrated that one member of the
 C, Harter, F. Wieland/ Biochimica et Biophysica Acta 1286 (1996) 75-93
rab family, Yptl, serves a function in assembly of ER-Golgi
SNARE complexes [247]. More generally, an interaction
of members of the Secl and rab families with SNAREs
might add an additional layer of specificity or serve as a
proofreading mechanism to vesicle docking and fusion;
(iii) two further components have been shown to affect
SNARE function in synaptic vesicles: synaptophysin, a
vesicle membrane protein, selectively forms a complex
with VAMP/synaptobrevin which excludes the t-SNAREs
syntaxin and SNAP-25 [262], and complexins, cytosolic
proteins found in neuronal (complexin I) as well as non-
neuronal (complexin II) tissue, which bind to the core
complex [263]. Binding of complexins to the SNARE
complex has been shown to occur via syntaxin, and to
compete with binding of a-SNAP (section 5.2.4.). This
finding was taken as an indication that complexins are
involved in regulation of membrane fusion. The presence
of a subpopulation of complexins (complexin II) in various
tissues supports a function of complexins also in constitu-
tive secretion. However, this hypothesis as well as the
precise mechanism of complexin action needs further ex-
perimental analysis.
5.2.3. NSF and SNAP
Once a vesicle has correctly docked to its target mem-
brane, general machinery now executes the fusion process.
Such general, widely distributed transport factors are NSF
and SNAP.
NSF was identified through its ability to reconstitute
vesicular transport between mammalian Golgi cisternae in
vitro after blockage by N-ethylmaleimide (NEM) [229,230].
Inactivation by treatment with NEM, an alkylating agent
that modifies SH groups of cysteine residues, results in the
accumulation of transport vesicles docked to the target
membrane. Besides a function in ER-to-Golgi transport
[264] and intra-Golgi transport [230], NSF is involved in
neurotransmission [233], endosome-endosome fusion
[265,266] and transcytosis [267]. Mutations in the yeast
homologue of NSF, Secl8p [268,269] results in the accu-
mulation of transport vesicles at various stages of the
secretory pathway, an indication for a role of NSF in
vesicle consumption [74]. NSF is a trimeric cytosolic
protein with each monomer containing a three domain
structure: an amino-terminal domain that has been pro-
posed to bind to the soluble NSF attachment proteins
(SNAPs) followed by two domains that contain consensus
ATP-binding sites [270,271]. One site, localized towards
the carboxy-terminus, is required for trimerization, while
the other domain catalyzes the hydrolysis of ATP [271],
NSF cannot bind to membranes directly, nor does it inter-
act with soluble SNAPs, but rather NSF attaches to SNAPs
once they are in their membrane-bound state. It is sup-
posed that membrane binding of SNAPs induces a confor-
mational change that results in the exposure of a NSF
attachment site [272].
Three different SNAPs have been identified: a-, /3- and
89
y-SNAP, each capable of mediating binding of NSF to
membranes and supporting intra-Golgi transport in vitro
[273]. a-SNAP and /3-SNAP are found in all cell types
and tissues analysed and are closely related (83% sequence
identity), y-SNAP, which is brain specific, is more dis-
tantly related (25%/23% sequence identity, respectively)
[274]. A homologue of a-SNAP has been identified in
yeast, Secl7, and has been demonstrated to genetically
interact with the yeast homologue of NSF, Secl8 [275].
Furthermore, in the SECI7 mutant transport vesicles are
accumulated, supporting a common function of Secl7p
with Sec 18p in vesicle consumption [74].
A complex composed of SNAREs, NSF and SNAP was
isolated from a detergent extract from brain that migrates
with a sedimentation constant of 20 S in velocity centrif-
ugation [233]. This complex is stable in the absence of
hydrolysable ATP (i.e., in the presence of ATPyS or ATP
in the absence of Mg 2+). In the presence of Mg2+/ATP,
NSF will dissociate the SNARE complex, thereby trigger-
ing membrane fusion [233] (Fig. 3). Although the precise
mechanism is still unknown, it is clear from genetic and
biochemical data that NSF and SNAP are essential players
in membrane fusion processes.
6. Conclusion
Reconstitution in vitro of single steps of the secretory
pathway, supported by genetic investigations has revealed
a basic frame of the molecular mechanisms that under[y
biosynthetic vesicular transport. Specifically, the general
machinery involved in the budding of a transport vesicle
and its fusion with a target membrane are now known at a
molecular level. Defining mechanisms for retrieval and
retention of resident proteins of the ER and the Golgi
apparatus has allowed new insights into the structural and
functional organization of these organelles. It remains to
be discovered how the many players identified work to-
gether in order to perform and balance anterograde and
retrograde vesicular transport. A most exciting challenge in
membrane research is to unravel the last step(s) that
allow(s) the immediate mixing of phospholipids of the v-
and t-membranes, i.e., bilayer fusion. However, this may
turn out to range within the experimental options of bio-
physics rather than of biochemistry.
Acknowledgements
We thank Tommy Nilsson for helpful critical reading of
the manuscript and Jochen Pavel for help with the figures.
Work in the lab of the authors is supported by grants from
the Deutsche Forschungsgemeinschaft (SFB 352), the state
of Baden-Wiirttemberg (Landesschwerpunkt) and by the
Human Frontier Science Program.
90 (2". Harter, 1=. Wieland / Biochimica et Biophysica Acta 1286 (1996) 75-93
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