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Chapter 17:

Vesicular Traffic, Secretion,


and Endocytosis

Molecular Cell Biology


Fifth Edition
Harvey Lodish • Arnold Berk • Paul Matsudaira •
Chris A. Kaiser • Monty Krieger • Matthew P. Scott •
Lawrence Zipursky • James Darnell

Hsou-min Li , 2006


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All transport steps within
*
the secretory pathway are
mediated by vesicle
transport (vesicle budding
from the donor
compartment, fusion with
the target compartment)
operated with similar *
mechanisms (coat and
SNARE)
Explain the 9 steps on the right *
Note: in vesicles and in
organelles, the cytosolic
side is always cytosolic, the
luminal side is always *
luminal or extracellular
*

*
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The two systems that defined the route of the secretory pathway
1. G. Palade (1960s) 3 min
ER 7 min
Pulse-labeling proteins by injecting radioactive amino acids into Golgi
the pancreas
Identify the compartment by autoradiography

37 min 117 min


immature mature
secretory secretory
granules granules

2. Yeast secretion mutant (R. Schekman, 1980s). 5 classes of yeast temperature- sensitive secretion (sec)
mutants. Pathway linerized by double mutant phenotypes (if AxB =A then A is in front to B)

budding fusion with


from TNG plasma3
membrane
Both systems got the same answer and can now be confirmed by GFP in live cells.

VSV-G protein
secretion to the plasma
membrane

The yeast-secretion-mutant system described above


and the isolated Golgi system (J Rothman’s group
1980s) on the next next slide identified and defined
most of what we know about vesicle budding and
fusion today. For example, treatment with GTP-r-S,
vesicles accumulate and it is used to isolate vesicles
and characterize the coat components. Treatment
with NEM, vesicles can’t fuse, and it is used to
isolate the fusion machinery.
Do you still think glycosylation is boring?

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I. Vesicle formation (budding): three kinds of known coats
COPI vesicles: Golgi → ER and between Golgi stacks.
COPII vesicles: ER → Golgi
Clathrin vesicles: from plasma
membrane or TGN to endosome

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General process of vesicle formation:
small GTPase activation and binding to donor membrane → coat assembly and
cargo recruiting → pinch off
They can all be reconstituted
in vitro using purified
components. Left: Sec23, 24,
13, 31 and 16, Sar1p-GTP and
artificial phospholipid vesicles.

There are three kinds of coated vesicles identified so far (coats for constitutive and regulated secretion are still not known).
Their components are listed below.

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COPII vesicle formation (ER to Golgi)

Serve as foundation for


coat assembly

Unknown signals (cargo protein


accumulating?) tell Sec12 to
activate Sar1

Sec24 binds to DxE signal on


the cytosolic tail of membrane
cargo (there are more than
one kinds of signals even for
ER)
Sec13/31

Sec16

Sar1 (or ARF) mutant that can’t hydrolyze GTP, or


treatment with GTP-r-S, cause the coat unable to
disassemble, and is used to isolate and study the coat
components.
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COPI vesicle formation: It has the same basic sequential steps as COPII vesicle formation.

The COPI coatomer complex can be divided


into two subcomplexes:
F-subcomplex (βδγζ)
B subcomplex (αβ’ε): some of the
subunits may bind cargo directly. For example
the α subunit binds the KKxx and KxKxx
motifs on cargo proteins.

for vesicle fusion,


our next topic

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Clathrin coated vesicles: for transport from TGN or plasma membrane to the
endosome

Dynamin, a big GTPase as a


molecular scissor, not required for
COPI and COPII vesicle formation.
ARF

Also initiated by ARF. It’s not know what determines which coat
(COPI, AP1,2,3, GGA) will assemble after ARF.

Tyrosine (Yxxφ ) signal recognized by adaptor subunit

coat
assembly

36 triskelion to form a
coat

with AP2 without AP2

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Coat disassembly after GTP hydrolysis by ARF and assisted by Hsp70
There are 4 kinds of adaptor molecules identified for the clathrin coat. These
adaptors mediate transport from different donor membranes. However, It’s not
known what determines which coat (COPI, AP1, 2, 3, or GGA) will assemble after
ARF.

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What does the cargo receptor recognize on the
soluble cargo?

What does the coat recognize on the membrane


cargo (must be cytosolic tail of membrane protein)?

Membrane receptors (for soluble cargo) and membrane cargo


e.g. KDEL
receptor

e.g. M6P
receptor

Soluble cargo

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Sometimes these vesicles
fuse among themselves to KKxx
form ERGIC (ER to Golgi
intermediate
compartment). ERGIC
then fuse with cis-Golgi.

COPI coats identified by


incubating isolated Golgi
COPII coats identified by membranes with cytosol,
incubating isolated ER ATP and GTP-r-S
membranes with cytosol,
ATP and GTP-r-S
DxE

Yeast mutants of COPI and COPII coats have the same phenotype (proteins accumulation in ER), why? → Without
v-SNARE, which need to be recycled back from cis-Golgi by COPI, COPII vesicle won’t bud from ER.
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Transport through the Golgi occurs by cisternal progression of the cisternae and retrograde transport of the
COPI vesicles.

It used to have two models:

contain
medial-Golgi
enzymes

TRENDS in Biochemical Sciences Vol.31 (2006) p601

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Live imaging of yeast Golgi cisternal
maturation Nature 2006 vol 441 p1007

Maturation of Golgi cisternae directly observed


TiBS 2006 vol 31 p601

Figure 3 | Three-dimensional observation of yeast Golgi cisternae.


Wildtype yeast cells expressing mRFP–Sed5 (cis, red) and Sec7–GFP
(trans, green) were observed under a high-speed confocal microscope
with scanning in the z axis. a, Five optical slices 1 mm apart were
taken at 0.5-s intervals to cover a whole cell. Three-dimensional
images were reconstructed into a movie (Supplementary movie 4), of
which representative views are shown. b, As in a except that seven
optical slices 1 mm apart were taken at 1-s intervals (Supplementary
movie 5). Scale bars, 2 mm.
Figure 2. Experimental observation of cisternal maturation. Representation of the experimentally observed time course of the maturation of a
greatly simplified Golgi stack, depicted in the first instance as two cisternae. To identify late cisterna, a late Golgi membrane protein has been
tagged with a red fluorescent marker; to identify an early cisterna, an early Golgi membrane protein has been tagged with a green fluorescent
protein. (a) During the time course of the experimental visualization, the late cisterna disappears as the red marker is extracted and transported
back to the early cisterna (a process represented by arrow 1). (b) The late cisterna, presumably, fragments into transport vesicles destined for
the plasma membrane (arrow 2). When the early cisterna receives the red marker it transiently appears yellow owing to the mixing of the
fluorescent signal from the green and red markers. Detailed studies show that this change can occur sequentially in sub-structures of a single
network like cisterna [7]. The green protein is then lost to a newly formed cisterna (arrow 3). (c) Transformation into a late cisterna is
completed and the cycle begins again. Concomitantly, new cisternae are formed from ER-derived vesicles and become visible as they receive
the green marker (arrows 3 and 4), which becomes the green, newly formed cisterna in (c). Cargo proteins (asterisks) were not visualized by
the techniques used in the experiments of Losev et al. [6] and Matsuura Tokita et al. [7] but are thought to remain in the cisterna during the
course of its maturation (and thus end the process in what has become a late cisterna having started in what was an early cisterna). The
transport intermediates, whether vesicles or narrow tubules, are also too small and faint to be seen clearly by these techniques [6,7] but, for
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simplicity, they have been depicted as vesicles here. It is the change in colour, and hence protein composition, of single continuously
cisternae that is the hallmark of cisternal maturation and the key observation in these experiments.
The basic formation sequence of events are similar for all three types of vesicles:
signaled (primed) by a small GTPase   adaptor or coat components bind to membrane receptor (recognize
specific sequences on the cytosolic tail of receptors for cargo or cargo itself)   coat assembly   budding   coat
dissociate   fusion

our next topic: vesicle fusion

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17.2 Molecular mechanisms of vesicle formation and fusion
I. The general process and molecular mechanism of
vesicle fusion: ducking through Rab-GTP interaction
with its effector--> Trans-SNARE complex assembly
through 4 α-helix interaction (sufficient to cause
membrane fusion alone)-->cis-SNARE complex
dissociation through hydrolysis of ATP by NSF.

Core complexes are represented by elongated coiled coils of


four intertwined, parallel α-helices, with each helix being
provided by a different SNARE motif. The centre of the
bundle contains 16 stacked layers of interacting side chains.
These layers are largely hydrophobic, except for a central ‘0’
layer that contains three highly conserved glutamine (Q)
residues and one highly conserved arginine (R) residue.
Accordingly, the contributing SNARE motifs are classified
into Qa-, Qb-, Qc- and R-SNAREs.
VAMP is resistant to Botulinum toxin cleavage after forming 16
the bundle.
Vesicle fusion --- The discovery

1984 Balch and Rothman et al.( Cell 39,


405-416), in vitro Golgi transport assay;
Isolated Golgi membrane fraction from
VSV infected mutant cells and
uninfected wild-type cells; assay for 3H- Vesicles containing N-acetylglucosamine
transferase I bud off from wild-type trans
labeled VSV G protein. They found that Golgi and fuse with mutant medial Golgi
transport needs : 37oC, cytosol, ATP
(by adding GTP-r-S, they found the
radiolabeled
COPI-coated vesicles) sugar nucleotide

Later found: NEM treatments of membranes → uncoated vesicles accumulated on Golgi membranes, no
fusion → add back cytosol, fusion ⇒ used to purify NSF (NEM-sensitive Factor) ⇒ NSF is the animal
homologue of Schekman’s yeast SEC18p, so the system is conserved from yeast to animal!
purified NSF can't bind to membrane → used to purify SNAP (soluble NSF attachment protein)
NSF+SNAP : have defined binding sites on Golgi membrane (saturable, resistant to alkaline extraction) →
make an affinity column with NSF+SNAP and tried to purify the receptor from solublized bovine brain
membranes ⇒ found SNARE (SNAP receptor) , a 7S complex of three proteins that's already known:
1. VAMP (vesicle associated membrane protein, synaptobrevin)
2. syntaxin (on plasma membrane))
3. SNAP 25 (synatosomal-associated protein 25kD) (on plasma membrane)
start with Golgi, end up with synapsis on plasma membrane??
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Different pairs of SNARE mediate fusion at different membranes (see below) with the same mechanism.
Use the same NSF and SNAP

“The first SNAREs isolated turned out to be some protein already known to be associated with
synapses (Scheller and colleagues)-----VAMP (on synaptic vesicles, also called synaptobrevin),
Syntaxin 1, SNAP25 (on PM). Here snap=synaptosomal associated protein of 25 kD. It is an
amusing coincidence that two molecules carrying an identical acronym, characterized
independently in different experimental systems, are later found to be functionally associated.
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So, started with in-between Golgi transport, end up with synapses (they used brain extracts).
Cell fusion occurs
physiologically. It is
Blue
Red vital for fertilization
nucleus
cytosol (sperm-egg),
development
(forming syncytial
tissues such as muscle),
and physiology (bone
resorption). Although
progress is being made,
the responsible fusion
proteins are still
unknown. However, it
is now easy to imagine
how DNA
rearrangements that
linked signal sequences
to SNAREs could have
triggered an
evolutionary process
that led to the creation
of proteins to mediate
physiologic cell fusion.

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SCIENCE VOL 300 13 JUNE 2003 1747
Application: botulinum neurotoxin: specific proteases that cut VAMP, syntaxin or SNAP25. They can be
used to treat spasmodic disorders or skin wrinkles (release the underlying muscle from spasmodism).
Peptides that interfere with interaction between SNARES will also work.

A million people have been


treated with BOTOX®
Cosmetic since FDA
approval in 2002. Not just
models and movie stars,
but people from all types
of professions.
The photos featured on this Web site are of actual BOTOX® Cosmetic use

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The role of ER and
Golgi in secretion.
Discovery of
ribosome (“Palade
granules”), rough ER
and the internal
structure of
mitochondria.

James Rothman
Randy Schekman

The “Signal Hypothesis”

The next Nobel prize on protein trafficking will be:


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Schekman and Rothman on vesicle formation and fusion.
17.4 Later stages of the secretory pathway: clathrin-coated vesicle formation and sorting at
trans-Golgi network
TGN → late endosome → lysosome
→ regulated secretion (e.g. hormone) What are the signals ?
→ constitutive secretion: apical vs. basolateral

lysosome
Mannose-6-phosphate (M6P) is the
sorting signal for soluble lysosomal
proteins (YxxØ for membrane
proteins). M6P is bound by M6P
receptor, which is bound by the AP
complex.

N-acetylglucosamine 22
still unknown
located at cis-Golgi
regulated secretion
Signal still unclear, some evidence suggest that proteins that can aggregate with chromogranin A, B and
secretogranin II under the TGN ionic condition (pH 6.5 and 1 mM Ca2+), are segregated in secretory granules.

constitutive secretion: coat still unknown


Signals: soluble proteins: default
Membrane proteins: divided into apical vs. basolateral plasma membrane in polarized cells
Polarized plasma membrane sorting
GPI anchor (glycosylphosphatidylinositol membrane anchor) is the only apical-basolateral sorting signal
identified so far, but it specifies apical sorting in MDCK epithelial cells and basolateral sorting in thyroid
cells.

This end won’t be interacting with


other cytosolic proteins or
cytoskeleton, so can diffuse more apical
freely. Why are all GPI-anchored
proteins extracellular surface proteins?

In hepatocytes and some other polarized cells, all plasma membrane proteins targeted to basolateral
membrane first. Apical proteins move back by transcytosis. 23
Transcytosis moves some endocytosed ligands, not to endosome, but across an epithelial layer .
Use pH differences to control receptor-ligand association-dissociation.

of intestine

apical

associate
dissociate

The receptor is no longer present in older kids, only present in young infants.

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17.5 Receptor mediated endocytosis: plasma membrane receptor exocytoplasmic domain binds to ligand,
cytosolic domain binds to AP2 → Internalized through clathrin-coated vesicle → uncoating → fuse with late
endosome → receptor and ligand dissociate due to low pH. (pinocytosis: no receptor, non-specific)
Various signals for endycytosis:
NPxY (LDL receptor); Yxxφ (M6P receptor); Leu-Leu; ubiquitin tag.

protonated
histidine

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How to deliver membrane proteins and organelles to the lysosomes to be degraded?

Endocytosed soluble proteins, to be


degraded. Membrane receptors are
recycled back to the plasma membrane.

What about membrane proteins that need


to be degraded (e. g. down-regulated
Lysosome resident proteins from TGN, receptors)? → multivesicular body
not to be degraded (multivesicular endosome)

Late endosomal “membrane” proteins are


sorted 3 ways:

Autophage (“eating oneself”): the


delivery of bulk amounts of cytosol or
entire organelles to lysosomes.

2. Lysosome resident membrane


proteins stay on the endosome
membrane
1. Budding “outward” for 3. Budding “inward” for
receptor recycling back to receptor membrane protein
plasma membrane degradation 26
The inward budding shares a common mechanism with
budding of enveloped virus (e.g. HIV) from the plasma
membrane.

like a
SNARE
complex?

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