Lodish • Berk • Kaiser • Krieger • Scott • Bretscher •Ploegh • Matsudaira
MOLECULAR CELL BIOLOGY
SIXTH EDITION
CHAPTER 14
Vesicular Traffic, Secretion,
and Endocytosis
©Copyright
2008 W.
H.©Freeman
andand
Company
2008
W. H. Freeman
Company
Outline:
1. Techniques for studying the secretory
pathway
2. Molecular mechanisms of vesicular
traffic
3. Vesicular trafficking in the early stages
of the secretory pathways
4. Protein sorting and processing in late
stages of the secretory pathways
5. Receptor mediated endocytosis
SEM of the formation of
clathrin-coated vesicles on the
cytosolic face of the plasma
membrane
6. Directing membrane proteins and
cytosolic materials to the lysosome
Secretory pathway: protein to various organelles by
transport vesicles
Anterograde: forward moving
Retrograde: backward moving
Trans position: farthest from the ER
Cis position: nearest the ER
Cisternal progression: cis-Golgi cisterna → cargo of protein →
move form cis → medial → trans ; anterograde transport
vesicle; normal
TGN (trans Golgi network): proteins not transport to ER or
Golgi, are destined for compartment to others (by different
types of vesicles)
1. from trans → fuses membrane → trnasport → exocytosis
2. from trans → stored inside → formation of secretory
vesicles; release by signal for exocytosis
3. from trans → late endosome → lysosome (intracellular
degradation of organelle) the mechanism not well know
endosome had endocytic pathway, from the plasma
membrane bringing membrane proteins and their bound
ligands into the cell
Overview of major protein-sorting pathways in eukaryote (protein targeting)
No signal peptide
Overview of secretory & endocytic pathways: Transport vesicles
transport vesicle
cargo proteins
same orientation
anterograde transport vesicles
retrograde transport vesicles
cisternal progression
trans-Golgi network (TGN)
secretory vesicle (regulated..)
constitutive secretion-exocytosis
transport vesicle-late endosome
endocytosis
Techniques for studying the secretory pathway:
Pulse-chase labeling & EM
autoradiography
Animal + radio AA → different
time → kill → chemical fix →
autoradiography
Tissue sections of pancreas acinar
cells -> a brief incubation (3 min) with
H3-Leucine -> transfer to unlabeled
medium & incubate for a period of
time (0, 7, 37, 117 min) -> cover tissue
sections with photographic emulsion > EM
Pulse-chase exp
脈搏
Low density lipoprotein receptor
補捉
To investigate the fate of a
specific newly synthesized
protein
Cell + isotope for 0.5h
↓ wash
Different time point
↓
Immunoprecipitation
↓
Specific protein
↓
SDS-PAGE
↓
degrade
PTM
Glyco..
<0.5h, protein convert to mature
Techniques for studying the secretory pathway:
Use of temperature-sensitive mutant proteins (e.g. vesicular stomatitis
virus 水疱口炎病毒 VSV G protein)
At restrictive temp. of 40oC, newly made G protein is misfolded &
retained within ER.
At permissive temp. of 32oC, accumulated G protein is correctly
folded & transported through secretory pathway.
Different time course → change Temp → misfolded → stop
transport
Palade’s early exp had found that in mammalian, vesicle mediated
transport of a protein molecule from ER to membrane about 30-60
min.
Techniques for studying the secretory pathway: by living cells
1. Transport of a protein through the secretory pathway can be
assayed in living cells:
1) Microscopy of GFP-labeled VSV G protein
2) Detection of compartment-specific oligosaccharide
modifications
2. Yeast mutants define major stages and many components in
vesicular transport
3. Cell-free transport assays allow dissection of individual steps in
vesicular transport
Microscopy of GFP-labeled VSV G protein
Plasma membrane
Use temperature-sensitive mutant,
VSVG-GFP.
40oC the protein in ER
32oC move → Golgi → plasma
membrane→
Form ER to Golgi about 60min
Protein transport through the secretory pathway can be visualized by
fluorescence microscopy of cells producing a GFP-tagged membrane
protein: VSV G protein
Techniques for studying the secretory pathway:
1. Transport of a protein through the secretory pathway can be
assayed in living cells:
1) Microscopy of GFP-labeled VSV G protein
2) Detection of compartment-specific oligosaccharide
modifications
2. Yeast mutants define major stages and many components in
vesicular transport
3. Cell-free transport assays allow dissection of individual steps in
vesicular transport
Transport of a membrane glycoprotein from the ER to golgi can be
assayed based on sensitivity to cleavage by endoglycosidaseD
電泳分離時
分子量大
分子量小
分子量大
分子量小
Cleavage by endoglycosidase D
Cell expression VSV G protein → at
Temp 40 → link radioactive aa and
protein keep in ER → Tem 32 C → VSV
G extracted → digested by
endoglycosidase (about cis Golgi
protein) → SDS electrophoresis
From ER to golgi about 60 min
Endoglycosidase can not cleavage ER’s
protein.
32 C: protein move from ER →
Golgi (modification) →
membrane
40 C: in ER not move. Did not
cleavage by endoglycosidase
Protein folding ok → move → golgi → can cleavage
ER to golgi
In ER
Addition & processing of N-linked oligosaccharides in R-ER of
vertebrate cells
酶的反應是有其專一性,其反應物必需是特定的,缺一不可
Add
Remove 2 mannose
Remove 3 mannose
In cis, specific glycosidase
Cleavage by endoglycosidase D.
• glycosidases (cis-)
• endoglycosidase D
Processing of N-linked oligosaccharide chains on glycoproteins within cis-,
medial-, and trans-Golgi cisternae in vertebrate cells
Techniques for studying the secretory pathway:
1. Transport of a protein through the secretory pathway can be
assayed in living cells:
1) Microscopy of GFP-labeled VSV G protein
2) Detection of compartment-specific oligosaccharide
modifications
2. Yeast mutants define major stages and many components in
vesicular transport
3. Cell-free transport assays allow dissection of individual steps
in vesicular transport
Yeast sec (secretion) mutants
protein
The temperature sensitive mutant → grouped into 5 classes
Combination of different mutant → for research of protein transport pathway, ie BD
→ protein in ER not Golgi → so ER is before, and Golgi is after. 利用到達的時間去計算
These studies confirmed that: cytosol → RER → ER-to Golgi transport vesiceles →
Golgi cisternce → secretory → exocytosed
Phenotypes of yeast sec mutants identified stages in the secretory pathway
Techniques for studying the secretory pathway:
1. Transport of a protein through the secretory pathway
can be assayed in living cells:
1) Microscopy of GFP-labeled VSV G protein
2) Detection of compartment-specific oligosaccharide
modifications
2. Yeast mutants define major stages and many
components in vesicular transport
3. Cell-free transport assays allow dissection of individual
steps in vesicular transport
Cell-free transport assay
To plasma membrane
Can not add
Protein transport from Golgi cisternae to another can be assayed in a cell-free system
Protein need modification in Golgi
Proof: golgi can retrograde vesicular
transport for modification
Normal expression
it demonstrated protein transport from one golgi cisterna to another
Tradional Model - Golgi is a static
organelle. Secretory proteins move
forward in small vesicles. Golgi
resident proteins stay where they are.
Two Models For Cis to
Trans-Golgi
Progression
“Radical” Model - Golgi is a
dynamic structure. It only exists
as a steady-state representation of
transport intermediates. Secreted
molecules move ahead with a
cisterna. Golgi resident proteins
move backward to stay in the
same relative position.
問題:
到底細胞內利用vesicle的方式的機轉是什麼?
Molecular mechanisms of vesicular traffic
Vesicle transport: from organelle (Donor)
target organelle
(a) Coated vesicle: From membrane interaction with integral
(b) Uncoated vesicle: Target membrane
vSNARE: Crucial to fusion of the vesicle with correct target membrane
tSNARE: specific joining of vSNARE
Overview of vesicle budding and fusion with target membrane
Assembly of a protein coat drives vesicle formation & selection of
cargo molecules.
A conserved set of GTPase switch proteins controls assembly of different
vesicle coats
Three types of coated vesicles have been characterized. All need GTP binding
antrograde
retrograde
To endosome
GTPase superfamily
ARF (ADP Ribosylation Factor)
Different coated proteins
Clathrin and adapter protein (AP): vesicles transport proteins from
the plasma membrane and trans-Golgi network to late endosomes
– With AP1: Golgi to endosome
– With AP2: Endocytosis (PM to endosome)
– With AP3: Golgi to lysosome and other vesicles
COPI: Golgi to ER (retrograde transport)
COPII: ER to Golgi (antrograde trnasport)
AP: complex consists of four different subunits
Vesicle buds can be visualized during in vitro budding reactions.
Coated vesicles
Artifical membranes and
purified coat protein (COP II)
→ polymerization of coat
protein onto the cytosolic face
of the parent membrane
A conserved set of GTPase switch proteins controls assembly
of different vesicle coats.
All three coated vesicles contain a small GTP-binding protein
COP I and clathrin vesicle: ARF (ADP-ribosylation factors)
COP II vesicle: Sar I protein
ARF and Sar I protein can switch GTP (GDP-protein → GTP-protein
active; GTPase)
There two sets of small GTP-binding proteins for vesicle secretion. One
is ARF and Sar I; another is Rab protein
ARF (ADP Ribosylation Factor) protein exchanges
bound GDP for GTP and then binds to its receptor
on Golgi membrane
A conserved set of GTPase switch proteins controls assembly
of different vesicle coats.
COPII coated formation
GTP → Sar1 conformational change →Sar1-GTP binding to membrane →
polymerization of cytosolic complexes of COPII subunit on the membrane →
formation of vesicle buds
Monomeric GTPase control coat assembly
Specific receptor
Cargo
protein
Sar1 attached to Sec23/24 coat protein
complex → cargo protein are recruited to the
formation vesicle bud by binding of specific
short sequence in their cytosolic regions to
sites on the Sec23/24 → assembly to second
type of coat complex composed of Sec13/31
→ completed → Sec23 promotes Sar1-GTP
hydrolysis → release Sar1-GDP →
disassembly of the coat → transport vesicle
Vesicle formation
Coat assembly controlled by monomeric G-protein (SAR1 or ARF) with
fatty acid tail
GDP-bound SAR1 or ARF are free in cytosol
Membrane-bound G-protein recruits coat protein subunits
Assembly of coat pulls membrane into bud Leads to exposure of fatty
acid tail membrane binding Donor membrane contains guanine
nucleotide-releasing factor -causes Sar1-GDP SAR1-GTP
Coated vesicles accumulate during in vitro budding reactions in the
presence of a nonhydrolyzable analog of GTP
Golgi membrane + COPI
coat proteins and GTP →
bud off
Non-hydrolyzable GTP
prevent disassembly of the
coat after vesicle release
Without
exchange
GTP GDP
by charperone
(hsp70)
Major coat protein: clathrin & adaptin
There are at least four types of adaptins,
each specific for a different set of cargo receptor.
Targeting sequence on cargo proteins make specific molecular contacts
with coat protein
Mistransport mechanism; retrograde
Different Rab GTPases & Rab effectors control docking of different
vesicles on target membranes: vesicle docking controlled by Rab
protein.
v-SNARE
Vesicle docking controlled by Rab
proteins
Monomeric GTPases attach to surface of
budding vesicle
Rab-GTP on vesicle interacts with Rab
effector on target membrane
After vesicle fusion GTP hydrolysed,
triggering release of Rab-GDP
Different Rab proteins found associated
with different membrane-bound
organelles
t-SNARE
Paired sets of SNARE proteins mediates fusion of vesicles with
target membranes.
Analysis of yeast sec mutants defective
in each of the >20 SNARE genes.
In vitro liposome fusion assay.
SNARE-mediated fusion → exocytosis
→ secretory protein
In this case, v-SNARE as VAMP
(vesicle associated membrane protein)
t-SNAREs are syntaxin
SNAP-25 attached to membrane by
hydorphobic anchor.
Formation of four-helix bundle:
VAMP (1), Syntaxin (1) and SNAP-25
(2)
But, in COPII with cis, each
SNARE has provide one helix
SNARE complex had
specificity
Dissociation of SNARE complexes after membrane fusion is driven
by ATP hydrolysis.
SNARE complex formation by
non-covalent interaction.
Dissociate → free SNARE →
can fuse next time
Two protein play important role
of dissociation or fusion with
a target membrane: NSF
(NEM-sensitive factor,
blocked by N-ethylmaleimide)
& α-SNAP (soluble NSF
attachment protein).
hexamer
Monomeric Rab-GTPases
A guanine nucleotide exchange factor
(GEF) recognizes a specific rab proteins
and promotes exchange of GDP for
GTP.
GTP bound Rabs have a different
conformation that is the “active” state.
Activated rabs release GDI, attach to the
membrane via covalently attached lipid
groups at their C-termini and are
incorporated into transport vesicles.
Rab-GTP recruits effectors that can
promote vesicle formation, vesicle
transport on microtubules, and vesicle
fusion with target membranes.
After fusion Rab-GTP hydrolyzes GTP
to GDP and is released from the
membrane. GTPase activating proteins
proteins accelerate hydrolysis, reducing
the avalability of active rabs.
Rab proteins (monomeric GTPase) help ensure the
specificity of vesicle docking
Soluble (i.e. cytoplasmic) Factors
NSF or n-ethylmaleimide (NEM) Sensitive Factor
SNAP- Soluble NSF Attachment Proteins
NSF + SNAP bind to target membranes (synaptic vesicle & plasma
membrane)
Receptors for NSF and SNAP are synaptobrevin (vesicle), SNAP25 (plasma membrane) and syntaxin (plasma membrane)
Membrane targets are called SNAREs (v- and t-) Soluble NSF
Attachment protein REceptors
SNAP-25- Synaptosome Associated Protein of 25 kDa
• Over-expression of truncated SNAP-25 blocks release
• Syntaxin, 15 kDa protein
• Sensitive to botulinum toxin A cleavage - release prevented
Synaptobrevin
Identified and cloned ~ 1988-1990
Originally called VAMP (Vesicle-Associated Membrane Protein)
and sometimes abbreviated as Syb
Cleaved by tetanus toxin (failure of exocytosis = death) 破傷風毒素
Spans vesicle membrane
~ 13 kDa
Inject antibodies to Synaptobrevin and release is blocked
Dissociation of SNARE complexes after membrane fusion is driven
by ATP hydrolysis.
ATP is not actually required
for release once
vesicles are docked, but is
thought to break
down the SNARE
complexes to promote
recycling.
拉上拉鍊
Rizo and Sudhof 2002
Nature Rev. Neurosci.
Rizo and Sudhof 2002
Nature Rev. Neurosci.
Membrane fusion reactions need to
overcome repulsive forces that take
over when membranes approach
within 3nm- hydration for ectoplasmic
and cytoplasmic leaflets as well as
charge repulsion in cytoplasmic
leaflets. Attractive hydrophobic forces
can be enhanced by membrane bending.
Rab proteins (monomeric GTPase) help ensure the
specificity of vesicle docking
Specificity of vesicle fusion
Need mechanism for selective vesicle trafficking -controlled by SNAREs and Rab
proteins
SNARE hypothesis proposes specific interactions between v- SNAREs and tSNAREs govern vesicle docking and fusion
Each organelle has specific SNAREs leading to specific vesicle fusion
Vesicle docking controlled by Rab proteins
Monomeric GTPases attach to surface of budding vesicle
Rab-GTP on vesicle interacts with Rabeffector on target membrane
After vesicle fusion GTP hydrolysed, triggering release of Rab-GDP
Different Rab proteins found associated with different membrane-bound organelles
Summary
Proteins moved between organelles of secretory pathway fully
folded, enclosed in vesicles -proteins only have to cross ER
membrane
Large amount of vesicular traffic between ER, Golgi, lysosomes
and plasma membrane
Vesicle budding is function of protein coats
Cargo selected by sorting/cargo receptors
Specificity of fusion controlled by Rabproteins, v-SNAREs and tSNAREs
Early stages of the secretory pathway
Vesicle-mediated protein trafficking between ER & cis-Golgi
Anterograde-COPII vesicle
Retrograde-COPI vesicle
Cargo protein
vSNAREs (yellow)
Rab important
Membrane specific receptor
bind to cargo → transport
Vesicle-mediated protein trafficking between the ER and cis-Golgi
Targeting sequence on cargo proteins make specific molecular contacts
with coat protein
COPII vesicles mediate transport from the ER to the Golgi
Formation of COPII vesicles:
triggered by Sec12 → induced
catalyzes the GDP for GTP of
Sar1 → binding Sar1 to ER
membrane → followed by binding
of Sec13/24 → formation of
complex →second complex
comprising Sec13 and 31 →
interact with fibrous proteins Sec
16 → coat polymerization
Sec24: interact with integral ER →
transport to Golgi
cytosol
ER lumen
Di-acidic sorting signal (Asp-X-Glu, or DXE).
3-D structure of ternary complex comprising the COPII coat proteins (Sec23, Sec24)
& Sar1-GTP.
CFTR: inherited disease cystic fibrosis
囊胞性纖維症
Mutation of CFTR receptor
(chloride channel) phenylalamine
508→ conformational change of
di-acidic sorting signal → did not
interaction with Sec24→ did not
formed COPII → did not transport
COPI vesicles mediate retrograde transport within the Golgi and from
the Golgi to the ER (ie mis-transport)
Most soluble ER-resident protein carry a
Lys-Asp-Glu-Leu (KDEL) sequence at
C-terminus.
KDEL signal & KDEL receptor:
retrieval of ER-resident luminal
proteins from Golgi.
Both COPI and II vesicle had KDEL
receptor.
Retrieval system prevented ER luminal
protein for folding.
KDEL binding affinity is sensitive pH.
It binding protein in Golgi, but release
in ER.
KDEL-receptors bind to KDEL-bearing
proteins in the low pH environment of the Golgi
and release that Cargo in the neutral pH of the
ER.
pH probably alters KDEL receptor
conformation - regulating cargo binding and
inclusion in COPI vesicles.
PH
high
COP I vesicles mediate retrograde transport for retrieval of ER
resident proteins (recycle protein)
necessary for soluble secretory proteins to move anterograde without loss of
ER resident proteins (e.g., PDI, BiP)
ER resident proteins possess ER retrieval signals
– KKXX at C-terminal end for ER membrane proteins interacts w/
COP1α/β (e.g., PDI)
– KDEL at C-terminal end for ER soluble proteins interacts w/ KDEL receptor
(e.g., BiP)
KDEL receptor serves to retrieve KDEL tagged proteins from cis-Golgi and
return them to ER
– KDEL receptors localized primarily to membranes of cis-Golgi itself and to
small vesicles that shuttle between ER and cis-Golgi
KDEL and KKXX signals are both necessary and sufficient for ER retention
Lys-Lys-X-X in KDEL receptor or membrane receptor( Retrieval of ER-resident
membrane proteins from Golgi)
At the very end of C-terminus, which faces the cytosol.
Binds to COPI α & β subunits and retrograde to ER.