MOLECULAR CELL BIOLOGY SIXTH EDITION

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MOLECULAR CELL BIOLOGY SIXTH EDITION

CHAPTER 14 Vesicular Traffic, Secretion, and Endocytosis

Lodish • Berk • Kaiser • Krieger • Scott • Bretscher •Ploegh • Matsudaira

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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 6. Directing membrane proteins and

cytosolic materials to the lysosome SEM of the formation of

clathrin-coated vesicles on the cytosolic face of the plasma membrane

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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

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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

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Overview of major protein-sorting pathways in eukaryote (protein targeting)

No signal peptide

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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

Overview of secretory & endocytic pathways: Transport vesicles

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Pulse-chase labeling & EM 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

Techniques for studying the secretory pathway:

Animal + radio AA → different time → kill → chemical fix → autoradiography

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Pulse-chase exp

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

Low density lipoprotein receptor

<0.5h, protein convert to mature

PTM Glyco..

脈搏補捉

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Use of temperature-sensitive mutant proteins (e.g. vesicular stomatitis virus 水疱口炎病毒 VSV G protein)

At restrictive temp. of 40^oC, newly made G protein is misfolded &

retained within ER.

At permissive temp. of 32^oC, 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.

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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

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Protein transport through the secretory pathway can be visualized by fluorescence microscopy of cells producing a GFP-tagged membrane

protein: VSV G protein

Use temperature-sensitive mutant, VSVG-GFP.

40oC the protein in ER

32oC move → Golgi → plasma membrane→

Form ER to Golgi about 60min Microscopy of GFP-labeled VSV G protein

Plasma membrane

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1) Microscopy of GFP-labeled VSV G protein

2) Detection of compartment-specific oligosaccharide modifications

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Transport of a membrane glycoprotein from the ER to golgi can be assayed based on sensitivity to cleavage by endoglycosidaseD

分子量大分子量小

電泳分離時

分子量大

分子量小

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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

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

From ER to golgi about 60 min

In ER ER to golgi

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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

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• glycosidases (cis-)

• endoglycosidase D

Processing of N-linked oligosaccharide chains on glycoproteins within cis-, medial-, and trans-Golgi cisternae in vertebrate cells

Cleavage by endoglycosidase D.

In cis, specific glycosidase

Remove 3 mannose Remove 2 mannose

Add

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Phenotypes of yeast sec mutants identified stages in the secretory pathway

Yeast sec (secretion) mutants

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

protein

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Protein transport from Golgi cisternae to another can be assayed in a cell-free system

Cell-free transport assay

Can not add

To plasma membrane

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Normal expression

Protein need modification in Golgi

Proof: golgi can retrograde vesicular transport for modification

it demonstrated protein transport from one golgi cisterna to another

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Two Models For Cis to Trans-Golgi

Progression

Tradional Model - Golgi is a static organelle. Secretory proteins move forward in small vesicles. Golgi

resident proteins stay where they are.

“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.

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問題:

到底細胞內利用vesicle的方式的機轉是什麼?

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Molecular mechanisms of vesicular traffic

Overview of vesicle budding and fusion with target membrane

(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

Vesicle transport: from organelle (Donor) target organelle

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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

GTPase superfamily antrograde retrograde

ARF (ADP Ribosylation Factor) To endosome

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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

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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

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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

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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

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Monomeric GTPase control coat assembly

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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 Cargo

protein

Specific receptor

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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

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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

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Major coat protein: clathrin & adaptin

There are at least four types of adaptins,

each specific for a different set of cargo receptor.

by charperone (hsp70)

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Targeting sequence on cargo proteins make specific molecular contacts with coat protein

Mistransport mechanism; retrograde

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Different Rab GTPases & Rab effectors control docking of different vesicles on target membranes: vesicle docking controlled by Rab

protein.

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

v-SNARE

t-SNARE

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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

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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

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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.

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Rab proteins (monomeric GTPase) help ensure the specificity of vesicle docking

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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), SNAP- 25 (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

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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) Spans vesicle membrane

~ 13 kDa

Inject antibodies to Synaptobrevin and release is blocked Synaptobrevin

破傷風毒素

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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.

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Rizo and Sudhof 2002 Nature Rev. Neurosci.

拉上拉鍊

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Rizo and Sudhof 2002 Nature Rev. Neurosci.

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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.

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Rab proteins (monomeric GTPase) help ensure the specificity of vesicle docking

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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 t- SNAREs 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

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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 t- SNAREs

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Early stages of the secretory pathway

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Vesicle-mediated protein trafficking between the ER and cis-Golgi

Anterograde-COPII vesicle Retrograde-COPI vesicle

Vesicle-mediated protein trafficking between ER & cis-Golgi

Cargo protein

vSNAREs (yellow)

Rab important

Membrane specific receptor bind to cargo → transport

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Targeting sequence on cargo proteins make specific molecular contacts with coat protein

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COPII vesicles mediate transport from the ER to the Golgi

3-D structure of ternary complex comprising the COPII coat proteins (Sec23, Sec24)

& Sar1-GTP.

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

Di-acidic sorting signal (Asp-X-Glu, or DXE).

ER lumen cytosol

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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

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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.

PH high 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.

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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.

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