lecture 16 vesicle transport and targeting in the secretory pathway cop coated vesicles snares...

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Lecture 16 sicle transport and targeting in the secretory path COP coated vesicles SNAREs otein sorting Secretion - Golgi to plasma membrane Retention in ER Golgi to lysosome

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Page 1: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

Lecture 16

Vesicle transport and targeting in the secretory pathwayCOP coated vesiclesSNAREs

Protein sorting Secretion - Golgi to plasma membraneRetention in ERGolgi to lysosome

Page 2: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

Transport between organelles is mediated by coated vesicles

Clathrin coated vesicles mainly involved in endocytosis (next time)

COP coated vesicles mediate ER to Golgi and back

Page 3: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

Transport between ER and Golgi compartments occurs via “COP-coated

vesicles”…

Collection of 4-7 “coat proteins” = “COPs”…(aka “Coatomers” )COP-coated vesicles function in transport between:

ER and GolgiGolgi and ER (retrieval)

intra-GolgiTGN and plasma membrane

Page 4: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

COP proteins

“cargo”

More COP proteins

Lipid bilayer

Sar1

COPII-coated vesicles - ER to Golgi-SarI in ER membrane

COPI coated vesicles - Golgi to ERARF (instead of Sar1) in Golgi

membraneWe will only consider Sar1

Cop coated vesicles contain many proteins

Page 5: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

Sar1:GTPase switch

on/off

ON: binds membranerecruits COP proteins

COP proteins then recruit specific cargo

Sar1 --Similar to RAN in nuclear import

Sar1 ARF triggers vesicle formation

Page 6: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

GAP

Large family (Ras) of proteinsMolecular “switches”

Sar1 GTPase

GTPSar1

GTPase

GDP

Pi

GDP

GTP

“on” “off”GEF

In cytoplasm, large amount in “off” form

cytoplasmic

Bound tomembrane

GTPase (GTP Binding Proteins)

Page 7: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

Sar1 activation exposes hydrophobic tail and membrane insertion

Sar 1 in membrane recruits COP proteins

Greasy foot

Page 8: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

The Ras “superfamily” of small GTPases…• Ras: signaling and regulating cell proliferation…

>30% of human tumors have Ras mutations…

Many (not all) Ras family members associated with membranes via covalent fatty acid tail (“greasy feet”)…

• EF-1/EF-Tu: translation…

• Ran: nuclear transport…

• Rho family (Rho, Rac, cdc42): actin assembly and organization (in a few lectures)

• Arf/Sar family of “Coat recruitment GTPases:” COP assembly and vesicle budding…

• Rab family: vesicle targeting and fusion (in a few minutes)

Page 9: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

Aside: G-proteins and ATPases as molecular switches

Translation:

IFs (GTP), EF-1/EF-Tu (GTP)

EF-2/EF-G (GTP)

Chaperones:

HSP70 family (ATP)

HSP60 (ATP)

SRP family:

SRP54 (GTP), SRP-R(GTP)

SRP-R(GTP)

Polymer dynamics:

Actin (ATP), Tubulin (GTP)

Dynamin (GTP)

Motors:

Myosin (ATP), Dynein (ATP)

Kinesin (ATP)

Signaling:

Heterotrimeric G proteins (GTP)

Ras family (GTP)

Cells make high-affinity transient molecular complexes as trigger or switch

GTP GDP + Pi

A paradox:

High-affinity/high-specificity = stable…

Energy input is required to dissociate high-affinity complexes…

(Example: to remove Sar 1 from membrane)

Bound UnboundBA BA +

Page 10: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

Summary of COPII-coated vesicle formation

COP subunits recruit specific cargo proteins…

Page 11: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

Vesicle transport is a complex process

3. Targeting and docking to specific compartment…

(ATP, GTP, and cytoplasmic protein factors…)1. Formation of

coated buds…

2. Formation of coated transport vesicle…

Target compartment

Coat proteins (“COPs”)

Donor compartment

SNAREs and Rabs

Page 12: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

The Snare hypothesis: v- and t-SNAREs target transport vesicles to the correct membrane

v-SNAREs

Budding Uncoating, targeting and docking

t-SNAREs

Specific pairing of V-SNAREs with T-SNAREs matches vesicle to target membrane compartment (>20 known snares in animals cells)

See ECB figure 15-20

Targeting and docking requires/is facilitated by specific Rab GTPase in vesicle and Rab effector in target (~30 known Rabs in animal cells)…

Cargo

Page 13: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

Bacterial toxins target the vesicle docking and fusion machinery of

neurons

Botulism A

Botulism B

Botulism C

Tetanus

SNAP25 (t-SNARE)

VAMP (v-SNARE)

Syntaxin (t-SNARE)

VAMP (v-SNARE)

A small subunit of the toxin acts as a specific protease that cleaves and inactivates targeting proteins

Net result is to block neuronal signaling by blocking neurotransmitter release (regulated secretory pathway)

Page 14: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

Vesicle transport is a multi-step process

3. Targeting and docking to specific compartment…

2. Formation of coated transport vesicle…

Donor compartment

1. Formation of coated buds…

(ATP, GTP, and cytoplasmic protein factors…)

Target compartment

4. Uncoating…

GTP

GDP + Pi

Coat proteins (“COPs”)

SNAREs and Rabs

GTPS and other non-hydrolyzable GTP analogs block uncoating, resulting in accumulation of docked, coated vesicles

GTP hydrolysis by Sar1 is required for uncoating

GTPS

Sar 1

Page 15: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

Vesicle transport is a multi-step process

3. Targeting and docking to specific compartment…

2. Formation of coated transport vesicle…

Sar1

Donor compartment

1. Formation of coated buds…

(ATP, GTP, and cytoplasmic protein factors…)

GEF and Sar1

Target compartment

4. Uncoating…

GTP

GDP + Pi

Coat proteins (“COPs”)

SNAREs and Rabs

GEF in donor membrane promotes nucleotide exchange, activating Sar1 @ ER, (ARF @ Golgi) and promoting coat assembly…

GTP hydrolysis serves as “timer” delaying uncoating (GAP in target membrane?)…

GTPase “cycle” provides directionality to vesicle coating/uncoating

Page 16: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

Vesicle transport is a multi-step process

3. Targeting and docking to specific compartment…

2. Formation of coated transport vesicle…

Coat recruitment GTPase

Donor compartment

1. Formation of coated buds…

(ATP, GTP, and cytoplasmic protein factors…)

GNRP/GEF and Coat recruitment GTPase

Target compartment

4. Uncoating…

GTP

GDP + Pi

Coat proteins (“COPs” or

“coatomer”)

SNAREs and Rabs

5. Fusion…SNARE plus other fusion

proteins

Page 17: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

SNAREs are necessary for membrane fusion

SNAREs bring two membranes into close apposition

Lipids flow between membranes - fusion

Other proteins cooperate with SNAREs to facilitate fusion and to pry SNAREs apart

ECB 15-21

Much still to learn!!!

Page 18: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

Lecture 16

Vesicle transport and targeting in the secretory pathwayCOP coated vesiclesSNAREs

Protein sorting/targetingSecretion - Golgi to plasma membraneRetention in ERGolgi to lysosome

How are proteins sorted to appropriate vesicles so that they are transported to proper location? What are the address label?

Page 19: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

Two secretory pathways; constitutive and regulated

Default pathway for ER/Golgi proteins

If no address label, then secrete

Signal required to trigger secretory granule fusionExample - neurotransmitter release

However, recent data suggests there may be ER exit sequences..For now, consider secretion default

ECB 15-28

Inside lumen is equivalent to outside of cell

15.9-secretory_pathway.mov

Page 20: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

Regulated secretion

Secretory granules containing insulin in pancreatic cells Signal for release is elevated glucose levels in blood

Page 21: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

If secretion is default, how are resident ER proteins retained?

C, M, T Golgi

ER

Plasma membrane

OutsideCGN TGN

Constituitive secretion

Secretory granuleRegulated secretion

Ex: BiP is a member of the HSP70 family that functions in the ER…They

aren’t!

BiP escapes from ER and must be “retrieved” from the Golgi…

C-terminal KDEL in BiP sequence functions as retrieval signal…

BiP KDEL

KKXX

KDEL-R

KDEL-receptors in Golgi direct retrieval/recycling…

KKXX at C-terminus of KDEL-R binds COPI coat and targets back to ER…

Page 22: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

Summary so far of protein targeting, revisited…

Cytoplasm

Secretion/membrane proteins

Secretory vesicles

RetrievalTransport

(constituitive secretion)

(regulated secretion)

Pro

tein

ta

rgeti

ng

Vesi

cle t

arg

eti

ng

RER

Golgi

Plasma membrane

See ECB figure 14-5

Default

Signal sequence (hydrophobic -helix)

KDEL (soluble proteins)

KKXX (membrane proteins)

Lysosomes

?Default

How are proteins targeted to the lysosome?

Page 23: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

Lecture 16

Vesicle transport and targeting in the secretory pathwayCOP coated vesiclesSNAREs

Protein sorting Secretion - Golgi to plasma membraneRetention in ERGolgi to lysosome

How are proteins sorted to vesicles leaving TGN for lysosome?

Page 24: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

Lysosomes degrade and recycle macromolecules…

Lysosomes in plant and animal cells contain acid hydrolases (hydrolytic enzymes) for degrading/recycling macromolecules

pH of lumen is about 5 - acidic!

How are hydrolases and other proteins targeted to lysosomes?

ECB 15-34

Page 25: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

I-cell disease helped decipher the signal for targeting proteins to the lysosome

• Recessive mutation in single gene…

• Fibroblasts of patients contain large inclusions (I-cells)…

• Lysosomes lack normal complement of acid hydrolases…

• All lysosomal enzymes secreted (secretion is the “default” fate for proteins in the ER-Golgi pathway)…

• Lysosomal enzymes of “wild-type” (normal) cells are modified by phosphorylation of mannose on oligosaccharide (forming mannose-6-phosphate)…

• Lysosomal proteins of I-cells lack M-6-P…

• Lysosomal targeting signal resides in carbohydrate!

Page 26: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

Mannose-6-P targets proteins from Golgi to lysosomeCis Golgi

Network (CGN)Trans Golgi

Network (TGN)

RER

M6P receptor recycling back to Golgi

Transport via clathrin-coated vesicles to…

Lysosome

M6P receptor in TGN directs transport of enzymes to lysosome via clathrin-coated vesicles

Addition of M6P to lysosomal enzymes in cis-Golgi

Patients with I-cell disease lack phosphotransferase needed for addition of M-6-P to lysosomal proteins in fibroblasts… secreted…

Lysosomal hydrolase (precursor

)

Addition of M6P

Removal of phosphate &proteolytic processing…

Maturehydrolase

M6P receptor

Clathrin coat

Uncoupling(pH 5)

Page 27: Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention

Protein targeting, revisited

Next lecture: endocytosis and clathrin coats

Cytoplasm

Secretion/membrane proteins

Secretory vesicles

Lysosomes

RetrievalTransport

(constituitive secretion)

(regulated secretion)

Pro

tein

ta

rgeti

ng

Vesi

cle t

arg

eti

ng

RER

Golgi

Plasma membrane

See ECB figure 14-5

Signal sequence (hydrophobic -helix)

KDEL (soluble proteins)

KKXX (membrane proteins)

M6P

Default or

signal?

Default or

signal?