Vesicle transport and targeting in the secretory pathwayCOP coated vesiclesSNAREs

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

Protein Trafficking

Protein Trafficking - Regulated transport to the trans-Golgi network

• Multimeric proteins (e.g., ion channels).

- KATP channels = 4 Kir6.1/6.2 subunits with 4 SUR1/2A/2B subunits in ER.

- NMDAR = combination of NR1, NR2A-D, or NR3A-B subunits.

- GABAAR: 16 different mammalian isoforms (α1-6, β1-3, γ1-3, δ, ε, π, and θ), making the total number of receptor combinations = 165; but only ~20-30 functionally distinct receptor types exist.

• During GABAAR assembly, chaperones, IgG-bp (BiP) and calnexin, interact with subunits.

• Association with ER depends on ER retention signals (KDEL).

• Hydrophobic residues.• Exact mechanism of ER retention involves

interaction with ER matrix, failure to be recruited for transport, or retrieved from the cis Golgi.

• Coatomer proteins (COPS) are involved in the selection of cargo for anterograde (COPII) or retrograde (COPI) transport between organelles.

Morphology• Diffusion barrier• Cytoskeleton = actin-spectrin-ankyrin

anchors membrane proteins (e.g., Nav channels).

• High [protein] crowding.• Soma, dendrites, axon - not 1 continuous

structure.• Inhibitory synapses vs excitatory synapses.

Development of Polarity

• Synapse Formation:

- GFP-PSD-95 visualized extension and maintenance of filopodia.

- Appeared to be translocated to filopodia as pre-assembled clusters, rather than as accumulating gradually.

- But occurs only when the postsynaptic scaffold/signaling complex is already there.

- Within 45 min, AMPA and NMDA receptors can be found postsynaptically.

Development of Polarity

• Axonal Development

- Nav channels cluster at the Nodes of Ranvier.

- Mechanism of how this occurs is unknown.

- In demyelinated axons, some form of anchoring occurs via Ankyrin G within the axon.

-In the PNS, paranodal Kv channels appear to cluster initially within nodes prior to lateral diffusion to their final destination.

Development of Polarity

-+

++

Dendrite

Axon

+-

--

mGluR2 mGluR7

Polarity Signals

• Dendrites hydrophobic motifs• Axons – GAP43

Postysynaptic Targeting

• mRNA Targeting• Protein targeting via lipid rafts• Specific transport pathways and proteins

- GABAA receptors

- NMDA receptors

- AMPA receptors

Transport between organelles is mediated by coated vesicles

Clathrin coated vesicles mainly involved in endocytosis

COP coated vesicles mediate ER to Golgi and back

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 Golgi

Golgi and ER (retrieval)

intra-Golgi

TGN and plasma membrane

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 membrane

We will only consider Sar1

Cop coated vesicles contain many proteins

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

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)

Sar1 activation exposes hydrophobic tail and membrane insertion

Sar 1 in membrane recruits COP proteins

Greasy foot

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

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

• Rab family: vesicle targeting and fusion (see below)

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-Ra (GTP)

SRP-Rb (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 +

Summary of COPII-coated vesicle formation

COP subunits recruit specific cargo proteins…

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

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)

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

Cargo

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)

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

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

GTP hydrolysis by Sar1 is required for uncoating

GTPgS

Sar 1

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

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

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!!!

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?

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

Inside lumen is equivalent to outside of cell

secretory_pathway.mov

Regulated secretion

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

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…

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 a-helix)

KDEL (soluble proteins)

KKXX (membrane proteins)

Lysosomes

?Default

How are proteins targeted to the lysosome?

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?

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?

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!

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)

Postsynaptic Removal of Receptors

• Specific endocytotic signals leads to recruitment of AP2 in the internalization of the plasma membrane.

• APs recruit clathrin, which instigates membrane invagination and endocytosis.

• Examples:- tyr-based signals recruit μ subunits of AP2.- dileu-based signals recruit β subunits of

AP2.- Arrestin binding to GPCRs facilitate

receptor internalization by its ability to assocociate with clathrin and AP2. - Ubiquitin may recruit AP2 or clathrin, release the receptor from anchoring in the membrane, or recruit receptors to the sites for endocytosis.

RER

GolgiTrans face

EE

LE

AP1

Lysosome

EE

(rapid)

(rapid)

COPI

COPII

AP2

Receptor Endocytosis• Agonist-dependent down-regulation of receptors has

been observed for a wide variety of ligands: e.g., GABAA receptors treated with GABA, BDZs, barbs, and neurosteroids; antidepressants and β-adrenergic receptors.

• Cell surface receptor number is a balance between 2 competing processes: delivery and removal of receptors.

• Synaptic strength is in part, determined by the number of surface AMPA receptors (LTP vs. LTD). BUT…

Evidence has shown that in response to NSF-GluR2 interaction, synaptic AMPA receptors are only internalized on the cytoplasmic face of the membrane and are not transported to the soma and degraded in the lysosomes.

• Insulin can also cause AMPA receptor down-regulation.

Protein targeting, revisited

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

Signal sequence (hydrophobic a-helix)

KDEL (soluble proteins)

KKXX (membrane proteins)

M6P

Default or

signal?

Default or

signal?

The modulation of synaptic strength by alterations in postsynaptic AMPA receptors. Early in development, most of the glu synapses are ‘silent’ at Vm. This results from the presence of NMDA, but not AMPA, receptors inthe postsynaptic membrane. Synapses become activated by a NMDA-dep-dent process, leading to the recruitment of AMPA receptors. Synapticmay be incr further, in response to high-freq activity (LTP), by the furtherrecruitment of AMPA receptors.