Lecture 13 - Membrane trafficking machinery

Question 1

Coated vesicles: what are they, what do they do, and what examples of them are there?

Answer 1

Budding vesicles that have a characteristic protein coat on their cytoplasmic surface

• Act as mechanical devices to induce membrane curvature
• Select components for inclusion in the vesicle

Question 2

Coated vesicles: what are they, what do they do, and what examples of them are there?

Answer 2

Budding vesicles that have a characteristic protein coat on their cytoplasmic surface

• Act as mechanical devices to induce membrane curvature
• Select components for inclusion in the vesicle

Clathrin, COPI, and COPII

Question 3

Clathrin, COPI, and COPII: where are they found/what movement do they facilitate?

Answer 3

Clathrin - plasma membrane, trans-Golgi network (early endosomes, secretory vesicles)

COPI - ER to Golgi intermediate compartment, Golgi apparatus

COPII - endoplasmic reticulum

Question 4

Clathrin: what is it, what does it do, what does it form, what is it composed of, how does its polymer form, and what determines its shape?

Answer 4

Paradigm for vesicle formation

Forms the main component of a coated vesicle with a basket-like structure

Each clathrin subunit- triskelion comprised of 3 heavy and 3 light chains

Assemble into a basket-like framework of hexagons and pentagons

Isolated triskelions can self-assemble to form cages in the absence of membranes - triskelions determine the geometry of the cage themselves

Question 5

Clathrin coat assembly: what are the subunits of clathrin and how do they impact the clathrin coat?

Answer 5

Globular domains at the end of chains - attach to the vesicles via the membrane

Heavy chains - create the base of the clathrin chain

Light chains - attach the heavy chains and globular domains (?)

Question 6

Clathrin adaptor proteins: what are they, what are they required for, and what types are there?

Answer 6

2nd major component of clathrin-coated vesicles

• Attachment of clathrin to the membrane
• Recruitment of cargo proteins into a vesicle

Monomeric adaptors:
* Dab2 (plasma membrane)
* AP180 (plasma membrane)
* GGA (trans-Golgi network)

Tetrameric adaptors
* AP2 (Plasma membrane)
* AP3 (Early endosome)
* AP4 (trans-Golgi network)

Question 7

AP complex structure: what subunits does it contain, what is the structure of AP2, and how does the AP complex link clathrin to membranes?

Answer 7

Heterotetramers with 2 large, 1 medium and 1 small subunit (adapting)

AP2:
* α/β2 - clathrin binding
* β2/µ2 domains - cargo binding

AP complex links clathrin to the membrane through binding to cargo and lipids

Question 8

Phosphoinositide lipids: what do they do, where are they found, how are different versions formed, and do they have distinct locations?

Answer 8

Phosphoinositides (PIs) can act as signals to recruit proteins to membranes or they can be hydrolysed to generate second messengers

Found only on cytoplasmic leaflet

Generated by differential phosphorylation of inositol ring at the 3, 4, and 5 positions

Yes, different PIs are enriched in different compartments and even domains within the same membrane compartment

Question 9

PI interconversions: what mediates them, how does each PI form associate with the membrane, what examples of PI forms are there, and what recruitments do they facilitate?

Answer 9

Interconversion between different PIs is mediated by specific kinases and phosphatases

PIs recruit effectors to the membrane in a specific manner

PI(4,5)P₂ - recruits AP2 to the plasma membrane, as well as other proteins
PI(3)P - recruits proteins to early endosomes

Question 10

AP2 recruitment: what facilitates it and what is the mechanism behind it?

Answer 10

PIP₂

• Binding to PIP₂ recruits AP2 to membrane
• Conformational change in AP2 induced
• Cargo protein binding permitted

Question 11

Dynamin: what is it, how does it polymerise, and what does it do?

Answer 11

High molecular weight GTPase

Polymerises into ring structure around the bud neck

• Recruits other proteins to the bud neck
• Uses GTP hydrolysis to pinch off vesicles - appears to function as a “pinchase”, causing vesicles to be released from donor membranes

Question 12

Clathrin curvature and uncoating: what facilitates these processes?

Answer 12

• BAR domain proteins help generate curvature during budding
• Mediated by the uncoating ATPase hsp70 and PIP₂ phosphatase

Question 13

COPI/COPII coats: what are they, how big are the vesicles they coat, what is their structure, what do they do, what subunits do they have, where does their budding occur, and how do they allow them to complete their function?

Answer 13

COat Proteins - multi-subunit protein complexes

Vesicles are ~50-70 nm in diameter

Cage systems - Triskelion structure of coatomer subcomplex

Interact with cargo and drive vesicle-budding

COPII - budding from the ER:
* Sec23/24 - cargo binding (ie adaptors in clathrin)
* Sec 13/31 - cargo assembly (ie AP2(?))

COPI - budding from the ERGIC and Golgi apparatus:
* Subunits: coatomer (7 subunit protein complex) - cargo binding and assembly

Question 14

Coat assembly: how do coats surround the donor membrane, and what substances promote their assembly?

Answer 14

Coat proteins are recruited from the cytosol onto the donor membrane - regulated by small GTPases

• Sar1 - COPII assembly on ER
• ADP Ribosylation Factor 1 (ARF1) - COPI assembly on ERGIC and Golgi
• Adaptor protein 1 (AP1) and Golgi-localized, gamma-ear-containing, ARF-binding protein (GGA) - Clathrin assembly on trans-Golgi network

Question 15

COPII coat formation: the process behind it

Answer 15

• Sar 1-GEF activates Sar 1-GDP by exchanging the GDP for a GTP
• Sar 1-GTP inserts its amphipathic helix into the donor membrane
• Sec 23 binds to Sar 1-GTP and Sec 24 binds to the cargo, forming the inner coat of the COPII-coated vesicle
• Sec 13/31 binds and forms a second outer coat, completing the COPII vesicle (after this is repeated to form a coat around the vesicle)

Question 16

GAP: what is it, why is it required and how does it do its function?

Answer 16

GTPase activating protein

Non-hydrolysable GTP analogues prevent uncoating, coats need to eventually be broken down

GAP hydrolysis GTP bound Sar1, forming GDP bound Sar1, causing Sar 1 to begin to dissociate, and causing the breakdown of the COPII/COPI coat

Question 17

Tubular carriers: do they differ from vesicles, what is the process of carrier formation, what example of tubule carriers is there, and how does it do its function?

Answer 17

Machinery for tubule formation is usually different to that for vesicles

Cargo -> growth -> stabilisation -> invagination -> scission

• SNX proteins - use the PX domain for membrane recruitment (binds PI3P) and the BAR domain to induce tubulation

Question 18

Targeting of transport carriers: how is targetting used and what ensures specificity?

Answer 18

Requires targeting - specific recognition used

Specificity is ensured by tethering factors, Rabs and SNAREs

Question 19

Tethering: what is the process of tethering, what factors are required, and what do they act together with?

Answer 19

Budding (vesicle ‘buds’ off) -> Tethering (long distance attachment to acceptor(?)) -> Docking (tight attachment to acceptor using SNAREs) -> fusion

Tethering factors required:
* Long flexible proteins
* Multisubunit complexes

Tethering factors act together with Rab GTPases

Question 20

Rab GTPases: what are they, what do they do, how prevalent are they, and where can they be found?

Answer 20

Monomeric, low molecular weight GTPases

key regulators of tethering - bind to specific effectors to activate proteins

> 60 Rabs in mammalian cells, a major class of Rab effectors are tethering proteins

Each Rab is localised to a particular compartment

Question 21

Rab 1: where is its subcellular location?

Question 22

Rab 2: where is its subcellular location?

Question 23

Rab 3A: where is its subcellular location?

Question 24

Rab 4/11: where is its subcellular location?

Question 25

Rab 5

Question 26

Rab 6

Question 27

Rab 7

Question 28

Rab 8: where is its subcellular location?

Question 29

Rab 9

Question 30

Tethering process

Question 31

Rab membrane recruitment

Answer 31

Membrane recruitment coupled to nucleotide exchange

(similar to Sar and ARF GTPases)

GDI chaperone protein

Question 32

Rab membrane release

Answer 32

GTP hydrolysis releases rab

from the target membrane

Cytsolic rab ready for another

round of transport

Question 33

Rab effector bidning

Answer 33

Tethering factors, but also:

Motor proteins, PI metabolising enzymes, GEFs and GAPs for other small GTPases……………

Rabs therefore regulate most aspects of membrane traffic

They are also important for maintaining organelle identity

Some effector interactions are cooperative allowing Rabs to form functional domains

Question 34

Membrane curvature bidning

Answer 34

ALPS motif binds to highly curved membrane of vesicle

GMAP210 is a Golgi tethering protein with an ALPS motif at one end

ALPS motif only binds to highly curved membranes

Question 35

Membrane docking and fusion

Answer 35

Small helical membrane proteins

> 30 SNAREs in a mammalian cell

Different SNAREs associated with different organelles

Each SNARE can only interact with its cognate partners

v-SNARE - vesicle SNARE
t-SNARE - target SNARE (syntaxin, Snap25)

Forms a 4 helical trans-SNARE bundle:
ie - synaptobrevin (v-SNARE - 1 chain), syntaxin (t-SNARE - 1 chain), and Snap25 (t-SNARE - 2 chains) forms a 4 helical trans-SNARE bundle

Question 36

Toxins - SNARE targetting

Answer 36

SNAREs mediate docking of synaptic vesicles with plasma membrane

Targets for neurotoxins (e.g. botulinum, tetanus)

Toxins proteolytically cleave SNAREs

Answer 37

• v- and t-SNAREs on apposing membranes interact to initiate formation of 4-helix bundle trans-SNARE complex
• SNAREs use energy released when interacting

helices wind up to pull membranes together

and expel water molecules

• Lipids then flow between outer leaflet of

membranes to form a connecting stalk

• Hemifusion - Lipids in inner leaflet contact each other for hemi-fusion (half-fusion). This widens the fusion zone to make new bilayer
• Fusion - Rupture of new bilayer results in full fusion

Question 38

SNARE dissociation

Answer 38

SNARE complexes are very stable-resistant to boiling

Must be dissociated for future transport - this is mediated by ATPase NSF

Accessory proteins along with ATPase NSF unwind SNAREs (using ATP obvs)

v-SNARE sent back to vesicle membrane in its own new membrane

Question 39

Viral membrane fusion

Answer 39

They fuse with the plasma membrane

• gp120 attaches to CD4, attaching HIV to target cell
• gp120 binds to chemokine receptor
• HIV fusion protein inserts into the membrane
• Conofromational change occurs
• Fusion occurs
• Entry of viral nucleocapsid occurs