Lecture 13 - Membrane trafficking machinery Flashcards
Coated vesicles: what are they, what do they do, and what examples of them are there?
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
Coated vesicles: what are they, what do they do, and what examples of them are there?
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
Clathrin, COPI, and COPII: where are they found/what movement do they facilitate?
Clathrin - plasma membrane, trans-Golgi network (early endosomes, secretory vesicles)
COPI - ER to Golgi intermediate compartment, Golgi apparatus
COPII - endoplasmic reticulum
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?
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
Clathrin coat assembly: what are the subunits of clathrin and how do they impact the clathrin coat?
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 (?)
Clathrin adaptor proteins: what are they, what are they required for, and what types are there?
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)
AP complex structure: what subunits does it contain, what is the structure of AP2, and how does the AP complex link clathrin to membranes?
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
Phosphoinositide lipids: what do they do, where are they found, how are different versions formed, and do they have distinct locations?
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
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?
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
AP2 recruitment: what facilitates it and what is the mechanism behind it?
PIP₂
- Binding to PIP₂ recruits AP2 to membrane
- Conformational change in AP2 induced
- Cargo protein binding permitted
Dynamin: what is it, how does it polymerise, and what does it do?
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
Clathrin curvature and uncoating: what facilitates these processes?
- BAR domain proteins help generate curvature during budding
- Mediated by the uncoating ATPase hsp70 and PIP₂ phosphatase
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?
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
Coat assembly: how do coats surround the donor membrane, and what substances promote their assembly?
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
COPII coat formation: the process behind it
- 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)
GAP: what is it, why is it required and how does it do its function?
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
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?
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
Targeting of transport carriers: how is targetting used and what ensures specificity?
Requires targeting - specific recognition used
Specificity is ensured by tethering factors, Rabs and SNAREs
Tethering: what is the process of tethering, what factors are required, and what do they act together with?
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
Rab GTPases: what are they, what do they do, how prevalent are they, and where can they be found?
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
Rab 1: where is its subcellular location?
Rab 2: where is its subcellular location?
Rab 3A: where is its subcellular location?
Rab 4/11: where is its subcellular location?
Rab 5
Rab 6
Rab 7
Rab 8: where is its subcellular location?
Rab 9
Tethering process
Rab membrane recruitment
Membrane recruitment coupled to nucleotide exchange
(similar to Sar and ARF GTPases)
GDI chaperone protein
Rab membrane release
GTP hydrolysis releases rab
from the target membrane
Cytsolic rab ready for another
round of transport
Rab effector bidning
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
Membrane curvature bidning
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
Membrane docking and fusion
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
Toxins - SNARE targetting
SNAREs mediate docking of synaptic vesicles with plasma membrane
Targets for neurotoxins (e.g. botulinum, tetanus)
Toxins proteolytically cleave SNAREs
- 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
SNARE dissociation
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
Viral membrane fusion
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
