Describe the common steps of vesicle formation.
- An initiation event occurs on the membrane which triggers the subsequent steps.
- Adaptor proteins interact with the initiator, the adaptors collect transmembrane cargo or cargo receptors (for soluble proteins), and a protein framework forms on top of the adaptors to form the coat.
- Fission - the bud is pinched off to separate the vesicle from the membrane.
- Uncoating - the coat is removed to allow vesicle targeting and fusion.
Which family of GTPases are responsible for COPI/II vesicle initiation? Which specific GTPases correspond to which vesicle?
Ras GTPases. Sar1 for COP-II and Arf1 for COP-I.
Describe the differences between Sar1-GDP and Sar1-GTP.
Sar1-GDP is inactive, soluble and cytoplasmic. Sar1-GTP is active, has an exposed amphipathic helix and partially embeds into the membrane.
Describe the roles of Sec13, Sec23, Sec24, and Sec31.
Sec23/24 are adaptor proteins (Sec23 binds Sar1-GTP, Sec24 then binds to Sec23), while Sec13/31 are coat proteins.
Sec13/31 do not require any energy source to shape & pinch the vesicle; the energy comes from protein-protein interactions.
Sec23/24 are adaptor proteins but also serve as GAPs. Elaborate.
They allow Sar1 to hydrolyze GTP. This process is still slow, and are therefore acting as a timer. When Sar1-GDP separates, this disassembles the adaptor proteins, which is a necessary step before vesicle fusion at the Golgi.
Describe the ER exit signals.
Di-phenylalanine (FF) exit signals for transmembrane proteins, at the C-terminus. Transmembrane proteins also have a DXE (acid flank) within the sequence. These are recognized by the Sec23/24 adaptor.
Lumenal proteins are recognized by various transmembrane cargo receptors (again, have the FF signals).
*ER proteins are transported to the Golgi by default, even w/o exit signals. Some are brought back to the ER (COP-I), while misfolded proteins are kept away (CNX/CRT).
Describe the ER retrieval signals.
These are involved in the COP-I vesicle pathway. KKXX at cytosolic C-terminus (Type I TM proteins), MXXRR at cytosolic N-terminus (Type II TM proteins).
Lumenal proteins would have a KDEL motif, recognized by TM KDEL receptors (recall: these have the KKXX motif).
Name the subunits and roles thereof in COP-I vesicle formation.
π½/πΎ and πΏ/π subunits (adaptors) collect cargo, and are GAPs for Arf1 to dissociate the coat.
πΌ/π½β subunits (COP-I coat) assemble on adaptors, shape and pinch off vesicle from the membrane.
What molecules serve as clathrin-coated vesicle initiators?
Phosphatidylinositol (PI). It can be phosphorylated to make phosphatidylinositol phosphates (PIPs).
PI(4)P recruits Golgi clathrin adaptors.
PI(4,5)P2 recruits plasma membrane clathrin adaptors.
What proteins serve as adaptors in the CCV formation process?
AP-1, AP-2 which bind to PIPs and cargo in the membrane, through cargo receptors. Coat proteins would then bind these adaptors.
Describe the role of clathrin triskelions in CCV formation.
Coat proteins, binding the adaptors. Consists of 3 heavy chains & 3 light chains.
What distinguishes Dynamin GTPase from other vesicle-related GTPases?
Facilitates separation of the vesicle from the membrane, and is NOT a member of the Ras family.
How does the clathrin coat disassemble?
Adaptor binding is weakened by PIP modifications (phosphatase). Auxillin (binds both clathrin & HSC70) is required for HSC70 recruitment (binds clathrin & auxillin).
HSC70 then induces a conformation change in clathrin, disaasembling into triskelions.
Clathrin then releases from HSC70 and is recycled to the membrane.
What are the roles of Rab GTPase proteins?
Rab GTPases provide specificity of vesicle targeting and attachment to acceptor membranes.
What are the two theories of Golgi transport? Which is correct?
Theory 1: vesicles transport cargo between layers (COP-I).
Theory 2: each layer matures into the next layer.
Both have evidence to suggest they are correct.
Describe two types of glycosylation relevant to Golgi transport.
N-linked glycosylation: removal of mannoses, addition of different (negative) sugars.
O-linked glycosylation: oligosaccharides attached to -OH groups of other serines, threonines.
Functions of glycosylation include:
Promotion of protein folding by increasing solubility of intermediates (preventing aggregation).
Protection from proteases & pathogens through rigidity of sugars.
Some modifications can alter receptor-ligand & cell-cell recognition.
Describe the role of proprotein convertases.
Conversion of protein prototypes into functional proteins.
Occurs by recognition of an amino acid sequence followed by cleavage of inhibitory regions.
Describe Rab anchoring.
Rabs have 2 prenyl lipid groups attached at their C-terminals. When GDP-bound, Rabs are soluble & the lipids groups are covered by GDP Dissociation Inhibitors (GDI).
When GTP-bound, lipid mods are exposed, allowing anchoring of Rab to the membrane.
Three primary functions of Rabs (specifically) include:
Connecting vesicles to motors on the cytoskeleton for transport;
Tethering of vesicles to acceptor membranes (specificity);
Recruitment of SNARE fusion proteins.
Describe the vesicle Rab cycle.
- Vesicle Rab-GDP is activated by GEF on the donor membrane & packed into vesicles.
- The vesicle is transported to the acceptor membrane; Rab-GTP interacts w/ motor proteins & tethers.
- After vesicle fusion, GAPs on acceptor membranes inactivate Rab, allowing recycling through the cytosol.
What is the first determinant of vesicle targeting specificity?
a) SNAREs
b) Rab-GTP
c) Tethers
d) Arf1
e) The cargo within the vesicle itself
c) Tethers
Describe the two types of tethers.
Coiled-coil tethers: primarily within the Golgi & on endosomes – dimers that remain assembled after use, and form the Golgi matrix.
Multisubunit tethers: other compartments
Describe the TRAPPI system.
Multisubunit complex acting as a GEF for Rab1 on the Golgi membrane.
Rab-GTP then binds a coiled-coil tether (p115) which then binds a vesicle and transfers it to the TRAPPI complex.
The TRAPPI complex helps to organize SNAREs for fusion. Thus, the TRAPPI complex has a vesicle-binding “domain”, a GEF and a SNARE-binding “domain”.
Important: this is from ER to Golgi, involves dissociation of adaptors & coat proteins.
