Vesicle Trafficking Flashcards
Formation of Vesicles
- coat protein assembly at bilayer begin to bend
- coat proteins select cargo to be packages into vesicle
- more coat protein binding results in formation of membrane sphere
- once coated, vesicles pinches off, coat detaches to reveal signal for transport
3 vesicle coat types
- COPII = ER to golgi
- COPII = golgi to ER
- Clathrin coated = plasma membrane
COPII coated vesicles
- sec12p is a GEF that exchanges GDP for GTP in sar1p
- sar1p activation by GTP leads to helix embedding in the ER membrane
- activated sar1p acquires sec 23/24p to form core inner coat of COPII coat
- outer coat scaffold complex acquired
- budding/pinching off of vesicle
- GTP hydrolysis by sar1p releases coat and vesicle
Key Proteins
Sar1p = activated by Sec12p & binds to sec 23/24p causing gathering
Sec 23/24p = distinguishes between cargo/non cargo proteins (samples membrane with inserted helix tail)
sec 13/31p = scaffold complex causing pinching off
COPi coated vesicles
Not well understood
7 polypeptides form the coat (coatamer)
Uses Arf1 GTPase similar to Sar1 from COPII system
Clathrin vesicle structure
More complex pathway
1. Arf GTPase recruits adaptor proteins (key component)
2. Clathrin layered onto coating to create clathrin coated vesicles composed of triskelions
3. Each triskelion composed of 3 heavy chains interacting at their C-terminal
4. Each heavy chain has a 25kDa light chain tightly bound
Heavy chains = structural backbone
Light chains - regulate formation and disassembly of lattice
Clathrin coated vesicle formation
- Arf1 GTPase initiates assembly and recruits cofactors
- adaptor proteins give specificity of destination, bind the clathrin coat to the membrane and select a specific set of transmembrane proteins
- dynamin pinches off vesicle - this is ATP dependent
- vesicle uncoating mediated by Hsc70/auxilin
Dynamin
GTP hydrolysis leads to power stroke and increased constriction
Fission of membrane where stress is largest
Disassembly of oligomer and dynamin recycling
* fly experiments show that dynamin inactivation causes paralysis because the nervous system no longer works *
SNARE hypothesis
Each transport vesicle has v-snares that bind to complementary t-snares on the target membrane
Binding between them creates stable 4 helix bundle
Snares both promote fusion (overcomes energy barrier) and ensures specificity of membrane fusion
*experimental evidence = using in-vitro liposomes with labelled snare proteins shows a light signal when vesicles fuse
Form trans-snare complex
Uses the energy released when helices wrap around each other to pull membranes together and squeeze water out
RAB Proteins
Monomeric GTPases
Guide vesicle targeting
Active form binds Rab effectors for movement/tethering vesicles
Specificity is ensured by matching surface markers on vesicles and receptors
membrane bound RAB gefs activate Rab in cytosol to bind to their membrane and then to Rab effectors
Activation of Rab creates membrane patch via positive feedback
RAB cascade can change an organelles identity
- rab domains can recruit a GEF for another rab. the new rab recruits a GAP for the previous rab
- this causes a new rab domain to form
Membrane Bending
Proteins such as BAR domains binds to and impose their shape on the underlying membrane via electrostatic attractions to the lipid head groups
Actin filaments and amphiphilic helical wedges help as well
Coat assembly
Monomeric GTPase controls coat assembly
- coat recruitment gtp-ases (like Arf and sar1) are responsible for the assembly of COPI and clathrin coats and the disassembly of COPII coats
- these proteins are usually in the cytosol in an inactive GDP bound state
- a GEF in the ER membrane binds to this inactive protein and promotes GTP uptake
- once activated the protein exposes amphiphilic helix and recruits adaptor coat protein subunits so budding begins
- reverse process (GTP hydrolysis) causes coat disassembly
Misfolded Proteins
Misfolded proteins remain in the ER where they bind to chaperone proteins like Bip
The proteins are transported back into the cytosol where they are degraded by proteasomes
Cells must make an excess of proteins to account for the select few that correctly fold
Distinguishing between the misfolded and folded proteins is done by the N-linked oligosacchrides
Misfolded proteins signal to the nucleus and cause unfolded protein response like a heat shock response to kickstart correct refolding
Protein Retrieval
Depends on ER retrieval signals
eg. KKXX sequence on resident ER membrane proteins binds to COPI coat
soluble ER proteins bind to KDEL receptors to be packaged into vesicles
ER resident proteins will also bind to each other at low affinity and high concentration in order to become too big to mistakenly enter the vesicles and leave in the first place
