SNARES 1 Flashcards
Why are proteins in the synaptic vesicle membrane needed?
How big are synaptic vesicles? and what are they loaded with?
- They are needed to control fusion with the plasma membrane
- 50nm and are loaded with NT. They form a cluster around the active zone of the pre-synaptic membrane
SNARES - what are examples of common neuronal SNAREs?
- proteins involved in membrane fusion therefore synaptic transmission
- SNAP25, Syntaxin1 and Synaptobrevin
- SNAREs mediate regulated secretion
Examples of cargo that undergo regulated secretion?
- hormones
- neuropeptides
- inflammatory mediators
Why doesn’t fusion occur all the time and what has to be overcome so that fusion can occur?
There is a negative charge at the phospholipid bilayer (which is what vesicles and the plasma membrane are comprised of).
This negative repellant force must be overcome so that membranes can fuse — this is done by SNARES!!
Steps 1-3 of fusion
1) Tethering - vesicle is tethered at the right place on the membrane
2) Docking - membranes are brought close together and the water is squished out
3) fusion initiation - membranes are bent to fuse them together. This means that membrane proteins are moved and the lipids are brought closer together
Steps 3-6 of fusion
4 - hemifusion - 1 leaflet from each phospholipid bilayer (from the plasma membrane and vesicle) fuse
5 - fusion pore opening - inner leaflets of the membranes fuse. This is needed for synaptic transmission
6 - collapse - membranes collapse depending on the size of the cargo being carried in the vesicle
What is kiss and run fusion and why is it useful?
Kiss and run fusion is when fusion pore opening occurs but the pore opens and snaps shut again pretty much instantly.
This method is useful as it ensures there is efficient membrane retrieval. Membrane retrieval is needed otherwise all nerve endings would increase in size and incorporate with each other when vesicle fusion occurs.
What are t snares, v snares and what is the cis snare complex?
V snares are snares localised to vesicles.
T snares are snares localised to the plasma membrane
V and t snares interact and once the membranes fuse all of the snares end up on one membrane – CIS SNARE COMPLEX
How were SNARES discovered?
Isolated by western blotting
What are NSF, SNAP proteins and SNAP receptors?
NSF = ATPase which uses energy from ATP–>ADP + Pi to start fusion
SNAP proteins are in the cell cytosol and come in 3 isoforms - a, B and y. They are involved in the transport from the ER to the Golgi
SNAP receptors are also known as SNAREs. They are binding sites for the SNAREs on membranes. SNAP proteins must bind to SNAPrs before NSF can
Why is the NSF/SNAP/SNAPr relevant?
Forms a very very stable construct which needs ATP–>ADP+Pi to occur
What were the main SNAREs first identified?
What are the 7S and 20S complex
Syntaxin A + B – target for botulinum (botox)
SNAP25
vAMP/Synaptobrevin
V (synaptobrevin and vAMP) and T (SNAP25 / syntaxin) snares go through protein interactions to form 7S complex
Then SNAP and NSF is added to form 20s complex
What was later shown about NSF ?
WHat did that tell us about the interaction between VAMP and syntaxin?
NSF (ATPase) is not required for vesicle fusion per se as
originally proposed –> required to dissociate trans and cis SNARE complexes and thereby regenerate free SNAREs for the next round of vesicle docking and fusion
The interaction between vAMP and Syntaxin was sufficient to allow SNAREs to come together
SNARE structure is highly conserved - why is knowing the structure useful?
- gives insight into how they work
- Common motifs (usually very important to function) are highly conserved — allow us to rationally design mutants when investigating SNARE function at a molecular level.
