Membrane Fusion Flashcards
what is the concept behind membrane fusion?
- cells are compartmentalized but need movement
- requires membrane fusion
- displacement of water from the hydrophillic surface of membrane is energetically unfavourable
- requires SNARE protein
why do membranes repel each other?
phosphates have a negative charge so phospholipids repel each other
what is membrane fusion?
the process where two separate lipid bilayers merge to become one
- need to be brought together closer than 1.5nm
what do membranes contains?
lipids and proteins
describe the vesicle budding
- contains part of the lumenal contents of the donor membrane
- contains membrane proteins and lipids from donor membrane
how does the vesicle fuse?
- contents mixes with the lumen of the accepting membrane or is released extracellularly
- proteins and lipids become part of the accepting membrane
what are the 3 steps of fusion?
- tethering - bridge between two membranes
- docking/the formation of a trans-SNARE complex
- fusion
what are the different stages of fusion?
- fusion initiation
- hemi fusion
- fusion pore opening
what are MTCs?
- multi subunit tethering complexes that provide a protein bridge
- tether one membrane to another
- bind a verity proteins on opposing membranes, this provides some specifity
what i the role of SNARE proteins?
- proteins that bring the membranes into close proximity
- allows membranes to overcome the repulsive forces
what is the structure of SNARE proteins?
- most are transmembrane
- contain a SNARE domain
- when it comes into contact another SNARE protein they form an alpha helical coil
what is a SNARE complex?
- a SNARE complex consists of 4 helical coils
- either 4 individual or a SNAP proteins and 2 others
- SNARE proteins are unstructured until they form a SNARE complex
- 4 helices are usually one helix from one membrane and 3 from another
- one of the strongest complexes
what is zippering?
zippering of the SNAREs overcomes the electrostatic repulsion of the phospholipids
what is post-tethering?
post tethering the SNAREs on opposite membranes starts to interact and then coil up forming the SNARE commplexing
- if you coil up a protein it becomes shorter, bringin the membranes into close proximity and overcoming the membrane repulsive forces
what is the centre of the SNARE complex?
- 4 amino acids, one from each helix pointing inwards
- either glutamine (Q) or arginine
- always 3Q’s and 1R
- Q SNAREs, Qa, Qb, Qc
- lots of hydrogen bonding and salt bridges
- hydrophobic central core
how is the SNARE complex specific?
- you need the right combinations of SNARE proteins for membrane fusion
- Qa,Qb,Qc and R SNARE are required for every membrane fusion event
how are SNAREs the minimal machinery for membrane fusion?
- experiment show that if you can make the SNAREs type I membrane proteins can get cells to fuse with each other
describe the process of SNARE proteins fusing (the experiment)
- SNARE domains need to be in the cytoplasm for membrane fusion
- N terminus in cytoplasm and C terminus in the lumen are type II membrane proteins
- converted them into type II membrane proteins
- flipped v SNARE (made the cytoplasm red)
- flipped t SNARE (made the nucleus blue)
- cells then fused - had multinucleated cells
why is the SNARE complex good for fusion?
once formed it is extremely stable
how are you abale to resuse the SNARE proteins/
- need to break the stable complex
- with a ALPHA-SNAP and NSF
what is NSF?
- hexamer
- ATPase, hydrolyses ATP
- Sensitive to NEM
what is alpha SNAP?
- required for NSF bidning to membranes
- binds and activates NSF ATPase activity
how do NSF and alpha SNAP work?
form a complex and with hydrolysis of ATP break open the SNARE complex
- SNARES bind to SNAP, they are SNAP receptors
what happens if you inhibit NSF?
you would still get one round of fusion
how are SNAREs regulated?
- sec1/Munc18 (SM proteins) and can regulate SNARE assembly
- when bound to sec1 it is in a closed conformation the SNARE protein would not be available to form a SNARE complex
- can clamp SNARE proteins
what are Rab proteins?
- prime membranes for fusion
- not fully understood
- family of 60 genes in humans
- interact reversibly with membranes and effectors in a GTP dependent manner
what is bound to inactive Rab proteins?
GDP
what is bound to active Rab proteins?
GTP
what do Rab proteins do?
it has low intrinsic GTPase activity, need GAP proteins (activating proteins)
what do you need to exchange GDP to GTP?
GEF proteins - exchange factor
what do Ras do?
makes membranes permissive
what do all fusion events have?
4 helix SNARE complex, an SM protein and a Rab protein
