Lecture 10 - Membrane fusion Flashcards
What are the stages of membrane fusion?
- Tethering - linking membranes >25nm membrane
- Docking - the formation of a trans-SNARE (SNAP receptor) complex
- Membrane fusion - which has its own substeps:
- Close apposition - the membranes need to be brought very close to each other - closer than 1.5nm
- Hemifusion - outer head groups joined
- Full fusion - both sets of head groups joined
What are tethering factors?
Multi-subunit tethering complexes (MTCs) provide a ‘protein bridge’ between two membranes i.e. they ‘tether’ one membrane to another. They bind a variety of proteins on opposing membranes.
Consist of 6-10 large proteins, provide that bridge
Different tethering complex for each type of membrane - provide some specificity
What are the key features of SNARE proteins?
SNARE proteins bring membranes into close proximity, which allows repulsion forces between membranes to be overcome, thereby allowing membranes to ‘fuse’
Nomenclature of SNARE proteins: A variety of names such as Syntaxin 8, Syntaxin 7, VAMP2, SNAP-25 etc.
Structure: Most are transmembrane and contribute one helical structure to a SNARE complex. Proteins such as SNAP-[no.] contribute two helical structures and are palmitoylated
A SNARE complex consists of 4 helical coils, so either 4 individual proteins or a SNAP proteins such as SNAP-25 and two other proteins. The SNARE proteins are unstructured until they form a complex.
Type 2 transmembrane protein has its N-terminus in the cytoplasm, type 1 has C-terminus in the cytoplasm. Most SNAREs are type 2. A few are palmitoylated and have a lipid anchor.
SNARE proteins of the plasma membrane and those in the vesicle are both type 2 - interact with each other and form complex.
For membrane fusion you need a SNARE complex
SNAP proteins have 2 SNARE domains, tend to be palmitoylated
Describe the structure of a SNARE complex
4 helices per complex (generally one helix from one membrane and three from the other)
‘zippering’ of the SNAREs overcomes the electrostatic repulsion of the phospholipids
Trans SNARE complex - SNAREs from two different membranes coming together
Generally 3 SNAREs on one membrane 1 on the other
SNARE domain at the N terminus of each SNARE - when they come together they start making that alpha helical coil, forming a trans SNARE complex. As they start coiling round each other the vesicles are brought in close apposition to 1.5nm overcoming the electrostatic forces, allowing those vesicles to fuse. As the SNARE proteins coil up and form a trans SNARE complex they physically bring the two different membranes to which they belong closer together
Describe how SNARE proteins are reused
Once the SNARE complex has formed – you have an extremely stable structure and if you want to use the SNARE proteins again you need to break them apart!
To break the SNARE complex apart you use alpha-SNAP and NSF
N.B. alpha-SNAP is not a SNARE protein (it’s just bad terminology)
SNAP-[no.] is a SNARE protein.
NSF and a-SNAP
NSF – N-ethyl maleimide Sensitive Factor
-hexamer
-ATPase (uses 6 molecules of ATP to break open the SNARE complex)
-sensitive to NEM (N-ethyl maleimide)
aSNAP – Soluble NSF Attachment Protein
-required for NSF binding to membranes (a-SNAP is the protein that binds the SNAREs and then binds to the NSF which uses the energy to break open the SNAREs)
-binds and activates NSF ATPase activity
NSF and aSNAP form a complex and with the hydrolysis of ATP break open the SNARE complex allowing the SNARE proteins to be re-used
SNARE - soluble NSF attachment protein receptors
Soluble NSF attachment protein is a-SNAP
After membrane fusion - a-SNAP and NSF come in and bind to the SNARE proteins, hydrolyse ATP and make all the SNARE proteins individual again. They then use these proteins in subsequent rounds of fusion
How do SM proteins regulate SNARE complex formation?
the syntaxin when bound to SM protein n-Sec-1 is in a closed conformation ie the SNARE domain would not be available to form a SNARE complex.
ie SM proteins can ‘clamp’ SNARE proteins in a non-fusogenic conformation
SNAREs are the minimum needed for fusion but it can be regulated using SM proteins and Rab proteins
What is the role of Rab proteins?
Rab Proteins Prime Membranes for Fusion
Rab proteins
a family of 60 genes in humans
‘license’ membrane fusion events (don’t really know how)
interact reversibly with membranes and effectors in a GTP dependent manner
Rab proteins are active bound to GTP, inactive bound to GDP. Use GEF and GAP for Rab cycle
Active Rab activates effector proteins that can then allow membrane fusion to proceed
