Lecture 10 - SNAREs 1: Membrane fusion machinery Flashcards
Describe how membrane fusion occurs all the time
- Synaptic vesicle fusion (communication between neurons and muscles
- Secretory granule fusion (endocrine & exocrine pancreas)
- Secretion of serum protein (albumin from hepatocytes and antibodies from plasma cells)
- Mucus secretion (epithelial mucosal cells
- Intracellular transport of proteins between organelles in all of your cells
What can secretory vesicles be visualized by?
electron microscopy - first developed in 1938
What 3 ways allow you to investigate the machinery of vesicle transport?
- Biochemical reconstitution
- Yeast Genetics
- Cloning
What can you use as an assay for biochemical reconstitution to determine the machinery of vesicle transport?
Intra-Golgi transport
Describe the identification of NSF (N-ethylmaleimide Sensitive Factor)
- N-ethylmaleimide (NEM) - alkylating agent that inhibits the activity of NSF, which is an ATPase involved in the disassembly of SNARE complexes during vesicle fusion.
- When membranes are salt washed NSF can no longer bind to membranes. Target purified and named SNAP (soluble NSF attachment protein)
- SNAPs bind to the membranes via hypothetical proteins called SNAP Receptors (SNAREs)
What does SEC17 encode?
a-SNAP
What does SEC18 encode?
NSF
Explain how machinery is conserved between yeast and human
- same machinery used
- Yeast Sec18 works in Golgi transport assay
- Used Sec17 to confirm a-SNAP required for transport
- When secretion blocked, cells changed density, mutants separated on density gradient based on floor polish (23 complementation groups).
Explain the process of cloning synaptic vesicle proteins
Antibodies were raised against synaptic vesicles purified from electric rays. The antibodies were then used to expression clone VAMP and Synataxin.
What cleaves VAMP/(synaptobrevin)?
Clostridial neurotoxins tetanus and botulinum B cleaves VAMP/(synaptobrevin)
- Botulism causes paralysis. Looking for target. Checked synaptic vesicles. Zinc protease. Tetanus toxin cause lockjaw cleave snare machinery
- VAMP a good candidate for being a component of membrane machinery
Describe the biochemical purification of SNAREs
Found they could purify a large complex that dissembles when ATP is hydrolyses
What is Rothman’s SNARE hypothesis?
1) SNAREs for each transport step within the cell
2) SNAREs should provide specificity to vesicle transport
3) SNAREs should be sufficient to drive lipid bilayer fusion
4) Proposed that NSF and ATP hydrolysis catalyses membrane fusion (this is wrong)
Are there SNAREs for every transport step?
38 SNAREs encoded in the human genome. Involved in various transport steps and fusion reactions. Few more SNAREs being identified but most found.
Describe the crystal structure of the neuronal SNARE complex
SNAREs zipper in a parallel coiled coil
VAMP + SNAP25 + Syntaxin
What is thought to provide energy to drive membrane fusion?
SNARE zippering is thought to provide energy to drive membrane fusion.
- bring 2 membranes together and fusing them is energetically unfavourable
What can SNAREs be divided into?
Q & R SNAREs
Describe the distribution of Q:R SNAREs
3 Qs to 1 R SNARE
- 3Q:1R ratio conserved in al complexes
Mutation of a Q/R SNARE inhibits SNARE activity
Do SNAREs provide the specificity of membrane fusion?
- Only get fusion with SNARE complexes which fit 3Q:1R ratio.
- SNAREs show some promiscuity but, on the whole, they predominantly interact with SNAREs from the appropriate membranes.
- Lots of additional machinery contribute to specificity (i.e. rabs/coat proteins and tethers)
Describe common features of SNARE proteins
- generally small 14-40 kDa
- all have at least 1 coiled-coil or SNARE motif
- generally C-terminally anchored
Explain how Recombinant SNAREs can drive membrane fusion of purified liposomes
Calcium increased fusion rate about 30-50 fold. However, removal of SNAREs or cleavage of SNAREs with toxins resulted in no fusion when Ca added.
What does NSF do?
recycles the SNAREs after fusion
- NSF not required for the fusion step
- The structure of the SNARE/NSF complex has been solved
Summarise SNAREs
1) SNAREs are required for all fusion steps in the endocytic/biosynthetic pathways
2) SNAREs are generally small C-terminal anchored proteins
3) SNAREs zipper up via their coiled-coil domains
4) SNAREs provide some of the specificity for membrane fusion
5) SNAREs can be classified into Q & R SNAREs
6) Most SNARE complexes contain a ratio of 1Q:1R SNAREs
7) Recombinant SNAREs (synthetically produced SNAREs)can fuse membranes
8) NSF acts to recycle SNAREs after fusion
What does SNARE stand for?
Soluble N-ethylmaleimide-sensitive Factor Attachment Receptor
