Lecture 19 Snares 1 Flashcards
When is membrane fusion occurring
In our bodies all the time
Examples of membrane fusion
- Synaptic vesicles fusion - communication between neurons and muscles at NMJ
- Secretory granule fusion - endocrine and exocrine pancreas e.g. insulin
- Secretion of serum proteins - albumin from hepatocytes and antibodies from plasma cells
- Mucus secretion - epithelial mucosal cells
- Intracellular transport of proteins between organelles in all of your cells
How can secretory vesicles be visualised
Electron microscopy (1938)
What does EM not tell you
The molecular machinery involved with vesicle fusion
Describe synaptic vesicles
50-100nm diameter
Fast fusion
ms
Describe zymogen granules
500nm diameter
slower fusions
second
3 approaches to identify machinery of vesicle transport
- biochemical reconstitution
- yeast genetics
- cloning
Give an example of biochemical reconstitution
Intra-Golgi transport assay
Describe Intra-Golgi transport assay
- break up…
- donor…
- marker…
- cell lines…
- mixed…
- vesicles budding off…
- gives quantification of…
- Break up cells to access the Golgi - add cytoplasm and ATP
- Donor Golgi-containing fraction from VSV-infected mutant - incorporate radioactive sugar (glucose) onto protein. This is a viral glycoprotein that normally traffics from ER to Golgi to PM so can use as marker to follow trafficking of membrane
- 1 cell line does not have the enzyme involved in glycosylation steps and other cell line contains the enzyme
- Mixed mutant cells and normal cells (acceptor Golgi-containing fraction from uninfected WT cells) with WT enzyme
- If get vesicles budding off, get incorporation of radioactive sugar onto VSV so can measure transfer of material between compartments
- Gives quantification of measure of IC trafficking
What was biochemical reconstitution used to identify
COPI vesicles
What was biochemical reconstitution used to identify (not COPI)
NSF
What happens in the assay as you increase levels of Golgi membrane
Increased amounts of signal
What inhibits the reaction of Intra-Golgi transport assay
• N-ethylmaleimide inhibits reaction (alkalyting reagent that reacts with free cysteine)
What target was purified from NSF inhibiting the reaction
N-ethylmaleimide Sensitive Factor (NSF)
What is NSF
ATPase
What occurs when Golgi membranes are salt washed
NSF can no longer bind to the membranes
Target purified from salt washing
SNAP
soluble NSF attachment protein
What were yeast genetics used to isolate and how
Made temp sensitive mutants which change their density when inhibit secretion.
They isolated sec mutants:
• Sec 1 – SNARE binding protein
• Sec 17 - encodes α-SNAP
• Sec18 – encodes NSF
Cloning of synaptic vesicle proteins identified and how
VAMP and Syntaxin
• Antibodies were raised against synaptic vesicles purified from electric rays (which have large axons)
• The antibodies were then used to expression clone VAMP and Syntaxin
What do Clostridial neurotoxins tetanus and botulinum B cleave
Vamp/ synaptobrevin
How did they know these toxins cleaved Vamp
Bands disappeared in SDS-PAGE gel.
Symptoms of tetanus/botulinum
- Tetanus to locked jaw
* Botulinum to floppy baby syndrome
What did they find in the biochemical purification of SNAREs
Found could purify a large complex that dissembles when ATP is hydrolysed
How does biochemical purification occur
- tagged..
- bind…
- bound…
- add…
- get…
- complex…
- Tagged NSF and made it recombinantly
- Bind NSF onto beads (believed this was an ATPase from immune affinity experiment)
- Bound to α/γ SNAP complex
- Add ATP that could be hydrolysed
- Got bands that could be sequenced
- Got syntaxin, VAMP and SNAP 25 complex
Describe the complex formed in biochemical purification
- VAMP on vesicle (T-SNARE)
- Syntaxin and SNAP25 on target membrane (V-SNARE)
- These interact to form a large complex that can be purified
- Hydrolysis of ATP fuses these things together
Rothman’s SNARE hypothesis
1) SNAREs for each transport step within the cell regardless of type of vesicle i.e. 38 SNAREs encoded in the human genome
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 bit is actually wrong!)
What do the VAMP and synataxin/SNAP 25 form
coiled-coil
How do snares form this coiled coil
zipper in a parallel coiled coil and in doing so they pull the vesicles close enough to provide the energy for the fusion of lipid bilayers
SNARE zipper provides…
energy to drive membrane fusion
What isn’t required for membrane fusion but is for recycling of snares
NSF
Trans-SNARE:
Trans-SNARE: When the SNAREs are coiled before fusion
Cis-SNARE:
Cis-SNARE: When the vesicles have fused and the SNAREs uncoil
Types of SNAREs
- Q type - Syntaxin-like/SNAP 25 (glutamine)
* R type - VAMP-like (arginine)
Ratio of types of snares
3Q:1R SNARE conserved in all complexes
Do SNAREs provide the specificity of membrane fusion?
- Only get fusion with SNARE complexes which fit 3Q:1R ratio (Mutation of a Q/R inhibits SNARE activity and fusion doesn’t work as well)
- SNAREs show some promiscuity but on the whole they predominantly interact with SNAREs from the appropriate membranes.
- Lots of additional machinery contribute to specificty (i.e. rabs/ coat proteins and tethers)
In Rothman’s SNARE hypothesis, he suggested SNAREs should provide specificity to vesicle transport. Was this correct?
PARTIALLY YES - SNAREs do provide specificity - but there are other things that also do
Common features of SNARE proteins
- Generally small 14-40kDa
- All have at least 1 coiled-coil or SNARE motif (except SNAP 25 have 2)
- Generally C-terminally anchored
How to see if the SNAREs the minimal fusion machinery?
- Synthetic vesicle, liposome, with VAMP on it
- Put dye onto vesicle
- Mix together and see if get fusion and is this sped up etc by adding other thing
- TIRF microscopy
Recombinant SNAREs can drive membrane fusion of…
purified liposomes
What happens in liposomes if add snare machinery on own
some fusion, but not much
What occurs if add calcium to liposomes
increases rate of vesicle fusion
What does syntaxin do
Regulates complex and inhibits ability of fusion - opens and closes the complex
Was Rothman correct in stating SNAREs were the minimum machinery for membrane fusion
yes - but other things like calcium assist
Role of NSF
snare recycling after fusion
Is NSF required for fusion
no
which complex does NSF act on
cis-SNARE complex
shape of nsf
ring
How does NSF work
- NSF uses ATP activity to unwind SNARE molecules i.e. unwind VAMP/Syntaxin coiled-coil after fusion
- The complex is unscrewed to separate the SNAREs
In Rothman’s SNARE hypothesis, he suggested NSF + ATP hydrolysis catalyses membrane fusion. Was this correct?
No – NSF recycles the SNAREs after fusion
