Lecture 18- SNAREs I Flashcards
Give some examples of when membrane fusion occurs?
- Synaptic vesicles fusion
- Secretory granule fusion
- Secretion of serum proteins
- Mucus secretion
- Intracellular transport of proteins between organelles
What are the 3 main approaches used to identify vesicle machinery?
- Biochemical reconstitution
- Yeast genetics
- Cloning
What was the aim of the intra Golgi transport assay (biochemical reconstitution)?
To study transport of proteins between stacks of the Golgi
What did the intra-Golgi transport assay allow the identification of?
NSF
What type of protein is NSF and how can this be confirmed?
NSF is an ATPase which can be confirmed by adding anti-NSF which prevents the binding of NSF to membranes
What do Sec17 and Sec18 encode?
Sec17 encodes alpha-SNAP
Sec18 encodes NSF
Which yeast genes were identified in yeast genetics that could cause defects in membrane trafficking and what do they encode?
- Sec1 (SNARE binding protein)
- Sec17- encodes alpha-SNAP
- Sec18- encodes NSF
What did the isolation of sec mutants (NSF and alpha-SNAP) in yeast genetics provide evidence for?
The importance of NSF in intracellular protein trafficking
How does SNAP and NSF interact?
SNAPs bind to SNAREs and recruit NSF (an ATPase).
NSF provides the energy to untangle the SNAREs
How were the synaptic vesicles (VAMP and syntaxin) cloned?
- Antibodies were raised against synaptic vesicles purified from electric rays
- The antibodies were then used to expression clone VAMP and syntaxin
What was discovered when purified synaptic vesicles were incubated with tetanus toxin?
Discovered clostridial neurotoxins tetanus and botulinum B would cleave VAMP
Outline the process of biochemical purification of SNAREs
- Used NSF to identify additional machinery involved in membrane fusion
- Incubated tagged NSF and cytosol and discovered NSF cleaved ATP
- Were then able to remove and identify gamma and alpha SNAP as these bound to the tagged NSF
- From the membrane extracts also got VAMP and syntaxin so could identify proteins they interact with
- Found they could purify a large complex that dissembles when ATP is hydrolysed
What is biochemical reconstitution?
Taking cellular processes, breaking them down to their fundamental components and reconstitute them
What is Rothman’s SNARE hypothesis?
- SNAREs for each transport step within the cell
- SNAREs should provide specificity to vesicle transport
- SNAREs should be sufficient to drive lipid bilayer fusion
- Proposed that NSF and ATP hydrolysis catalyses membrane fusion (INCORRECT)
• CORRECT: V-SNARE is on the vesicle and t-SNARE is on the target membrane and in the presence of NSF, this drives membrane fusion
How many SNAREs have been identified?
38 SNAREs- involved in various transport steps and fusion reactions
Are there SNAREs for every transport step and are they tissue specific?
There are SNAREs for each transport step and each step uses a different combination of SNAREs
SNAREs can be tissue specific
Outline the crystal structure of the neuronal SNARE complex
- SNAREs zipper in a parallel coiled coil
- 1 coil from VAMP and syntaxin
- 2 coils from SNAP25
- When they zipper together, they bring the vesicle closer to the target membrane, allowing lipid bilayer fusion to occur
How does SNARE zippering occur?
- Two SNAREs zipper together forming a tight trans-SNARE complex
- Two bilayers merge in a process called hemifusion
- Fusion-pore opens
- Fusion-pore enlarges to form a cis-SNARE complex
- SNARE zippering is thought to provide the energy to drive membrane fusion as it is energetically unfavourable
Divide VAMP, syntaxin and SNAP25 into R and Q SNARES and explain why
- VAMP contains arginine so is an R SNARE
* Syntaxin and SNAP25 both contain glutamine so are Q SNAREs
Explain how the 3Q:1R ratio is composed
- 4 coil domains make up a stable SNARE complex
- 1 x VAMP (R SNARE)
- 1 x syntaxin (Q SNARE)
- 2 x SNAP25 (Q SNARE)
Outline some common features of SNARE proteins
- Generally small (14-40kDa) as usually membrane associated
- All have at least 1 coiled coil or SNARE motif
- Generally C-terminally anchored
- R and Q SNAREs usually have TM domain
- Often have additional regulatory domains (N-terminal domains) which help regulate SNARE complex function
What types of fusion are SNAREs not required for?
Mitochondrial fusion
What can recombinant SNAREs drive?
Drive membrane fusion of purified liposomes
What is required for the SNAREs to open and close confirmation?
Requires calcium and regulation of the regulatory domain
