Lecture 18- SNAREs I

Question 1

Give some examples of when membrane fusion occurs?

Answer 1

1. Synaptic vesicles fusion
2. Secretory granule fusion
3. Secretion of serum proteins
4. Mucus secretion
5. Intracellular transport of proteins between organelles

Question 2

What are the 3 main approaches used to identify vesicle machinery?

Answer 2

1. Biochemical reconstitution
2. Yeast genetics
3. Cloning

Question 3

What was the aim of the intra Golgi transport assay (biochemical reconstitution)?

Answer 3

To study transport of proteins between stacks of the Golgi

Question 4

What did the intra-Golgi transport assay allow the identification of?

Answer 4

NSF

Question 5

What type of protein is NSF and how can this be confirmed?

Answer 5

NSF is an ATPase which can be confirmed by adding anti-NSF which prevents the binding of NSF to membranes

Question 6

What do Sec17 and Sec18 encode?

Answer 6

Sec17 encodes alpha-SNAP

Sec18 encodes NSF

Question 7

Which yeast genes were identified in yeast genetics that could cause defects in membrane trafficking and what do they encode?

Answer 7

• Sec1 (SNARE binding protein)
• Sec17- encodes alpha-SNAP
• Sec18- encodes NSF

Question 8

What did the isolation of sec mutants (NSF and alpha-SNAP) in yeast genetics provide evidence for?

Answer 8

The importance of NSF in intracellular protein trafficking

Question 9

How does SNAP and NSF interact?

Answer 9

SNAPs bind to SNAREs and recruit NSF (an ATPase).

NSF provides the energy to untangle the SNAREs

Question 10

How were the synaptic vesicles (VAMP and syntaxin) cloned?

Answer 10

• Antibodies were raised against synaptic vesicles purified from electric rays
• The antibodies were then used to expression clone VAMP and syntaxin

Question 11

What was discovered when purified synaptic vesicles were incubated with tetanus toxin?

Answer 11

Discovered clostridial neurotoxins tetanus and botulinum B would cleave VAMP

Question 12

Outline the process of biochemical purification of SNAREs

Answer 12

• 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

Question 13

What is biochemical reconstitution?

Answer 13

Taking cellular processes, breaking them down to their fundamental components and reconstitute them

Question 14

What is Rothman’s SNARE hypothesis?

Answer 14

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 (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

Question 15

How many SNAREs have been identified?

Answer 15

38 SNAREs- involved in various transport steps and fusion reactions

Question 16

Are there SNAREs for every transport step and are they tissue specific?

Answer 16

There are SNAREs for each transport step and each step uses a different combination of SNAREs

SNAREs can be tissue specific

Question 17

Outline the crystal structure of the neuronal SNARE complex

Answer 17

• 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

Question 18

How does SNARE zippering occur?

Answer 18

1. Two SNAREs zipper together forming a tight trans-SNARE complex
2. Two bilayers merge in a process called hemifusion
3. Fusion-pore opens
4. Fusion-pore enlarges to form a cis-SNARE complex
5. SNARE zippering is thought to provide the energy to drive membrane fusion as it is energetically unfavourable

Question 19

Divide VAMP, syntaxin and SNAP25 into R and Q SNARES and explain why

Answer 19

• VAMP contains arginine so is an R SNARE

* Syntaxin and SNAP25 both contain glutamine so are Q SNAREs

Question 20

Explain how the 3Q:1R ratio is composed

Answer 20

• 4 coil domains make up a stable SNARE complex
• 1 x VAMP (R SNARE)
• 1 x syntaxin (Q SNARE)
• 2 x SNAP25 (Q SNARE)

Question 21

Outline some common features of SNARE proteins

Answer 21

• 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

Question 22

What types of fusion are SNAREs not required for?

Answer 22

Mitochondrial fusion

Question 23

What can recombinant SNAREs drive?

Answer 23

Drive membrane fusion of purified liposomes

Question 24

What is required for the SNAREs to open and close confirmation?

Answer 24

Requires calcium and regulation of the regulatory domain

Question 25

Which SNAREs are found on the cell membrane and which SNAREs is attached to the vesicle?

Answer 25

VAMP attached to vesicle

Syntaxin and SNAP25 are positioned on membrane