Lecture 7 - Mechanisms and experiments

Question 1

Draw the graph of incorporation of radioactive GlcNac vs. time with just cytosol added (i.e. no ATP), addition of the latter plus NEM and then addition to cytosol from ATP-treated extracts to NEM treated membranes. Also say what type of vesicles accumulate in each case, if this occurs.

Answer 1

For graph refer to notes.

• No ATP has lowest level. Accumulation of uncoated vesicles occurs.
• NEM is next lowest level. Also get accumulation of uncoated vesicles
• Addition of cytosol from ATP-treated extracts to NEM treated membranes leads to accumulation transport rescue.

Question 2

If NEM is only added to either the donor or acceptor membrane, what occurs?

Answer 2

Still get cycling on the other membrane.

Question 3

What were the steps in identification of NSF and purification of NSF?

Answer 3

1. Treated donor and acceptor membranes with NEM –> accumulation of uncoated vesicles
2. Rescue transport by adding cytosol from ATP treated exgtracts
3. Fractionate cytosol (velocity sedimentation with glycerol gradient then ion-exchange FPLC)
4. Run out on SDS-PAGE and excise band
5. Identified 76 kDa protein

Question 4

Describe SNAP and why its important for NSF and why the researchers did another experiment to find a SNAP receptor^

Answer 4

SNAP is a factor that acts at the same step as NSF and is necessary for NSF association to membranes. SNAP stays associated to membranes even when ATP is added but is released by 1M KCl –> peripheral membrane protein.
Researchers are looking for a putative integral membrane protein to link these.

Question 5

Describe the identification of a SNAP receptor.

Answer 5

1. Purify myc-tagged NSF from E. coli and couple to anti-myc agarose beads
2. Add purified SNAPs
3. Add detergent extracted membranes (theorize that these contain putative receptor)
4. Wash
5. Elute
6. Run out on SDS-PAGE
7. Excise band and sequence
8. Identify SNAREs, (these were already identified as synapse components and immunolocalization studies indicate that v-SNAREs associate with vesicles and t-SNAREs associate with target)

Question 6

What was the original SNARE hypothesis and why was it wrong?

Answer 6

v-SNARE and t-SNARE associate via NSF + SNAPs
The model was later modified when it was shown that NSF and SNAPs are not required for the SNARE interaction.
Later shown that NSF and SNAPs are required after fusion for separating SNAREs.

Question 7

What is the modified SNARE hypothesis pathway, including Rab tethering?

Answer 7

1. Specific Rabs are localized by GEFs, (Rab-GTP has hydrophobic region that inserts into membrane)
   - Rab effector is on target membrane
2. Rab-GTP on vesicle binds to Rab effector
3. Tethering brings SNAREs close together
4. v-SNAREs and t-SNAREs associate to mediate docking and fusion by wrapping around each other in a coil
5. Fusion occurs whereby vesicle contents spill into target compartment and vesicle membrane proteins are now in the target membrane and formation of the trans-SNARE complex
6. SNAPs bind to trans-SNARE complex and recruit NSF
7. NSF hydrolyzes ATP and mediates SNARE dissociation