Lecture 10 - Membrane Fusion

Question 1

Why does membrane fusion require SNARE proteins?

Answer 1

1. The fusion of two membrane requires the displacement of water from the hydrophilic surface.
2. Phosphate heads are negative = repel each other. SNAREs can overcome these repulsive forces.

Question 2

Define the term ‘membrane fusion’.

Answer 2

Membrane fusion is the process whereby two separate lipid bilayers merge to become one.

Question 3

Membranes must be in close proximity for fusion to occur. Number?

Answer 3

1.5nm.

Question 4

Brief description of membrane fusion.

Answer 4

Vesicle membrane fuses and becomes part of the acceptor membrane. The vesicle contents mix with the lumenal contents of the accepting membrane.

Question 5

Brief description of budding.

Answer 5

Vesicle ‘buds’ from a donor membrane. Budding vesicle will contain part of the lumenal contents of the donor membrane.

Question 6

3 key steps of membrane fusion?

Answer 6

1. Tethering - linking the membranes, 25nm apart.
2. Docking/formation of a trans-SNARE complex.
3. Membrane fusion.

Question 7

Step 1: tethering. How are membranes loosely attached to each other?

Answer 7

Multi-subunit tethering complexes (MTCs). They consist of 6-10 proteins.

Question 8

MTCs differ for each type of membrane. Why?

Answer 8

Provides specificity.

Question 9

Main function of SNARE proteins?

Answer 9

To bring membranes in close proximity, allowing repulsion forces to be overcome, thus permitting membranes to fuse.

Question 10

Structure of a SNARE protein?

Answer 10

Most are TM type II - N terminus is in the cytoplasm when on a vesicle.

Question 11

For membrane fusion to occur we need a SNARE complex. Describe this structure.

Answer 11

4 alpha helical domains that bind to each other. Each SNARE protein has a SNARE domain that forms a helical structure when associated with others.

Therefore, most SNARE complexes consist of 4 SNARE proteins = one SNARE domain per protein.

Question 12

Some SNARE proteins have 2 SNARE domains, and so only 2 are required to form the complex. Give an example.

Answer 12

SNAP-25.

Question 13

How do these SNARE domains come together?

Answer 13

Should be noted that generally one helix comes from one membrane; the other three from the other membrane.

The N terminus of each SNARE domain coil around each other, pulling vesicles together (1.5nm). This is the ZIPPERING OF SNAREs and overcomes repulsive forces.

Question 14

Trans-SNARE complex?

Answer 14

v-SNARE (vesicle) and t-SNARE (target membrane).

Question 15

Key features of a SNARE complex?

Answer 15

1. Within the bundle hydrophobic AAs point into the central core.
2. 3 helices provide glutamine (Q); 1 arginine (R).
3. There will always be 3 Q SNAREs and 1 R SNARE.
4. Q SNAREs are further classified = Qa = syntaxin, Qb = SNAP-N, Qc = SNAP-P.
5. Complex is v stable due to H bonds and salt bridges.

Question 16

Q-SNAREs and R-SNAREs provide specificity to fusion events. Why?

Answer 16

A Qa, Qb, Qc and R are all required for fusion to occur.

Question 17

An experiment was performed that confirmed that SNAREs are the minimal machinery required for membrane fusion. Explain this experiment.

Answer 17

Flipped V SNAREs and T SNAREs so that they were Type I. Using fluorescent proteins found they could get the cells to fuse with each other since the domains are on the outside. Nothing else required.

Question 18

SNARE proteins can be recycled by breaking the SNARE complex. What is required to perform this?

Answer 18

Alpha-SNAP and NSF (note: alpha-SNAP is not a SNARE protein).

Question 19

What does NSF stand for?

Answer 19

NEM Sensitive Factor. NEM = N-ethyl Maleimide.

Question 20

What does alpha SNAP stand for?

Answer 20

Soluble NSF Attachment Protein.

Question 21

How does NSF and alpha SNAP function?

Answer 21

NSF is a hexamer that uses ATP to break the complex (ATPase). Alpha-SNAP is required for NSF binding, and activates the ATPase activity of the NSF.

Question 22

Sec1/Munc18 (SM) proteins regulate SNARE complex assembly. How?

Answer 22

The binding of an SM protein controls whether the SNARE complex is in an open or closed conformation. When bound = closed = unable to form complex.

Question 23

Rab proteins prime membranes for fusion. How?

Answer 23

Rab proteins reversibly bind GTP and GDP using GEF and GAP. GTP-Rab will activate effector proteins.

Question 24

SUMMARY: all membrane fusion events have?

Answer 24

1. A 4 helix SNARE complex.
2. SM protein.
3. Rab protein + effector proteins.