Membrane fusion

Question 1

SNARE proteins

Answer 1

account for specificity of membrane fusion, form a tight complex that brings synaptic vesicle and PM together

Question 2

3 classes of SNARE

Answer 2

1. ) Vamp
2. ) Syntaxin
3. ) SNAP-25

Question 3

VAMP protein

Answer 3

Class of snare. Vesicle associated membrane protein.
Sits on synaptic vesicles.
1 C terminal domain and huge helical middle part (transmem).

Question 4

Syntaxin

Answer 4

Similar structure to VAMP, transmembrane part at end. 3 amphipathic helices.
Lives on plasma membrane.

Question 5

SNAP-25

Answer 5

2 amphipathic helices, NO transmembrane domain.

has cysteine AA residues in middle that covalently anchor via palmitoyl side chains to cytosolic mem. face.

Question 6

SNARE complex

Answer 6

4 alpha helical bundle (coiled coil). Secondary structure, bury uncharged residues inside. Can overcome repulsive forces to get fusion.
Syntaxin 1 alpha, VAMP 1 alpha, SNAP-25 2 alpha

zipper from N-termini to C-termini of SNARE complex.

Question 7

Recycling SNAREs

Answer 7

SNARE complexes are highly stable, so cell has NSF protein set and alpha-SNAP to disassemble SNARE with ATP hydrolysis.

Question 8

NSF

Answer 8

involved in disassembly/recycling of SNARE complex. Homologue of helicase.
Triple ATPase.
HExamer, every step hydrolyzes 6 NSF’s and it twists and binds to end of snares, as it twists it hydrolyzes ATPs and unwinds coiled coil
Uses adaptor protein alpha-snap.

Question 9

How many ATPs to disassemble a SNARE complex?

Answer 9

6 ATPs

Question 10

Alpha-SNAP

Answer 10

NOT related to SNAP-25. Adaptor protein.

Binds to SNARe and recruits NSF so can unwind SNARE complex.

Question 11

Sec1 protein disassembly

Answer 11

After SNARE complex disassembled it helps to refold them to active conformation.
It binds and changes syntaxin into active conformation.

Question 12

Sec1 protein assembly

Answer 12

Also regulates when SNARe complex forms
Nsec1 binds syntaxin but syntaxin forms complex with itself without Nsec1 bound, its inactive… Nsec1 binds this and stabilizes it until signal comes in and Nsec releases so can get SNARE complex

Nsec1 knockout mice have hyperfusion (don’t wait for signal)

Question 13

Types of viruses that use fusion

Answer 13

Enveloped viruses (HIV, influenza, ebola). Use similar machinery to SNARE.

Question 14

Virus fusion

Answer 14

have TM domain at one end (inserted into viral mem) and highly hydrophobic fusion domain (folded and hidden until signal cuases its exposure so it inserts into target cell mem and allows fusion).

Question 15

Viral fusion proteins

Answer 15

Fusogenic viral proteins (not SNARE), used coiled coil to drive it.
ex: gp41 of HIV.

Question 16

gp41 of HIV

Answer 16

example of viral fusogenic protein.
2 alpha amphipathic helices and TM domain on one side and fusogenic hydrophobic protein… as it floats inactive b/c these buried.
They make hairpin w/ epxosed peptide with anti-parallel coiled coil from alpha helices, peptide gets embedded in PM.
Same fusion principle, diff proteins.

Question 17

Influenza activation

Answer 17

Low pH activates fusogenic protein so it opens up and tries to form coiled coil so outside cell never active b/c pH too high. It binds and gets internalized (cell may be trying to destroy but it just gets activated in lysosome)

Question 18

HIV activation

Answer 18

fuses with PM. gp120 gp41. Sits on gp41 and hides it, binds to t cell receptor and after this it reveals gp41… activated by receptor binding.
PBD and PFD (pocket binding domain).

Question 19

HIV hiding

Answer 19

Hides in dendritic cells (CD4), normally dendritic cell takes up foreign, to lysosome, degraded, display for CD4. But HIV doesn’t fuse so it can’t get degraded (hides until you take drugs away).