Sept 17 - Mechanisms of Vesicular Trafficking lecture

Question 1

v-snare

Answer 1

on a vesicle membrane - VAMP in neurosecretory vesicles

Question 2

t-snare

Answer 2

on a target membrane (syntaxin, snap-25 on a neuronal plasma membrane

Question 3

Docking

Answer 3

interactions between v- and t- snares

Question 4

How snares work

Answer 4

Coiled coil interactions of four helices form stable complex-driven membranes together and destabilize bilayers. t-snare complex provides 3 helices, vsnare provides 4th

Question 5

NSF

Answer 5

Uses ATP hydrolysis to break otherwise stable t-snare/vsnare pairs.
This is required to disrupt pre-existing SNARE pairs on the target membrane. Frees up t-SNAREs to interact with new v-SNAREs
REquired in a priming step prior to docking and fusion.
This is required to disrupt pre-existing SNARE pairs on the target membrane.

Question 6

Rab proteins

Answer 6

Ras like small GTPases, attach to membrane via C-terminal prenyl group.
IN ADDITION TO GEFS AND GAPS, THERE ARE GDIS: Not used by Arf/Sar family but used by Rho and Rac. Binds to Rab GDP and sequesters it from membrane bound GEFS.

Question 7

GDI

Answer 7

Not used by Arf/Sar family but used by Rho and Rac. Binds to Rab GDP and sequesters it from membrane bound GEFS.

Question 8

SM proteins

Answer 8

Named for yeast Sec1 and worm Munc18 - coiled coil proteins, each interacts with specific t-SNARE on target membrane. Facilitates SNARE-mediated fusion after trans-SNARE assembly - enhances zippering of membrane proximal region of SNARE domains. Some SM proteins may also prevent tSNARE assembly until the right time/place.

Question 9

Tethering

Answer 9

Grabbing a vesicle by the target membrane. Initiated by GTP-bound rab proteins, which recruit soluble tethering proteins from cytosol. One end binds vesicle, the other binds target membrane. Each tether specific for a given vesicle.

Question 10

How is specificity of membrane fusion defined?

Answer 10

By a combination of SNAREs, Rabs, SM proteins, tethers, and other regulators.

Question 11

VAMP

Answer 11

Vesicle associated membrane protein - on synaptic vesicle

Question 12

Syntaxin

Answer 12

on pre-synaptic membrane

Question 13

SNAP-25

Answer 13

Synaptosome associated protein - associates with syntaxin

Question 14

What does NSF stand for

Answer 14

NEM sensitive factor.

Question 15

What does SNAP stand for

Answer 15

Soluble NSF attachment protein

Question 16

NEM

Answer 16

treatment with NEM caused:
- accumulated 70nm vesicles
• vesicles are uncoated
• many vesicles are locked onto larger membranes

Question 17

Epistasis

Answer 17

when the effect of one gene depends on the presence of one or more ‘modifier genes’ (genetic background).

Question 18

What happens when you add GTP gamma S to golgis? How does this differ from NEM treatment?

Answer 18

Coats get locked on due to blockage of Arf. GTP hydrolysis is required to remove the coats.
For NEM treatment, there was accumulation of vesicles, which were uncoated, many of them locked onto larger membranes.

Question 19

Sec mutants

Answer 19

Secretion is ok at permissive temperature (25 degrees)

Secretion is defective at non-permissive temperature (37 degrees)

Question 20

Invertase

Answer 20

The protein they chose to study which is exocytosed in yeast.

Question 21

Genetic epistasis

Answer 21

Combining mutants - it will tell you in which order things happen!

Question 22

Some way to identify defective genes

Answer 22

1. complementation by transfection with a genomic library - select for clones that grow at restrictive temperature (so like a rescue, sorta)
2. Combining mutants - genetic epistaxis