SNAREs Flashcards
(32 cards)
What membrane fusion processes happen in our bodies?
Synapatic vesicle fusion
Secretory granule fusion
Secretion of serum proteins
Mucus secretion
What are the three approaches to identifying vesicle machinery?
Biochemical reconstitution
Yeast genetics
Cloning
How is biochemical reconstitution done to find SNAREs?
Cells are broken open and put back together, reconstituting vesicles
Inhibitors and fractionation are used to identify pathways and molecules being inhibited
How is glycosylation in CHO cells followed?
CHO cells missing glycosylation had radioactive sugars added.
Enzymes catalysing glycosylation bud off golgi of another cell. The vesicles can bud and fuse to the other golgi, leading to transfer of the radioactive sugar.
How does NSF stimulate vesicle fusion?
Binds to SNAP receptors catalysed by ATP
When membranes are salt washed, NSF no longer binds to membranes
What do SEC1, 17 and 18 encode?
1- SNARE binding protein
17- α-snap
18- NSF
What does tetanus toxin cleave in protein?
VAMP
Outline the SNARE hypothesis
- Involved in each transport step within the cell
- Should provide specificity for vesicle transport
- Should be sufficient to drive lipid bilayer fusion
- NSF and ATP hydrolysis catalyse vesicle fusion
What sort of structure do SNAREs form?
A zip in a coiled coil
What is syntaxin’s function
Drives proteins closer together and allows membrane fusion
What is in the zero layer of SNAREs
Hydrophilic electrostatic interaction between 3 Gly and 1 Arg. This 3Q:1R ratio is conserved
What is the different between Q and R SNAREs?
R- Arg at 0
Q- Syntaxin 1a at 0
Describe SNAREs
Generally 14-40kDa
At least 1 coiled-coil SNARE motif
Generally C-terminally anchored
Recombinant SNAREs can drive fusion of purified liposomes
What recycles SNAREs after fusion?
NSF
Outline the steps leading to budding
Nucleation
Zippering
Fusion-pore opening
20s complex formation with SNAPs and NSF
Budding
What make up the trans SNARE?
SNAP25 and syntaxin
Why is VAMP2 expressed in high levels in the endocrine pancreas?
Absorb nutrients
What happens when VAMP2 is knocked out?
Death at birth due to loss of synaptic transmission
What happens when SNAP25 is knocked out?
Death at birth due to loss of synaptic transmission
What disease is VAMP2 associated with?
Neurodevelopment disorder. Floppy baby syndrome (Hypertonia), autistic features and can have hyperkinetic movements
What disease is SNAP25b associated with?
Neurodevelopment disorder. Seizures, speech delay and cerebellar ataxia
What disease is SNAP29 associated with?
Cerebral dysgenesis, neuropathy, ichthyosis and palmoplantar keratoderma syndrome (CEDNIK syndrome)
What disease is syntaxin 11 associated with?
Familial hemophagocytic lymphohystiocytosis
What does the S75P mutation in VAMP2 do and why?
Slows rate of liposome fusion as proline disrupts coiled coils
This was measured with a fluorescent dye on a synthetic membrane
