SNAREs and membrane fusion

Question 1

Examples of membrane fusion in the body

Answer 1

synaptic vesicle fusion
secretory granule fusion
secretion of serum proteins
mucus secretion
intracellular transport

Question 2

How can secretory vesicles be visualised?

Answer 2

electron microscopy

Question 3

What are the 3 main approaches to identify machinery of vesicle transport?

Answer 3

biochemical reconstitution
yeast genetics
cloning

Question 4

What is Rothman’s SNARE hypothesis?

Answer 4

SNAREs for each transport step within the cell
they should provide specificity to vesicle transport
they should be sufficient to drive lipid bilayer fusion
proposed that NSF and ATP hydrolysis catalyses membrane fusion- this was wrong

Question 5

How many SNAREs are encoded in the human genome?

Answer 5

38

Question 6

SNARE zipping mechanism

Answer 6

VAMP from vesicle membrane and syntaxin zipper up in a parallel fashion
coils the alpha helices together which disrupts lipid bilayer conformation

Question 7

How is energy provided for the zipping of SNAREs?

Answer 7

fusing membranes is an energetically unfavourable so the coiling of domains provides this energy
ATP is needed to take SNARE complexes apart once zipped

Question 8

What are the two types of SNAREs?

Answer 8

R- VAMP molecules
Q- syntaxin and all target SNAREs

Question 9

Bioichemical reconsitution

Answer 9

Intra-golgi transport assay- following VSV-G protein get glycosylated
ER and golgi used to follow transport of different sugars
Cho functionally haploid

Question 10

NSF

Answer 10

N-ethylmaleimide is an ATPase
cycles on and off membranes in an ATP dependent manner

Question 11

Clostridial neurotoxins

Answer 11

tetanus and botulinum B
cleave VAMP, causing paralysis or lockjaw due to being an important component of membrane machinery

Question 12

How were SNAREs biochemically purified?

Answer 12

moved from cho cells to brain
used recombinant snap and NSF
found they could purify a large complex that dissembles when ATP is hydrolyzed

Question 13

What is the ratio of Q:R SNAREs?

Answer 13

3:1
conserved in all complexes and seems to be important

Question 14

Common features of SNAREs

Answer 14

generally small from 14-10kDa
all have a least 1 coiled coil alpha helix or SNARE motif
generally C-terminally coded
lots of additional regulatory domains

Question 15

TIRF microscopy

Answer 15

another type of biochemical reconstitution that allows looking at things very close to the surface of membranes
was used to see if SNAREs could drive membrane fusion completely independently

Question 16

What ion drives the fusion of membranes?

Answer 16

Calcium

Question 17

SNARE recycling

Answer 17

NSF recycles the SNAREs after fusion but isnt required for fusion step
have to be recycled or VAMP and SNARE will be on same membrane
NSF acts on cis-SNARE complex to unwind them to be recycled

Question 18

What disease is caused by VAMP2 mutations?

Answer 18

neurodevelopmental disorder with hypotonia and autistic features with or without hyperkinetic movements

Question 19

What disease is caused by SNAP25b mutations?

Answer 19

neurodevelopmental disorder with seizures, intellectual disability, severe speech delay and cerebellar ataxia

Question 20

What diseases are caused by mutations in SNAP29?

Answer 20

cerebral dysgenesis
neuropathy
ichthyosis
palmoplantar keratoderma syndrome

Question 21

What disease is caused by mutations in syntaxin 11?

Answer 21

familial hemophagocytic lymphohistocytosis type 4
rare disease of immune system, predominantly infants
overproliferation of T cells, killer cells, B cells + macrophages
can die from infection due to defective killing by T cells

Question 22

How does STX11 mutation cause FHL4?

Answer 22

reduced levels of STX11
defective degranulation from cytotoxic T cells by an unclear mehanism

Question 23

How do clostridial neurotoxins effect the nervous system?

Answer 23

can cause muscle spasms that can fracture your own bones
make entire body go floppy and lose all feeling

Question 24

Clostridial toxin mechanism

Answer 24

zinc dependetn proteases that cleave SNAREs
part of them allows access to the neuron and have a translocation domain to get into the endosome
very specific as will only cleave one protein

Question 25

What are the differences between botulinum and tetanus?

Answer 25

noth work the same way but have different targets
botulinum- targets excitatory neurons and has many cosmetic/clinical uses
tetanus- targets inhibitory neurons, no uses and most are vaccinated against it