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Cell and molecular extras > Lecture 19 Snares 1 > Flashcards

Lecture 19 Snares 1 Flashcards

1
Q

When is membrane fusion occurring

A

In our bodies all the time

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2
Q

Examples of membrane fusion

A
  • Synaptic vesicles fusion - communication between neurons and muscles at NMJ
  • Secretory granule fusion - endocrine and exocrine pancreas e.g. insulin
  • Secretion of serum proteins - albumin from hepatocytes and antibodies from plasma cells
  • Mucus secretion - epithelial mucosal cells
  • Intracellular transport of proteins between organelles in all of your cells
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3
Q

How can secretory vesicles be visualised

A

Electron microscopy (1938)

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4
Q

What does EM not tell you

A

The molecular machinery involved with vesicle fusion

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5
Q

Describe synaptic vesicles

A

50-100nm diameter
Fast fusion
ms

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6
Q

Describe zymogen granules

A

500nm diameter
slower fusions
second

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7
Q

3 approaches to identify machinery of vesicle transport

A
  1. biochemical reconstitution
  2. yeast genetics
  3. cloning
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8
Q

Give an example of biochemical reconstitution

A

Intra-Golgi transport assay

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9
Q

Describe Intra-Golgi transport assay

  • break up…
  • donor…
  • marker…
  • cell lines…
  • mixed…
  • vesicles budding off…
  • gives quantification of…
A
  • Break up cells to access the Golgi - add cytoplasm and ATP
  • Donor Golgi-containing fraction from VSV-infected mutant - incorporate radioactive sugar (glucose) onto protein. This is a viral glycoprotein that normally traffics from ER to Golgi to PM so can use as marker to follow trafficking of membrane
  • 1 cell line does not have the enzyme involved in glycosylation steps and other cell line contains the enzyme
  • Mixed mutant cells and normal cells (acceptor Golgi-containing fraction from uninfected WT cells) with WT enzyme
  • If get vesicles budding off, get incorporation of radioactive sugar onto VSV so can measure transfer of material between compartments
  • Gives quantification of measure of IC trafficking
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10
Q

What was biochemical reconstitution used to identify

A

COPI vesicles

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11
Q

What was biochemical reconstitution used to identify (not COPI)

A

NSF

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12
Q

What happens in the assay as you increase levels of Golgi membrane

A

Increased amounts of signal

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13
Q

What inhibits the reaction of Intra-Golgi transport assay

A

• N-ethylmaleimide inhibits reaction (alkalyting reagent that reacts with free cysteine)

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14
Q

What target was purified from NSF inhibiting the reaction

A

N-ethylmaleimide Sensitive Factor (NSF)

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15
Q

What is NSF

A

ATPase

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16
Q

What occurs when Golgi membranes are salt washed

A

NSF can no longer bind to the membranes

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17
Q

Target purified from salt washing

A

SNAP

soluble NSF attachment protein

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18
Q

What were yeast genetics used to isolate and how

A

Made temp sensitive mutants which change their density when inhibit secretion.

They isolated sec mutants:
• Sec 1 – SNARE binding protein
• Sec 17 - encodes α-SNAP
• Sec18 – encodes NSF

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19
Q

Cloning of synaptic vesicle proteins identified and how

A

VAMP and Syntaxin
• Antibodies were raised against synaptic vesicles purified from electric rays (which have large axons)
• The antibodies were then used to expression clone VAMP and Syntaxin

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20
Q

What do Clostridial neurotoxins tetanus and botulinum B cleave

A

Vamp/ synaptobrevin

21
Q

How did they know these toxins cleaved Vamp

A

Bands disappeared in SDS-PAGE gel.

22
Q

Symptoms of tetanus/botulinum

A
  • Tetanus to locked jaw

* Botulinum to floppy baby syndrome

23
Q

What did they find in the biochemical purification of SNAREs

A

Found could purify a large complex that dissembles when ATP is hydrolysed

24
Q

How does biochemical purification occur

  • tagged..
  • bind…
  • bound…
  • add…
  • get…
  • complex…
A
  1. Tagged NSF and made it recombinantly
  2. Bind NSF onto beads (believed this was an ATPase from immune affinity experiment)
  3. Bound to α/γ SNAP complex
  4. Add ATP that could be hydrolysed
  5. Got bands that could be sequenced
  6. Got syntaxin, VAMP and SNAP 25 complex
25
Q

Describe the complex formed in biochemical purification

A
  • VAMP on vesicle (T-SNARE)
  • Syntaxin and SNAP25 on target membrane (V-SNARE)
  • These interact to form a large complex that can be purified
  • Hydrolysis of ATP fuses these things together
26
Q

Rothman’s SNARE hypothesis

A

1) SNAREs for each transport step within the cell regardless of type of vesicle i.e. 38 SNAREs encoded in the human genome
2) SNAREs should provide specificity to vesicle transport
3) SNAREs should be sufficient to drive lipid bilayer fusion
4) Proposed that NSF and ATP hydrolysis catalyses membrane fusion (this bit is actually wrong!)

27
Q

What do the VAMP and synataxin/SNAP 25 form

A

coiled-coil

28
Q

How do snares form this coiled coil

A

zipper in a parallel coiled coil and in doing so they pull the vesicles close enough to provide the energy for the fusion of lipid bilayers

29
Q

SNARE zipper provides…

A

energy to drive membrane fusion

30
Q

What isn’t required for membrane fusion but is for recycling of snares

A

NSF

31
Q

Trans-SNARE:

A

Trans-SNARE: When the SNAREs are coiled before fusion

32
Q

Cis-SNARE:

A

Cis-SNARE: When the vesicles have fused and the SNAREs uncoil

33
Q

Types of SNAREs

A
  • Q type - Syntaxin-like/SNAP 25 (glutamine)

* R type - VAMP-like (arginine)

34
Q

Ratio of types of snares

A

3Q:1R SNARE conserved in all complexes

35
Q

Do SNAREs provide the specificity of membrane fusion?

A
  • Only get fusion with SNARE complexes which fit 3Q:1R ratio (Mutation of a Q/R inhibits SNARE activity and fusion doesn’t work as well)
  • SNAREs show some promiscuity but on the whole they predominantly interact with SNAREs from the appropriate membranes.
  • Lots of additional machinery contribute to specificty (i.e. rabs/ coat proteins and tethers)
36
Q

In Rothman’s SNARE hypothesis, he suggested SNAREs should provide specificity to vesicle transport. Was this correct?

A

PARTIALLY YES - SNAREs do provide specificity - but there are other things that also do

37
Q

Common features of SNARE proteins

A
  • Generally small 14-40kDa
  • All have at least 1 coiled-coil or SNARE motif (except SNAP 25 have 2)
  • Generally C-terminally anchored
38
Q

How to see if the SNAREs the minimal fusion machinery?

A
  • Synthetic vesicle, liposome, with VAMP on it
  • Put dye onto vesicle
  • Mix together and see if get fusion and is this sped up etc by adding other thing
  • TIRF microscopy
39
Q

Recombinant SNAREs can drive membrane fusion of…

A

purified liposomes

40
Q

What happens in liposomes if add snare machinery on own

A

some fusion, but not much

41
Q

What occurs if add calcium to liposomes

A

increases rate of vesicle fusion

42
Q

What does syntaxin do

A

Regulates complex and inhibits ability of fusion - opens and closes the complex

43
Q

Was Rothman correct in stating SNAREs were the minimum machinery for membrane fusion

A

yes - but other things like calcium assist

44
Q

Role of NSF

A

snare recycling after fusion

45
Q

Is NSF required for fusion

A

no

46
Q

which complex does NSF act on

A

cis-SNARE complex

47
Q

shape of nsf

A

ring

48
Q

How does NSF work

A
  • NSF uses ATP activity to unwind SNARE molecules i.e. unwind VAMP/Syntaxin coiled-coil after fusion
  • The complex is unscrewed to separate the SNAREs
49
Q

In Rothman’s SNARE hypothesis, he suggested NSF + ATP hydrolysis catalyses membrane fusion. Was this correct?

A

No – NSF recycles the SNAREs after fusion

Cell and molecular extras (16 decks)

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