Sept 25 lec

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13 Apr 2011
of 4
bPg 3slide 3:
-sth in cytosol mediating this process. Sth uses ATP. ATPase involved in this
The donor is inacapable of glysolating protein. The acceptor can glycosylate but
doesnt have proteins.
G protein is sensitive to endo H. golgi extracts. Longer u leave the WT protein, the
more resistant the protein is.
PG 4 slides 1:
-fraction wid highest activity NsF. If trated wid poison.. it kills the activity. It
attacks ATP in cytosol. NSF might b an ATPase. NEM kills the activity of cytosol.
So he takes poisoned cytosol and adds fractions ofnon poisoned cytosol. One protein,
called NSFprotects the cytosol from
One proteinit had all the activity in it. He made an antibody against it. So
antibody can deplete the activity. So the actual protein is NSF in active form.
-first component of fusion machinery
Complementary approaches:
- eg. Fusion machinery must include sth on synaptic vesicles. Others were using
yeast genetics. Mutation in yeast that didnt secrete properly.
-its atp ase.
-2 binding domains. D1, D3. NSF acts as heximer. N terminis domain is protein
protein interaction.
NSF binds to membranes vis aSNAP.
-ATP ase can only bind to membranes in presence of non hydrolysable atp …and sth
-sth on the membranes that NSF and SNAP stick to. Sth in the membranes thats a
SNAP receptor…so he called them the he wanted to find snares.
-presence of ATP and presence of non-hydrolysable atp
Identification of SNAP receptors
-(chose brain cuz richer source of snares)
-everything bound toNSF is guna fall off.
Imp for fusion step. SNAP-25. This cud b the fusion machinery.
Genertic and boiochemical approaches identified the same proteins!
- all are conserved thru evolution. Mediate
SNARE proteins are the target of Botox
- botox are proteases.
-tetanus toxin.-prevents motor neurons from turning off.
-light chain is taken out into cytoplasm
-Botox- Bot ASNAP25nerve terminal doesnt return from its terminal. It
remains intact wid nerve terminal protein. Nerves dont repair themselves.
SNARE cleavage by neurotoxins proved that they were essential for
Botox effects.
SNARE Proteins: machinery Implicated in membrane fusion
-they’re functional complex. N forma structure called coiled coil (helical proteins
interact in a way that they wrap arnd eachother, parallel /antiparealle. Hydrophobic
aas every 3-4 reesidues. In helix theres 3.5 aa per turn.).snap25 gives 2 helices.
Anchored in themembrane. These vesicles can b brought into close contact wid Pm
cuz of these
Coiled coils assemble in a parallel complex. Both VAMP and Syntaxin have
transmembrane anchors but dont go to lumen at all. This interaction wud happen in
parallel. From N to C terminal. Acting s a zipper. Closure to a membrane….fuse.
-layers interactionmiddle layer is not ahydrophobic interaction but charged
-all other layers are hydrophobic only the middle one is not.
SNARE Nomenclature.
-vSNARE (vesicle snare), tSNARE (target snare)
-all vamp related had R ,
-syntaxin itself if Qa
SNARE Domains:
-SNARE: interaction domain is close to TM domain.
SNARE proteins can be of 3 maorcategories.
-Qa: 3 helices. They can form a coiled coilcan interact wid snare protein in a closed
-Qb/Qc in b/w.
Multiple isoforms of SNARES
-VAMP on vesicles.
STX1234- in PM.
-homologs in intracellular.
-do the lipids fuse? Put molecules/content into lipid circles and see if the contents
fuse or not
Content mixing assay:
-florescence lost==fusion of molecules.
Steps involved in vesicle fusion.
-specific sites in the cell.
-60Rabs in trafficking.
-Rabs are GTPases.
-GDP-inactive form.
GTP- active
low ability to exchange nucleotides.
GEF allows it to attach to effector.
GAP causes hydrolysis of nucleotide. To b reused, its taken back to its origin GDI-
binds to GDP form and extracts it from lipid bilayer (outa membrane) and puts it
into cytoplasm.
Exocyst complex:
-mutations in this protein stops secretion in yeast.
Tethering complex is are found at a specific sites in the cell.
-fcn of tethering: facilitate attachment of other molecules like snares etc.
CD spectroscopy: Syntaxin- many helices.
Snap 25- not many helices. So, syntaxin binds wid snap25 and start to form helicle
structures (snare domain). Snap+syntax = helix=vamp protein joins inand forms 4
helix complex.