chapter 3 - exocytosis

Question 1

3.1. [exocytosis] What are SNAREs? which snares are involved in vesicle exocytosis?

Answer 1

SNARE proteins mediate vesicle fusion
- SNARE proteins: proteins containing a SNARE domain

• SNARE= soluble NSF (N-ethylmaleimide sensitive factor)-attachment protein receptor
  SNARE proteins are targets of clostridial botulinum and tetanus toxins; these neurotoxins enter the presynaptic terminal and act as highly specific proteases, leacing to a selective block of presynaptic membrane fusion

Three SNARE proteins are essential for SV fusion:
* the vesicular SNARE protein synaptobrevin/VAMP (vesicle-associated membrane protein)
* SNAP-25
* syntaxin-1
* The synaptic SNARE complex (synaptobrevin-SNAP-25-syntaxin-1)
forms a parallel four-helix bundle

Question 2

3.2. [exocytosis] draw the functional domains of snares involved in sv exocytosis and the assembled snare complex

Answer 2

see slides + figure.

• SNARE proteins contain a SNARE motif, a characteristic sequence of 70-80 residues
  -the linker sequence of SNAP-25
  serves for membrane anchoring via palmitoylation
  palmitoylation: covalent attachment of fatty acids, e.g. palmitic acid, to cysteine (and less frequently to serine or threonine) residues of proteins.
• the energy released during Synaptic SNARE complex assembly fuels membrane fusion, likely by a simple mechanical force

Question 3

3.3. [exocytosis] describe SNARE/SM protein cycle and draw it

Answer 3

see figure

1. assembly/priming - chaperones: CSPalpha/beta/gamma+ alpha/beta/gamma synucleins - N to C terminal zippering of trans-snare complexes
2. fusion pore opening- partial trans snare proteins / sm protein assembly
3. fusion pore expansion- trans-snare proteins are converted onto cis-snare complexes (i.e. snare complexes on a single membrane)
4. recycling - snare complex disassembly and vesicle recycling - ATPase NSF and SNAPs dissociate cis-snare complexes into monomers. SNF is also the ATPase that loads snare proteins with energy

chaperones - proteins that assist the confirmation folding/unfolding of proteins as well as the assembly/disassembly of multi protein complexes

sm proteins

-evolutionary conserved cytosolic proteins; essential partners for SNARE proteins in fusion
-The N-terminus of syntaxin-1 tethers the SM protein Munc18, and this interaction is absolutely essential for fusion in vivo
-SNARE- and SM-protein complexes may stabilize the attachment of vesicles to the target membrane, thereby participating in docking

Question 4

3.4. [exocytosis] how do chaperones support snare protein function? draw it as well

Answer 4

check figure

Chaperones are proteins that assist the confirmational folding/unfolding of proteins as well as the assembly or disassembly of multi-protein complexes

Two types of chaperones support SNARE protein function

1. the classical chaperone complex SPa (cysteine string protein a, a SV protein), Hsc70, and SGT (small glutamine-rich tetratricopeptide repeat protein). This complex binds to SNAP-25 on the target membrane, thereby supporting the functional competence of SNAP-25 to engage in SNARE complexes.
2. the nonclassical chaperones a/B/y-synucleins, which are bound to phospholipids and synaptobrevin/VAMP on vesicles, and bind to assembling SNARE complexes to support their folding.

Question 5

3.5 [exocytosis] how do dysfunctional chaperones result in neurodegeneration? Draw it.

Answer 5

Defective chaperone function is implicated in neurodegeneration
* Both a-synuclein and CSPa are linked to neurodegeneration:
* a-synuclein mutation or duplication causes familial Parkinson’s disease
* many neurodegenerative diseases feature Levy bodies, which contain a-synuclein (e.g. Parkinson’s disease, Lewy body dementia)
* deletion of CSPa in mice has no immediate effect on neurotransmitter release, but leads to increased ubiquitination and degradation of SNAP-25 and to reduced SNARE-complex assembly, resulting in fulminant neurodegeneration that kills mice after 2-3 months
- abnormal exposure of neurons to misfolded SNAREs and/or abnormal SNARE complex assembly impairs neuronal survival

UBIQUITINIFICATION = covalent attachment of ubiquitin to proteins that need to be degraded