Temperature-sensitive paralytic mutations demonstrate that synaptic exocytosis requires SNARE complex assembly and disassembly.
The neuronal SNARE complex is formed via the interaction of synaptobrevin with syntaxin and SNAP-25. Purified SNARE proteins assemble spontaneously, while disassembly requires the ATPase NSF. Cycles of assembly and disassembly have been proposed to drive lipid bilayer fusion. However, this hypothesis remains to be tested in vivo. We have isolated a Drosophila temperature-sensitive paralytic mutation in syntaxin that rapidly blocks synaptic transmission at nonpermissive temperatures. This paralytic mutation specifically and selectively decreases binding to synaptobrevin and abolishes assembly of the 7S SNARE complex. Temperature-sensitive paralytic mutations in NSF (comatose) also block synaptic transmission, but over a much slower time course and with the accumulation of syntaxin and SNARE complexes on synaptic vesicles. These results provide in vivo evidence that cycles of assembly and disassembly of SNARE complexes drive membrane trafficking at synapses.
Littleton
J T
JT
Laboratory of Genetics, University of Wisconsin, Madison 53706, USA.
Chapman
E R
ER
Kreber
R
R
Garment
M B
MB
Carlson
S D
SD
Ganetzky
B
B
D000595:Amino Acid Sequence; D000818:Animals; D046508:Culture Techniques; D004330:Drosophila / Q000235:genetics*; D005089:Exocytosis / Q000502:physiology*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D018377:Neurotransmitter Agents / Q000378:metabolism; D010243:Paralysis / Q000235:genetics*; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis; D050600:SNARE Proteins; D017386:Sequence Homology, Amino Acid; D013569:Synapses / Q000502:physiology*; D013572:Synaptic Vesicles / Q000502:physiology; D013696:Temperature; D033921:Vesicular Transport Proteins
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins