Calcium-dependent dissociation of synaptotagmin from synaptic SNARE complexes.
The formation of the synaptic core (SNARE) complex constitutes a crucial step in synaptic vesicle fusion at the nerve terminal. The interaction of synaptotagmin I with this complex potentially provides a means of conferring Ca2+-dependent regulation of exocytosis. However, the subcellular compartments in which interactions occur and their modulation by Ca2+ influx remain obscure. Sodium dodecyl sulfate (SDS)-resistant core complexes, associated with synaptotagmin I, were enriched in rat brain fractions containing plasma membranes and docked synaptic vesicles. Depolarization of synaptosomes triggered [3H]GABA release and Ca2+-dependent dissociation of synaptotagmin from the core complex. In perforated synaptosomes, synaptotagmin dissociation was induced by Ca2+ (30-300 microM) but not Sr2+ (1 mM); it apparently required intact membrane bilayers but did not result in disassembly of trimeric SNARE complexes. Synaptotagmin was not associated with unstable v-SNARE/t-SNARE complexes, present in fractions containing synaptic vesicles and cytoplasm. These complexes acquired SDS resistance when N-ethylmaleimide-sensitive fusion protein (NSF) was inhibited with N-ethylmaleimide or adenosine 5'-O-(3-thiotriphosphate), suggesting that constitutive SNARE complex disassembly occurs in undocked synaptic vesicles. Our findings are consistent with models in which the Ca2+ triggered release of synaptotagmin precedes vesicle fusion. NSF may then dissociate ternary core complexes captured by endocytosis and recycle/prime individual SNARE proteins.
Leveque
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C
Institut National de la Santé et de la Recherche Médicale, Unité 464, Institut Jean Roche, Faculté de Médecine Secteur Nord, Université de la Méditerranée, Marseille, France. leveque.c@jean-roche.univ-mrs.fr
Boudier
J A
JA
Takahashi
M
M
Seagar
M
M
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000502:physiology*; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000037:antagonists & inhibitors; D002462:Cell Membrane / Q000378:metabolism; D004351:Drug Resistance; D005033:Ethylmaleimide / Q000494:pharmacology; D005089:Exocytosis / Q000502:physiology; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D052066:N-Ethylmaleimide-Sensitive Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051381:Rats; D050600:SNARE Proteins; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013569:Synapses / Q000378:metabolism*; D013572:Synaptic Vesicles / Q000378:metabolism; D050863:Synaptotagmin I; D050857:Synaptotagmins; D033921:Vesicular Transport Proteins
D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050600:SNARE Proteins; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D033921:Vesicular Transport Proteins; D050857:Synaptotagmins; C022571:adenosine 5'-O-(3-thiotriphosphate); D012967:Sodium Dodecyl Sulfate; D000255:Adenosine Triphosphate; D052066:N-Ethylmaleimide-Sensitive Proteins; C498737:Nsf protein, rat; D005033:Ethylmaleimide; D002118:Calcium