synaptotagmin mutants reveal essential functions for the C2B domain in Ca2+-triggered fusion and recycling of synaptic vesicles in vivo

Synaptotagmin has been proposed to function as a Ca2+ sensor that regulates synaptic vesicle exocytosis, whereas the soluble N-ethylmaleimide-sensitiv e factor attachment protein receptor (SNARE) complex is thought to form the core of a conserved membrane fusion machine. Little is known concerning th e functional relationships between synaptotagmin and SNAREs. Here we report that synaptotagmin can facilitate SNARE complex formation in vitro and tha t synaptotagmin mutations disrupt SNARE complex formation in vivo. Synaptot agmin oligomers efficiently bind SNARE complexes, whereas Ca2+ acting via s ynaptotagmin triggers cross-linking of SNARE complexes into dimers. Mutatio ns in Drosophila that delete the C2B domain of synaptotagmin disrupt clathr in AP-2 binding and endocytosis. In contrast, a mutation that blocks Ca2+-t riggered conformational changes in C2B and diminishes Ca2+-triggered synapt otagmin oligomerization results in a postdocking defect in neurotransmitter release and a decrease in SNARE assembly in vivo. These data suggest that Ca2+-driven oligomerization via the C2B domain of synaptotagmin may trigger synaptic vesicle fusion via the assembly and clustering of SNARE complexes .