<i>synaptotagmin</i>Mutants Reveal Essential Functions for the C2B Domain in Ca²⁺-Triggered Fusion and Recycling of Synaptic Vesicles<i>In Vivo</i>

Synaptotagmin has been proposed to function as a Ca²⁺ sensor that regulates synaptic vesicle exocytosis, whereas the soluble<i>N</i>-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is thought to form the core of a conserved membrane fusion machine. Little is known concerning the functional relationships between synaptotagmin and SNAREs. Here we report that synaptotagmin can facilitate SNARE complex formation<i>in vitro</i> and that <i>synaptotagmin</i> mutations disrupt SNARE complex formation <i>in vivo</i>. Synaptotagmin oligomers efficiently bind SNARE complexes, whereas Ca²⁺ acting via synaptotagmin triggers cross-linking of SNARE complexes into dimers. Mutations in <i>Drosophila</i>that delete the C2B domain of synaptotagmin disrupt clathrin AP-2 binding and endocytosis. In contrast, a mutation that blocks Ca²⁺-triggered conformational changes in C2B and diminishes Ca²⁺-triggered synaptotagmin oligomerization results in a postdocking defect in neurotransmitter release and a decrease in SNARE assembly <i>in vivo</i>. 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.