Membrane Penetration by Synaptotagmin Is Required for Coupling Calcium Binding to Vesicle Fusion In Vivo

The vesicle protein synaptotagmin I is the Ca2+ sensor that triggers fast, synchronous release of neurotransmitter. Specifically, Ca2+ binding by the C2B domain of synaptotagmin is required at intact synapses, yet the mechanism whereby Ca2+ binding results in vesicle fusion remains controversial. Ca2+ -dependent interactions between synaptotagmin and SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment receptor) complexes and/or anionic membranes are possible effector interactions. However, no effectorinteraction mutations to date impact synaptic transmission as severely as mutation of the C2B Ca2+ -binding motif, suggesting that these interactions are facilitatory rather than essential. Here we use Drosophila to show the functional role of a highly conserved, hydrophobic residue located at the tip of each of the two Ca2+ -binding pockets of synaptotagmin. Mutation of this residue in the C2A domain (F286) resulted in a _50% decrease in evoked transmitter release at an intact synapse, again indicative of a facilitatory role. Mutation of this hydrophobic residue in the C2B domain (I420), on the other hand, blocked all locomotion, was embryonic lethal even in syt I heterozygotes, and resulted in less evoked transmitter release than that in sytnull mutants, which is more severe than the phenotype of C2BCa2+ -binding mutants. Thus, mutation of a single, C2B hydrophobic residue required for Ca2+ -dependent penetration of anionic membranes results in the most severe disruption of synaptotagmin function in vivo to date. Our results provide direct support for the hypothesis that plasma membrane penetration, specifically by the C2B domain of synaptotagmin, is the critical effector interaction for coupling Ca2+ binding with vesicle fusion.