Calcium/otoferlin enables t-SNAREs, which have not been preassembled into heterodimers, to drive membrane fusion. (A) Fusion assays were conducted using reconstituted syntaxin 1 and synaptobrevin 2 vesicles in the presence of 15 µM soluble SNAP-25 and 4 µM otoferlin. (B) Either half of otoferlin, C2ABC, or C2DEF was sufficient to accelerate membrane fusion using soluble SNAP-25. In the absence of calcium or otoferlin, fusion was not observed. E indicates that the experiment was performed under EGTA conditions.