Otoferlin stimulates SNARE-mediated proteoliposome fusion in a calcium-dependent manner. Fusion experiments were performed using donor v-SNARE proteoliposomes and t-SNARE acceptor proteoliposomes with varying concentrations of recombinant otoferlin fragments. The components were incubated together for 20 min at 37°C in the presence of 0.1 mM EGTA. At t = 20 min, calcium was added to a final concentration of 1 mM. (A and B) Both C2ABC (A) and C2DEF (B) stimulated SNARE-mediated proteoliposome fusion in response to calcium. (A and B) E indicates that the experiment was performed under EGTA conditions. (C and D) Mean value for the extent of fusion expressed as a percentage of maximum fluorescence intensity after addition of detergent for 10 µM C2ABC (C) and C2DEF (D) under the indicated conditions. Fusion was attenuated by addition of cd-syb or cd–t-SNARE. Error bars represent SD (n = 3).