Physiological densities of FL syt regulate fusion via a SNARE-dependent mechanism. (A) Titration of reconstituted FL syt in Vr alters the rate and extent of Ca²⁺-promoted membrane fusion. The experiments were performed as described in Fig. 1 B; the average copy number of syt per vesicle is indicated. (B) Samples from A were subjected to SDS-PAGE and stained with Coomassie blue. Mr, molecular mass. (C) Ca²⁺ dose response from fusion assays containing 30 copies of syt in Vr and 3% PIP₂ in Tr. The corrected data were fitted with a sigmoidal curve; the [Ca²⁺]_1/2 was 250 µM, and the Hill coefficient was 1.5. Experiments were repeated twice, and similar results were obtained. (D–F) The cytoplasmic domains of SNAREs block syt-promoted fusion. (D and E) Increasing concentrations of cd t-SNAREs (D) or cd syb (E) were preincubated with Tr and Vr-syt. Membrane fusion was monitored using the same protocol as shown in Fig. 1 B. (F) The final extent of fusion at 80 min was plotted as a function of [cd SNARE] (n = 3). All data shown are represented as mean ± SEM.