Reconstitution of active FL syt. (A) A schematic diagram of the in vitro FL syt-regulated fusion assay. (B) Titration of PIP₂ in Tr. Vesicles bearing FL syt and syb (Vr-syt) were preincubated with Tr, which harbored the indicated amount of PIP₂, for 20 min in the presence of 0.2 mM EGTA before the addition of 1 mM Ca²⁺. Fusion was monitored for another 60 min after Ca²⁺ injection. (C and D) Increasing [PIP₂] resulted in increases in the extent (C) and initial rate (D) of regulated fusion. (E) In reactions lacking FL syt, membrane fusion was only slightly promoted by Ca²⁺, and this small effect occurred when the PIP₂ levels were ≥1%. (B–E) Experiments were repeated twice, and similar results were obtained. (F) Optimization of the preincubation time. Fusion was monitored as in B. The duration of the preincubation step was varied as indicated. (G and H) The extent (G) and initial rate (H) of regulated fusion were plotted versus the preincubation time (n = 4). The data shown are represented as mean ± SEM.