Figure 5.

Ca²⁺ and synaptotagmin-1 induce a drastic increase in the rate of SNARE-mediated lipid mixing that is abolished by the R398Q,R399Q mutation. (a) Lipid mixing between synaptobrevin- and syntaxin-1/SNAP-25-containing proteoliposomes with 1:200 (black) or 1:500 (green) P/L ratios. Equal amounts of the proteoliposomes were mixed, and lipid mixing was detected by monitoring lipid fluorescence dequenching. In all experiments, the syntaxin-1/SNAP-25 liposomes were preincubated with a C-terminal synaptobrevin fragment (residues 49-96). F/F₀ represents the fluorescence relative to the starting point. The pink trace shows an experiment performed with a 1:200 P/L ratio in the presence of 1 μM C₂AB fragment and 100 μM Ca²⁺. (b-h) Lipid-mixing assays were performed as in (a) with a 1:500 P/L ratio plus different additions as indicated. Synaptotagmin-1 fragments (1 μM) were added at the beginning of the fusion reaction. In (b), Ca²⁺ or Mg²⁺ was added after 250 s of reaction (black arrows) as indicated. In (c-h), 100 μM Ca²⁺ was added at 250 s (black arrows). In (d), subsequent additions of 200 μM and 1 mM Ca²⁺ for a reaction with the C₂AB-R398Q,R399Q mutant are indicated by pink arrows. WT and mutant C₂B domains were purified using our standard protocol³⁶, except for one experiment where the WT C₂B domain was purified under less stringent conditions [indicated as C₂B*; blue triangles in (f)]. Each set of experiments shown in each panel was performed on the same day with the same proteoliposome preparations.