Fig. 3.

The +1 and +2 layers of the v-SNARE are essential to fusion reactions containing the cognate SM protein or the Vc peptide. (A) Normalized initial lipid-mixing rates of reconstituted fusion reactions containing 5 µM WT Munc18-1, 5 µM WT synaptic exocytic t-SNAREs, and 1.5 µM WT or mutant VAMP2. Data are presented as the percentage of initial lipid-mixing rate of WT reactions. The reconstituted fusion reactions contained 100 mg/mL of the macromolecular crowding agent Ficoll 70. In the presence of macromolecular crowding agents, the SNARE-SM–mediated fusion reaction was faithfully recapitulated without requiring a low-temperature preincubation step (11). Error bars indicate SD. (B) Diagrams of WT VAMP2 and VAMP2-derived Vc peptide. (C) Normalized initial lipid-mixing rates of reconstituted fusion reactions containing 5 µM Vc peptide, 5 µM WT synaptic exocytic t-SNAREs, 1.5 µM WT or mutant VAMP2, and 100 mg/mL Ficoll 70. Data are presented as the percentage of initial lipid-mixing rate of WT reactions. Error bars indicate SD. (D) The basal SNARE-mediated fusion reaction involves the +3 to +8 layers in CTDs, whereas all of the CTD layers (+1 to +8) are required for the Munc18-1– or Vc peptide-stimulated fusion reaction.