Fig. 6. Effects of Munc18-1 and syntaxin-1 mutations on liposome fusion.

(A) Diagram summarizing the content mixing between T-liposomes containing syntaxin-1 and SNAP-25, which were preincubated with Munc18-1, NSF, and αSNAP, and V-liposomes containing synaptobrevin in the presence of Munc13C. V-liposomes contain trapped Cy5-strepatavidin, and T-liposomes contain trapped PhycoE-biotin. (B to D) Content mixing between V-liposomes and T-liposomes was monitored from the increase in the fluorescence signal of Cy5-streptavidin caused by FRET with PhycoE-biotin. Assays were performed with WT or mutant Munc18-1 (B), or WT or mutant syntaxin-1 and WT (C) or D326K mutant (D) Munc18-1, as indicated. Experiments were started in the presence of 100 μM EGTA, and Ca²⁺ (600 μM) was added at 300 s.