Figure 8.

P335A mutant of Munc18-1 can substantially bypass the requirement of Munc13-1 in lipid and content mixing assays. A–D, Lipid mixing (A, C) between V and T liposomes was measured from the fluorescence dequenching of Marina Blue–labeled lipids, and content mixing (B, D) was monitored from the development of FRET between PhycoE-Biotin trapped in the T liposomes and Cy5-streptavidin trapped in the V liposomes. In A and B, the assays were performed in the presence of NSF-αSNAP, Munc13-1 C₁C₂BMUNC₂C, synaptotagmin-1 C₂AB fragment, and WT or mutant Munc18-1s as indicated. In C and D, the assays were performed in the presence of NSF-αSNAP, synaptotagmin-1 C₂AB fragment, and WT or mutant Munc18-1s as indicated. Experiments were started in the presence of 100 μm EGTA and 5 μm streptavidin, and Ca²⁺ (600 μm) was added after 300 s. E, Quantification of content mixing in the experiments of D. Bars represent averages of the normalized fluorescence observed after 800 s (500 s after Ca²⁺ addition) in experiments performed at least in triplicate. Error bars represent SDs.