Figure 7.

Munc18-1 P335A and Y337A are gain-of-function mutants stimulating lipid mixing in a reconstituted SUV-GUV fusion assay. A, Incubation scheme: syntaxin-1 GUVs (20 pmol syntaxin-1, 14 nmol lipid) containing PI(4,5)P₂ (2 mol%) were preincubated in the absence or presence of the indicated Munc18-1 constructs (42 pmol) for 30 min at room temperature. Soluble SNAP-25 (177 pmol) and Syt1/syb-2 SUVs (2.1 pmol Syt1, 7.5 pmol syb-2, 2.5 nmol lipid) were added and incubated for 10 min on ice to allow vesicle docking. Membrane fusion was measured in a final volume of 104 μl at 37°C. B, Melting temperatures show that both mutants are correctly folded. C, Lipid mixing was monitored by the increase in Atto 488 fluorescence for 30 min. Data were normalized to the maximum fluorescence after liposome lysis by detergent. Error bars indicate SEM. N = 3.