The R65Q mutation inhibits membrane fusion in vitro. (A) Schematic depicting the liposome fusion reaction. (B) T- and labeled v-liposomes were preincubated on ice for 2 hours (hs) with control buffer, 2.5 μM Munc18-1^WT, 2.5 μM Munc18-1^R65Q, or 2.5 μM cdV2. Subsequently, the temperature was increased to 37°C, and the fluorescent signal was read every 2 minutes (min) for a total of 120 minutes. (C) Dose-dependent inhibition of fusion by Munc18-1^R65Q. The liposome fusion reaction was performed as in panel B but with increasing concentrations of Munc18-1^R65Q. (D) Dose-dependent activation by Munc18-1^WT. The liposome fusion reaction was performed as in panel B but with increasing concentrations of Munc18-1^WT. (E) Competition experiments using Munc18-1^WT and Munc18-1^R65Q. T- and v-liposomes were preincubated on ice for 2 hours in the presence of 2.5 μM Munc18-1^WT and control buffer or increasing concentrations of Munc18-1^R65Q (1.2-5.0 μM). Subsequently, the temperature was increased to 37°C, and the fluorescent signal was read every 2 minutes for a total of 120 minutes. (F) Competition experiments using Munc18-1^R65Q and Munc18-1^WT. T- and v-liposomes were preincubated on ice for 1 hour in the absence (control) or presence of 2.5 μM Munc18-1^R65Q. Munc18-1^R65Q-treated liposomes were incubated on ice for 2 hours with increasing concentrations of Munc18-1^WT (2.5-10.0 μM). The temperature was increased to 37°C, and fusion was monitored as described above. (G) T-liposomes were incubated in the absence (control) or presence of Munc18-1^WT, Munc18-1^R65Q, or cdV2 for 2 hours on ice, followed by the addition of v-liposomes. (H) T- and v-liposomes were preincubated for 2 hours on ice to allow the assembly of SNARE complexes, followed by the addition of control buffer, Munc18-1^WT, or Munc18-1^R65Q. Subsequently, the temperature was raised to 37°C, and lipid mixing was measured. (I) T- and v-liposomes were preincubated 2 hours on ice, and then cdV2 was added to the reaction and incubated for 1 hour on ice. After that, control buffer, Munc18-1^WT, or Munc18-1^R65Q was added, and the temperature was increased to 37°C to induce lipid mixing. In panels B-I, the blue curve shows the effect when the strong inhibitor cdV2 was added from the beginning of the fusion reaction. Graphs in the insets of panels B-I show the fold activation of at least 3 independent experiments using different liposome preparations. The fold activation was calculated as a ratio of the difference of the initial rate of membrane fusion at 60 minutes in the presence of Munc18-1 minus the initial rate of fusion in the absence of Munc18-1 divided by the initial rate of fusion in the absence of Munc18-1. *P < .05 compared to control level.