Figure 4. Effects of Munc18-1 mutations that alter synaptobrevin binding on membrane fusion in reconstitution assays.

Lipid mixing (A,C,E) between V- and T-liposomes was measured from the fluorescence de-quenching of Marina Blue-labeled lipids. Content mixing (B,D,F) 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,B), the assays were performed in the presence of NSF-αSNAP with or without WT Munc18-1 (M18) and/or Munc13-1 C₁C₂BMUNC₂C (M13) as indicated. In (C,D), assays were performed in the presence of NSF-αSNAP, Munc13-1 C₁C₂BMUNC₂C and WT or mutant Munc18-1s as indicated. In (E,F), 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.

DOI: http://dx.doi.org/10.7554/eLife.24278.010

Figure 4—figure supplement 1. Effects of Munc18-1 mutations that alter synaptobrevin binding on membrane fusion in reconstitution assays.

(A,B) Quantification of content mixing in the experiments of Figure 4D. Bars represent averages of the normalized fluorescence observed after 150 s (A) and after 500 s (200 s after Ca²⁺ addition) (B) in experiments performed at least in triplicate. Error bars represent standard deviations. (C,D) Lipid and content mixing assays performed under the same conditions as those shown in Figure 4C,D but containing in addition synaptotagmin-1 C₂AB fragment.

DOI: http://dx.doi.org/10.7554/eLife.24278.011

Figure 4—figure supplement 2. Effects of Munc18-1 mutations that alter synaptobrevin binding on membrane fusion in reconstitution assays.

(A,B) Lipid and content mixing assays performed under the same conditions as those shown in Figure 4E,F but in the absence of the synaptotagmin-1 C₂AB fragment. The gray curves show a reference experiment performed in the presence of NSF-αSNAP, Munc13-1 C₁C₂BMUNC₂C and WT Munc18-1. (C) Quantification of content mixing in the experiments shown in Figure 4F. 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 standard deviations.

DOI: http://dx.doi.org/10.7554/eLife.24278.012