Fig. 4

The TMR mutation supported Syb2 binding to isolated Munc18-1. A Illustration of Munc18-1-promoted reconstituted liposome fusion between v-liposomes bearing Syb2 (1–116) and t-liposomes containing Syx1 (1–288)/SNAP-25. V-liposomes were prepared by including NBD-PE and Rhodamine-PE, and NBD fluorescence at 538 nm was monitored. B Traces of lipid mixing reactions promoted by Munc18-1. Traces represented are from one of three independent replicates. All reactions were performed at 30 °C. C Quantification of the results shown in B. Data are presented as mean ± SD, n = 3. D Interaction between Munc18-1 and Syb2 detected by GST pull-down assay. The representative gel shown was from one of three replicates. E Quantification of integrated densities of Munc18-1 bands. The band densityy of WT Munc18-1 was taken as 1. Data are presented as mean values ± SD, n = 3. F Effect of TMR mutation on the Munc18-1 binding SNARE complex detected by GST pull-down assay. The SNARE complex was assembled by incubating GST-SN1, SN3, the cytoplasmic domain of Syb2 (29–93) and Syx1 (2–253) overnight at 4 °C. Bands of Munc18-1 and the SNARE complex are indicated in the figure. Representative gel displayed is from one of three independent replicates