Fig. 1

Residues T323/M324/R325 are essential for SNARE complex assembly from Munc18-1/Syx1 catalyzed by the MUN domain. A Crystal structure of Munc18-1/Syx1 (PDB ID: 3C98) and positions indicating mutation sites (T322, T323, M324 and R325). Mutation sites are highlighted in red, and the residue sequence (317–323) without electron density is shown as dashed lines. B The triple mutation T323A/M324A/R325A (TMR) completely abolished the transition from Munc18-1/Syx1 to the SNARE complex catalyzed by the MUN domain, as shown by native PAGE. C Quantification of the results shown in B. Data are presented as mean values ± SD; n = 3. D The TMR mutation had no effect on the interaction between Munc18-1 and Syx1, as detected by GST pull-down assay. E Effect of TMR mutation on fusion between liposomes containing Munc18-1/Syx1 and liposomes reconstituted with Syb2 in the presence of SNAP-25, the Syt1 C2AB fragment, and 1 mM CaCl₂, with or without the Munc13-1 C1C2BMUN fragment. F The quantification results of data shown in E. Data are presented as mean values ± SD; n = 3. Representative gel displayed is from one of three independent replicates. M18-1, Munc18-1; Syx1, Syntaxin-1; M13, Munc13-1 C1C2BMUN domain; SN25, SNAP-25; Syb2, Synaptobrevin-2