Intracellular Vesicle Fusion Requires a Membrane-Destabilizing Peptide Located at the Juxtamembrane Region of the v-SNARE

Rathore, Shailendra S.; Liu, Yinghui; Yu, Haijia; Wan, Chun; Lee, MyeongSeon; Yin, Qian; Stowell, Michael H.B.; Shen, Jingshi

In a liposome co-flotation assay, WT and mutant VAMP2 bound equally well to the t-SNAREs ( Figure S3 ). Thus, the juxtamembrane motif is dispensable for SNARE complex assembly, consistent with the ability of the VAMP2 mutant to drive normal basal fusion ( Figures 2C and 2D ). Next, we examined whether the juxtamembrane motif regulates SNARE-Munc18-1 association. Using isothermal titration calorimetry (ITC), we observed that the affinity of Munc18-1 binding to the WT SNARE complex was similar to its binding to the mutant SNARE complex in which the juxtamembrane motif of VAMP2 was mutated ( Figure 3B ). We then further examined SNARE-Munc18-1 binding using a glutathione S-transferase (GST) pull-down assay. We observed that mutation of the juxtamembrane motif did not impair the interaction of GST-Munc18-1 with the SNARE complex ( Figure 3C ), in agreement with the ITC results. Thus, although selectively required for the SNARE-Munc18-1-mediated fusion reaction, the juxtamembrane motif of VAMP2 is dispensable for formation of the SNARE-Munc18-1 complex.