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

To examine the function of the juxtamembrane motif in membrane fusion, synaptic exocytic SNAREs (syntaxin-1, SNAP-25, and VAMP2) and SM protein (Munc18-1) were reconstituted into a liposome fusion assay. The kinetics of the liposome fusion reactions were measured using lipid and content mixing assays (Yu et al., 2019) . The conserved residues of the juxtamembrane motif were mutated into alanines, and the VAMP2 mutant was reconstituted into proteoliposomes at the same density as the wild-type (WT) protein (Figures 2A, 2B , and S1A). We observed that the SNARE-Muncl8-1-mediated fusion reaction was strongly inhibited when the juxtamembrane motif was mutated (Figures 2C-2E, S1B, and S2). In contrast, the basal SNARE-driven fusion (without Munc18-1) was not affected by the mutation (Figures 2C-2E and S2). The selective involvement of the juxtamembrane motif in the SNARE-Munc18-1-mediated fusion reaction is consistent with the discovery that basal SNARE-driven fusion is fundamentally distinct from the SM protein-assisted fusion pathway (Baker et al., 2015; Jiao et al., 2018; Yu et al., 2018) . The SNARE-SM-mediated fusion reaction, but not basal SNARE-driven fusion, recapitulates intracellular vesicle fusion Yu et al., 2015 Yu et al., , 2018 .