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 all v-SNAREs, the SNARE motif is directly connected to the juxtamembrane motif without extra residues between them ( Figure 1A ). Insertions of flexible linkers between the SNARE and juxtamembrane motifs of VAMP2 strongly inhibit exocytosis in vivo (Deák et al., 2006; DeMill et al., 2014; Kesavan et al., 2007) . However, it was unclear whether and how the linker insertions directly influence the vesicle fusion reaction. Next, we introduced helix-breaking glycine and serine residues between the SNARE motif and juxtamembrane motif of VAMP2 ( Figure 7A ). We observed that these linker insertions had little effect on the basal SNARE-driven liposome fusion reaction ( Figure 7B ). Insertion of 21 residues only moderately reduced the basal SNARE-driven liposome fusion, whereas shorter insertions had no effect on the fusion kinetics ( Figure 7B ). We then examined how the linker insertions affect the SNARE-Munc18-1-mediated fusion reaction. We found that insertion of two residues resulted in normal fusion kinetics ( Figure 7B ). However, further extension of the linker strongly inhibited SNARE-Munc18-1-mediated liposome fusion ( Figure 7B ). With seven or more helix-disrupting residues added, the liposome fusion reaction was essentially reduced to the basal level ( Figure 7B ), similar to mutations of the juxtamembrane motif (Figures 2 and 4) .