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

The juxtamembrane motif of VAMP2 is comprised of basic and hydrophobic residues that directly interact with lipids ( Figures 1B and 4A ; Bowen and Brunger, 2006; Brewer et al., 2011; Ellena et al., 2009; Han et al., 2016) . It has been speculated that electrostatic interactions between the basic residues and lipid head groups are involved in the vesicle fusion reaction (Montal, 1999; Williams et al., 2009) . Direct evidence for this model, however, has been lacking. Next, we examined the functional role of a conserved stretch of basic residues (K85/R86/K87) in the juxtamembrane motif ( Figure 4A ). We observed that SNARE-Munc18-1-mediated liposome fusion was strongly inhibited when the K85/R86/K87 (KRK) stretch was deleted or mutated into alanines ( Figure 4B ), indicating a critical role of these residues in the fusion reaction. We reasoned that, if the KRK stretch promotes membrane fusion through electrostatic interactions with lipids, then its activity should rely on the overall charge rather than specific amino acids. To test this possibility, the KRK sequence was substituted with triple lysines (KKK) or triple arginines (RRR) ( Figure 4A ), both of which retained the overall charge of the juxtamembrane motif. Indeed, VAMP2 variants bearing the KKK or RRR substitutions were fully active in mediating SNARE-Munc18-1-mediated liposome fusion ( Figure 4B ). We then replaced the KRK sequence with six histidine residues (His 6 ), which are weakly basic at pH 7.4 ( Figure 4A ). We found that the VAMP2 variant bearing the His 6 substitution also supported SNARE-Munc18-1mediated liposome fusion, albeit with a lower efficiency ( Figure 4B ), consistent with partial protonation of histidine side chains at neutral pH. By contrast, substitution of the KRK stretch with acidic residues (EDE) abrogated SNARE-Munc18-1-mediated liposome fusion ( Figure 4B ). None of these mutations significantly affected basal SNARE-driven fusion ( Figure 4B ), confirming the dispensability of the juxtamembrane motif in the basal fusion reaction. These data demonstrate that the function of the juxtamembrane motif in membrane fusion requires the basic residues.