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

Next, we sought to determine whether the juxtamembrane motif of VAMP2 directly influences the integrity of membrane bilayers. A synthetic peptide encompassing the VAMP2 juxtamembrane motif was added to protein-free liposomes ( Figure 5A ). Using a dequenching-based liposome leakage assay, we observed that the juxtamembrane motif peptide induced significant content release from the population of liposomes (Figures 5B and 5C) . In contrast, peptides derived from other SNARE sequences, including the Npeptide of syntaxin-1 and the Vc peptide of VAMP2, did not induce content release ( Figures 5B and 5C ). Because membrane disruption is often accompanied by lipid mixing (Bailey et al., 1997; Thorén et al., 2005) , we next examined whether the SNARE-derived peptides can induce liposome lipid mixing. Indeed, the juxtamembrane motif peptide, but not the Npeptide or Vc peptide, induced lipid mixing of protein-free liposomes ( Figure S4 ), correlating with the membrane-disrupting activity of the juxtamembrane motif peptide ( Figures 5B and 5C ). Next, we used negative staining electron microscopy to visualize the effect of the juxtamembrane motif peptide on liposome morphology. We observed that protrusions emanated from liposomes incubated with the juxtamembrane motif peptide but not the N-peptide or Vc peptide ( Figure 5D ), consistent with the ability of the juxtamembrane motif peptide to remodel membrane bilayers.