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

Proteoliposome reconstitution-To reconstitute t-SNARE liposomes for lipid-mixing assays, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoylsn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3phosphoserine (POPS) and cholesterol were mixed in a molar ratio of 60:20:10:10. To prepare v-SNARE liposomes for lipid-mixing assays, POPC, POPE, POPS, cholesterol, (N-(7-nitro-2,1,3-benzoxadiazole-4-yl)-1,2-dipalmitoyl phosphatidylethanolamine (NBD-DPPE) and N-(Lissamine rhodamine B sulfonyl)-1,2-dipalmitoyl phosphatidylethanolamine (rhodamine-DPPE) were mixed at a molar ratio of 60:17:10:10:1.5:1.5. SNARE proteoliposomes were prepared by detergent dilution and isolated by Nycodenz density gradient flotation Yu et al.,2018) . Detergent was removed by overnight dialysis using Novagen dialysis tubes against the reconstitution buffer (25 mM HEPES [pH 7.4], 100 mM KCl, 10% glycerol, and 1 mM DTT). To prepare sulforhodamine B-loaded liposomes for content-mixing assays, v-and t-SNAREs were reconstituted in the presence of 50 mM sulforhodamine B. Free sulforhodamine B was removed by overnight dialysis followed by liposome flotation on a Nycodenz gradient. The protein: lipid ratio of v-SNARE liposomes was 1:200, similar to VAMP2 densities on native synaptic vesicles (Takamori et al., 2006) , while the protein: lipid ratio of t-SNARE liposomes was 1:500. SNARE mutants were reconstituted into liposomes at the same molar densities as their respective WT proteins. Protein-free liposomes were prepared in a similar way as SNARE liposomes except that proteins were omitted.