All lipids were obtained from Avanti Polar Lipids Inc. For t-SNARE reconstitution, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glyc-ero-3-phosphoserine (POPS) and cholesterol were mixed in a molar ratio of 60:20:10:10. For v-SNARE reconstitution, POPC, POPE, POPS, cholesterol, (N-(7-nitro-2,1,3-benzoxadiazole-4-yl)-1,2-dipalmi-toylphosphatidylethanolamine (NBD-DPPE) and N-(Lissamine rhod-amine B sulfonyl)-1,2-dipalmitoylphosphatidylethanolamine (rhod-amine-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 on a Nycodenz density gradient flotation.13 (link) Complete detergent removal was achieved by overnight dialysis of the samples in 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-loaded liposomes, t- or v-SNARE liposomes were reconstituted in the presence of 50 mM sulforhodamine B (Sigma). Free sulforhodamine B was removed by overnight dialysis followed by liposome flotation on a Nycodenz gradient. The protein: lipid ratio was at 1:200 for v-SNAREs and at 1:500 for t-SNARE liposomes.