Reconstitution of Proteoliposomes for SNARE and Ypt7 Studies

Proteoliposomes were prepared (Zick et al., 2014 (link)) from mixed micellar solutions (containing 50 mM β-octyl-glucoside) by detergent dialysis (20-kDa cutoff membrane) in RB150/Mg²⁺ (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid⋅NaOH, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 10% glycerol [vol/vol]) with individual SNAREs and prenylated Ypt7 at various molar protein:lipid ratios. Lipids dissolved in chloroform were mixed in vials containing β-octyl-glucoside at the following proportions. For vacuolar mixed lipids (16:0 18:1), 44.8–47.6 mol% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 18 mol% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 18 mol% soy l-α-phosphatidylinositol (PI), 4.4 mol% 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine, 2 mol% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate, 1 mol% 16:0 1,2-dipalmitoyl-sn-glycerol, 8 mol% ergosterol, and 1 mol% PI(3)P. For VML (18:2) compositions, as for RPLs used in Figures 3–7, lipids were mixed in the same proportions as described but with the dilinoleoyl forms of PC, phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidic acid (PA). All proteoliposomes throughout this study contained 0.2 mol% Marina-Blue-PE or 3 mol% NBD-PE.