Proteoliposomes for content-mixing assays were prepared by detergent dialysis in RB150/Mg2+ (20 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 10% glycerol [vol/vol]) as described (Zucchi and Zick, 2011 (link)) from lipid mixes mimicking the vacuolar composition (43.6 mol% 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 18 mol% 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 18 mol% soy l-α-phosphatidylinositol, 4.4 mol% 1,2-dilinoleoyl-sn-glycero-3-phospho-l-serine, 2 mol% 1,2-dilinoleoyl-sn-glycero-3-phosphate, 1 mol% 16:0 1,2-dipalmitoyl-sn-glycerol [all from Avanti Polar Lipids, Alabaster, AL]; 8 mol% ergosterol [Sigma-Aldrich, St. Louis, MO], 1 mol% each of di-C16 phosphatidylinositol 3-phosphate and phosphatidylinositol 4,5-bisphosphate [Echelon Biosciences] and 3 mol% 7-nitrobenz-2-oxa-1,3-diazole [NBD]–1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine [DPPE; Life Technologies, Carlsbad, CA]) for donor liposomes or 3 mol% Marina-Blue-DPPE for acceptor, subsets of the four vacuolar SNAREs, and Ypt7p, entrapping Cy5-labeled streptavidin or biotinylated R-phycoerythrin. Molar protein:lipid ratio was 1:2500 for SNAREs and 1:2000 for Ypt7p. Isolation after reconstitution was achieved by floatation on a three-step Histodenz gradient (35, 25% Histodenz [wt/vol] and RB150/Mg2+). Histodenz (Sigma-Aldrich) solutions were prepared as 70% stock solution in modified RB150/Mg2+ with a reduced concentration (2% [vol/vol]) of glycerol to compensate for the osmotic activity of the density medium; lower-concentration solutions were obtained by dilution with RB150/Mg2+. RPLs for experiments based on lipid dequenching were prepared with 1.5 mol% of NBD-DPPE and rhodamine-DPPE for donor RPLs and without fluorescent lipids for acceptor RPLs. RPLs for experiments based on tethering via streptavidin were prepared with 0.1 mol% 18:1 Biotinyl Cap PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) [Avanti Polar Lipids]) and without entrapped content markers.