(A) Fusion of proteoliposomes bearing Ypt7p and 4-SNAREs (1:5000 molar ratio to lipid phosphate), prepared with either the complete vacuole lipid mix (squares) or missing PE (diamonds), PE and DAG (triangles), or PE, DAG, and ERG (circles). Fusion was assayed as the FRET between lumenal fluorescent proteins in the continuous presence of an excess of external nonfluorescent streptavidin. (B) Proteoliposomes were analyzed for their protein composition by SDS-PAGE and Coomassie blue staining. Lipid composition: Lane 1, complete vacuolar lipid mix; lane 2, PE omitted; lane 3, PE and DAG omitted; lane 4, PE, DAG, and ERG omitted. In each case, the percentage of PC was increased to account for the omitted lipid(s). (C) Similar protease-accessibility of SNAREs and Rab across proteoliposomes preparations. Proteoliposomes (1.2 mM lipid) were incubated in 15 µl RB150 with 60 mM HEPES/NaOH pH 8.0 for 10 min at 27°C with either no addition of protease, with 60 µg/ml of proteinase K which had been preincubated for 10 min with 1 mM PMSF prior to proteoliposome addition (indicated by asterisk), with fully-active proteinase K which had not been preincubated with PMSF, or with fully active proteinase K and 1% (wt/vol) β-octylglucoside. After this incubation, PMSF was added to samples which had fully active proteinase K and the incubation continued for an additional 10 min. All samples were then mixed with SDS sample buffer, heated to 95°C for 5 min and subjected to SDS-PAGE. Gels were stained with Coomassie blue, and bands corresponding to Vam3p, Nyv1p, and Ypt7p quantified by scanning with a Microtek Bio-5000 scanner (Microtek Lab, Inc., Santa Fe Springs, CA) and UN-SCAN-IT gel 5.3 software (Silk Scientific, Orem, UT). For each of these three proteins, the intensity of the band from samples which never saw proteinase K was set to 100%. Dark bars correspond to VML proteoliposomes, light bars to proteoliposomes prepared without PE, DAG, and Erg. Shown is the average of two experiments +/− standard deviations. (D) The size distribution of various proteoliposome preparations was analyzed by dynamic light scattering with a Zetasizer nano ZS (Malvern Instruments Inc., Westborough, MA) through non-invasive back-scatter at 173°. For each liposome preparation, at least four samples (400 µl at a lipid concentration of 20 µM) were measured in low volume disposable sizing cuvettes at 25°C. Shown is the average diameter (+/− standard deviation) of independent proteoliposome preparations composed of the complete vacuolar lipid mix (dark bars) or without PE, ERG, DAG (light bars) and bearing either Nyv1 (1R; n = 3), all four SNAREs (4SNARE; n = 2), Vam3 and Vti1 (2Q; n = 1), or Nyv1, Vam3, and Vti1 (RQab; n = 2).