Figure 1. Comparison of lipid mixing and content mixing assays.

(A) Assays used to study membrane fusion in vitro and possible outcomes. (B) Description of proteoliposomes as used in the following figures. (C–E) Two populations of 4SNARE-RPLs (with 1.5 mol% Marina-Blue-phosphatidylethanolamine [PE] and NBD-PE containing biotinylated R-phycoerythrin, or with no fluorescent lipids containing Cy5-labeled streptavidin; 250 µM lipid each) were incubated at 27°C with combinations of Sec17p, Sec18p, and HOPS, and assayed for 10 min for: lipid dequenching (C), lumenal content mixing protected from 5 µM external streptavidin (D), and mixing of lumenal content markers in the absence of external streptavidin, thus measuring both fusion and lysis (E). (F–H) Incubations were as in (C–E), but with RPLs bearing either the R-SNARE Nyv1p or the 3Q-SNAREs Vam3p, Vti1p, and Vam7p. Final concentrations of proteins were: Sec17p (600 nM), Sec18p (200 nM), and HOPS (100 nM). The mean and standard deviations of three experiments are shown.

DOI: http://dx.doi.org/10.7554/eLife.03251.003

Figure 1—figure supplement 1. Representative kinetic data for panels C–H in figure 1.

DOI: http://dx.doi.org/10.7554/eLife.03251.004

Figure 1—figure supplement 2. Sensitivity of lipid dequenching and lipid quenching assays to simulated rounds of fusion.

(A) Mixed micellar solutions of DLPC and β-octylglucoside, containing the fluorescently labeled lipids Marina-Blue-phosphatidylethanolamine (PE) (0.2 mol%) and NBD-PE (1.5 mol%), were mixed with unlabeled mixed micellar solutions prior to liposome formation. To simulate 0, 1, 2, 3, 4, or 5 rounds of fusion, 1 part of labeled lipid solution was mixed with 0, 1, 2, 3, 4, or 5 parts of unlabeled lipid solution. After liposome formation by dialysis, the fluorescence intensity of these liposomes (250 µM lipid) was determined in the presence or absence of detergent (0.1% [wt/vol] Thesit). The intensity measured in the absence of detergent is expressed as percentage of the signal intensity measured in the presence of detergent. (B) As described in (A), but 1 part of Marina-Blue-PE (0.2 mol%) mixed micellar solution was mixed with 0–5 parts of NBD-PE (3 mol%) mixed micellar solution prior to liposome formation.

DOI: http://dx.doi.org/10.7554/eLife.03251.005