Lipid-mixing fusion assay

Fusion assays were performed as previously described¹³ with the modifications of the lipid compositions of donor and acceptor liposomes described above. Briefly, Sey1p and Yop1p were reconstituted at the indicated protein:lipid ratios into donor and acceptor liposomes in the presence of A100 buffer containing 0.1% DDM at room temperature. Donor vesicles contained NBD-PE and rhodamine-PE. After detergent removal by Bio-Beads SM-2 Resin (Bio-Rad), samples were spun once to remove insoluble material. Lipid concentration was determined based on rhodamine or dansyl fluorescence. Donor and acceptor liposomes were mixed in a 1:3 ratio in the presence of A100 buffer containing 5 mM MgCl₂. Data were subsequently collected every minute using a Flexstation III (Molecular Devices) at 37 °C. Pre-fusion data were collected during the first 10 min. The average of the pre-fusion data was set as the baseline fluorescence value. Buffer or 1 mM GTP was added to the reactions and the dequenching of NBD fluorescence data caused by the fusion of donor and acceptor vesicles was followed for 40 min. Triton-X 100 was then added to a final concentration of 2.5% and the reactions further incubated for 10 min to determine maximum fluorescence. The difference between the average of the maximum fluorescence data and the baseline value is defined as “total fluorescence”. The difference between the fusion data and the baseline value was then expressed as the percentage of the total fluorescence.