FIGURE 2:

The D127N DATL variant promotes both inner and outer leaflet lipid mixing. (A) Sample raw fluorescence trace monitoring inner leaflet mixing. Sodium dithionite was added twice to wild-type DATL donor and acceptor vesicles to ensure complete outer leaflet NBD quenching prior to the start of the reaction. After obtaining a flat baseline indicating no further quenching of the outer leaflet, 1 mM GTP was added to start the reaction. After completion of fusion, 0.5% Anaope X-100 was added to obtain maximum fluorescence. (B–E) Similar inner leaflet mixing kinetics were observed for wild type (B, D) and D127N (C, E) DATL at both saturating (B, C) and limiting (D, E) GTP. For reference, the data for each are plotted relative to total (inner plus outer) lipid mixing by the same variant at the same protein and GTP concentrations. All lipid mixing was performed at a 1:1000 protein/lipid ratio and a 1:2 donor/acceptor vesicle ratio. All data are the average of two technical replicates and similar results obtained with two biological replicates.