FIGURE 1:

The hydrolysis-deficient D127N DATL variant promotes only one round of lipid mixing. (A–E) Wild type or D127N DATL reconstituted into donor and acceptor vesicles and fusion monitored over time as the dequenching of NBD-labeled lipid in donor vesicles upon mixing with unlabeled lipid in acceptor vesicles at 37°C. (A) Lipid mixing by wild type or D127N DATL after the addition of 1 mM GTP. (B) Lipid mixing by wild-type DATL after the addition of the indicated final concentrations of GTP. (C) Lipid mixing by wild-type DATL after the addition of 1 µM GTP followed by two additional spikes of 1 µM GTP at the indicated times. (D) Lipid mixing by wild-type DATL preincubated with 5 mM ATP after the addition of 1 µM GTP with or without 1 U nucleoside diphosphate kinase. (E) Lipid mixing by D127N DATL after the addition of the indicated final concentrations of GTP. All lipid mixing was performed at a 1:1000 protein/lipid ratio, a 1:2 donor/acceptor vesicle ratio, and DATL present at 0.6 µM (final concentration). All data are the average of two technical replicates and similar results obtained with at least two biological replicates.