FIGURE 3:

GTP hydrolysis by D127N DATL does not account for fusion. (A) Steady state inorganic phosphate release by full-length wild type and D127N DATL during membrane fusion. (B-G) GTP hydrolysis by full-length and D127N DATL during membrane fusion. GTP hydrolysis by wild type (B, E) or D127N (C, F) DATL at 1 µM (B, C) or 5 µM (E, F) ³²P-α-GTP. (D) Quantification of B, C shows little or no hydrolysis by D127N DATL within the initial 60 s period of membrane fusion at 1 µM GTP. (G) Quantification of E, F shows little or no hydrolysis by D127N DATL within the first 120 s of membrane fusion at 5 µM GTP, though it is detected at later times (inset shows an enlarged view of the first 120 s). All assays were performed under fusion assay conditions with full-length DATL incorporated into liposomes at a 1:1000 protein/lipid ratio and DATL present at 0.6 µM (final concentration). In B, C, E, and F, reactions were acid quenched at the indicated time points and resolved by TLC. All TLC plates were exposed for 20 h to ensure maximal sensitivity for detecting GDP.