Nucleotide binding and GTPase activity of hLsg1. (A) GTP binding of hLsg1. Nucleotide binding was measured as described in Materials and Methods. BSA was used as control, while Sar1p-WT and the GDP-restricted Sar1p mutant (Sar1p-T39N) were used as positive and negative GTP binding controls, respectively. The graph is the sum of three separate experiments. (B) GTPase activity of purified recombinant and immunoprecipitated hLsg1. Elution times of GDP and GTP standards are indicated (top panel). GTPase activities of purified recombinant Sar1-WT and hLsg1 are shown (middle panel) as well as GTPase activities of immunoprecipitated Sar1p and hLsg1 (lower panel). Incubation times were identical (18 h) except for the hLsg1 precipitate (4 h). (C) Hydrolysis of GTP by hLsg1. GTPase activities of purified recombinant hLsg1 were analyzed by HPLC as described in Materials and Methods. A solution containing 5 μM hLsg1 and 200 μM GTP was incubated at 37°C. Samples were taken at different time-points and analyzed for percentages of GTP and GDP.