OATL1 functions as a GAP specific for Rab33B. (A) Rab33B is a specific substrate of OATL1. The Rab-GAP activity of OATL1 toward indicated Rab proteins is summarized. The GTPase activity of each Rab protein in the presence of BSA or OATL1 is shown. A significant p-value is shown (Student’s unpaired t test). (B) Dose dependency of the GAP activity of OATL1 is shown. GTP hydrolysis by Rab33B in the presence of BSA or the concentrations of OATL1 indicated was measured. (C) A conserved catalytic arginine residue in the TBC domain of OATL1 is essential for its GAP activity. GTP hydrolysis by Rab33B in the presence of BSA, OATL1, or OATL1-RK was measured. A significant p-value is shown (Student’s unpaired t test). (B and C) The results are expressed as the amount of the GTP-bound form of Rab33B after the reaction as a percentage of the amount before the reaction. Error bars represent the means ± SEM of data from three determinations. (D) OATL1 is the only TBC protein that possesses strong Rab33B-GAP activity. Total lysates of COS-7 cells expressing FLAG-Rab33B alone (control; lanes 1, 9, and 27) or FLAG-Rab33B and the T7-tagged TBC proteins indicated (lanes 2–8, 10–26, and 28–43) were incubated with glutathione–Sepharose beads that had been coupled with purified T7–GST-Atg16L1 used as a trapper of active Rab33B. Total lysates (input; top and bottom) and proteins trapped by the Atg16L1 beads (IP, immunoprecipitation; middle two panels) were analyzed by immunoblotting with anti-FLAG tag antibody (top two panels) and anti-T7 tag antibody (bottom two panels). Input means 1/60 volume of the reaction mixture. Black lines indicate that intervening lanes have been spliced out.