Fig. 4. TBC1D20 is a GAP for Rab1 and Rab2. Biochemical assays for GTP hydrolysis with the Rabs and GAPs indicated were performed as described previously (Fuchs et al., 2007; Haas et al., 2005). All reactions were carried out for 60 minutes. (A) To determine the specific activity towards a range of GTPases, 0.5 pmoles TBC1D20 were tested against 100 pmoles of the Rab GTPases indicated, as well as ARF1 and Sar1. The basal GTP hydrolysis seen with a buffer control was subtracted for each GTPase. (B) 5 pmoles of wild-type and R105A mutant TBC1D20 full-length or the TBC-domain only (amino acids 1-317) were tested against 100 pmoles wild-type Rab1 or the Rab1 Q67L hydrolysis-defective mutant. Basal GTP hydrolysis seen with buffer is plotted as open bars, and GAP-stimulated GTP hydrolysis as grey bars. (C) Buffer (control) or 0.5 pmoles of the GAP indicated were tested against 100 pmoles Rabs 1 and 2. Basal GTP hydrolysis seen with buffer is plotted as open bars, and GAP-stimulated GTP hydrolysis as grey bars.