Rab3GAP is a Rab18 GEF. (a) Rab3GAP complexes were used for GEF assays toward a representative group of Rab GTPases. Error bars indicate the standard deviation of the mean (n = 3). Rab3GAP complexes and individual subunits (GAP1 and GAP2) were analyzed on protein gels stained with colloidal Coomassie brilliant blue stain. (b) Mant-GDP kinetic GEF assays were performed using the Rab3GAP complex and Rab18 as a substrate to derive catalytic efficiency (k_cat/K_m). Initial rates of nucleotide exchange were derived from three independent experiments and are plotted as a function of Rab3GAP concentration. (c) Rab3GAP complexes and individual subunits were used for GEF assays toward Rab1b, Rab2a, and Rab18. Error bars indicate the standard deviation of the mean (n = 3). (d) HeLa cells were cotransfected for 20 h with GFP-Rabs and Myc-tagged Rab3GAP1 in the presence and absence of the Tom70-FLAG-Rab3GAP2 mitochondrial-targeting fusion. The cells were fixed and then stained with FLAG and Myc antibodies; Rabs were visualized using GFP fluorescence. Bars are marked in the figure.