Disease-associated mutations in Rab3GAP1 and Rab3GAP2 result in loss of Rab18 GEF activity. (a) A schematic of the Rab3GAP complex showing the Rab3 GAP domain and the conserved N-terminal region of Rab3GAP1. Pathological missense mutations in Rab3GAP1 and Rab3GAP2 are marked. Wild-type and disease mutant Rab3GAP complexes were used for GEF assays toward Rab1b, Rab2a, and Rab18. Error bars indicate the standard deviation of the mean (n = 3). (b) Wild-type Rab3GAP1, Rab3GAP1 and Rab3GAP2, or (c) wild-type and disease mutant Rab3GAP1 were used for GTPase assays with Rab3a or Rab3b. Error bars indicate the range (n = 2). This is a subset of the full screening data presented in the inset bar graph panel of Fig. S1 a. (d) HeLa cells were cotransfected for 20 h with GFP-Rabs, wild-type and disease mutant Myc-tagged Rab3GAP1, and the Tom70-FLAG-Rab3GAP2 mitochondrial-targeting fusion as indicated in the figure. The cells were fixed and then stained with FLAG and Myc antibodies; Rabs were visualized using GFP fluorescence. Bars are marked in the figure.