Rab5A mutants with impaired nucleotide exchange are not recruited by Rabex-5. (A) Rab5A mutants showing impaired nucleotide exchange induced by Rabex-5–VPS9. The indicated Rab5A mutants exhibit impaired nucleotide exchange. In this experiment, 1 µM Rab5A–GDP mutants were incubated with 50 nM Rabex-5–VPS9. The nucleotide exchange reaction was monitored by the decrease in intrinsic tryptophan fluorescence after addition of 100 µM GppNHp (all values have been normalized to the initial Rab5A–GDP fluorescence). (B–D) Confocal images of Cos-7 live cells expressing the mitochondrially localized ^mCherry-FRBActA cotransfected with ^FKBP-eGFPRabex-5 and ^mCitrineRab5A_A56D/Y82 (B), ^mCitrineRab5A_Y82A (C), or ^mCitrineRab5A_A56D (D). Addition of 1 µM A/C heterodimerizer (arrow) 24 h after transfection induces recruitment of Rabex-5 to mitochondria followed by Rab5A relocalization. The recruitment was quantified by calculating the PCC between ^mCherry-FRBActA and ^FKBP-eGFPRabex-5 or the ^mCitrineRab5A variants. Bars, 10 µm. Overlay images and defined time points are shown in Fig. S5 (A–C). (E) Catalytic efficiencies (k_cat/K_M) of Rabex-5–VPS9 toward Rab5A, Rab5A_A56D/Y82A, Rab5A_Y82A, and Rab5A_A56D (note the logarithmic scale of the y-axis). (F) Comparison between Rab5A, the double mutant Rab5A_A56D/Y82A, and the single mutants Rab5A_Y82A and Rab5A_A56D in localization to mitochondrial membranes by Rabex-5. A minimum of 50 cells in three independent experiments were assessed for the mitochondrial localized Rab5A variant. Data are shown as means ± SD.