Fig. 1. DJ-1 is localized to sub mitochondrial membrane vesicles enriched in ATP synthase (SMVs).

a Coomassie staining of the protein bands present in submitochondrial vesicles (SMVs) is shown in the left panel. The right panel shows an immunoblot using an anti-DJ-1 antibody against solubilized protein from SMVs. The blot confirms that DJ-1 is present in the SMV subcellular fraction. b WT and mutant DJ-1 immunolocalize with ATP synthase β-subunit. Extracts from HEK293 cells expressing flag-tagged WT or mutant DJ-1 were prepared for coimmunoprecipitation analysis with anti-FLAG antibody. Proteins were revealed with the indicated antibodies in western blots. Note that endogenous ATP synthase β-subunit is copurified with WT DJ-1 and also with mutant DJ-1. c Purified WT and mutant DJ-1 immunolocalize with ATP synthase β-subunit in vitro. Purified flag-ATP synthase β-subunit protein was incubated with purified DJ-1. The mixture was immuno-precipitated with anti-DJ-1 antibody. The protein interaction was revealed by western blotting with the indicated antibodies. d The ability of SMVs to sequester H⁺ is improved by recombinant WT DJ-1 protein. Shown are ACMA (H⁺ indicator) fluorescence intensity changes induced in response to ATP in the presence of rat SMVs. A decrease in indicator intensity represents sequestration of H⁺ into the lumen of SMVs. Data were acquired every 30 s for 5 min in the presence of BSA control protein or recombinant protein (all proteins were added at 10 µg/100 µl final). For WT DJ-1 (n = 6 samples), M3 DJ-1 (n = 8) or M5 DJ-1 (n = 8). WT DJ-1 protein maximizes the ability of SMVs to sequester H⁺ whereas mutant DJ-1 proteins have no effect. **p < 0.01, *p < 0.05, recombinant DJ-1 protein versus BSA control (n = 6). Paired, two-tailed t-test. Error bars represent mean ± SEM. e The ability of SMVs to sequester H⁺ is compromised in DJ-1^−/− SMVs. WT (n = 7) and DJ-1^−/− (n = 6). **p < 0.01, *p < 0.05, Error bars represent mean ± SEM