Fig. 4.

DJ-1 localizes to the outer mitochondrial membrane after oxidative stress. (a) M17 cells were transfected with V5-tagged DJ-1 variants as indicated and stained for DJ-1 (V5, green) and mitotracker (red). After exposure to PQ²⁺ (Lower), DJ-1 colocalized with mitochondria much more extensively than under control conditions (Upper), as evidenced by the yellow color where staining overlaps. The C106A and C106D variants did not relocalize to mitochondria, whereas C53A and C46A did. This finding is quantified in b by counting the proportion of V5-positive cells that showed strong colocalization with mitotracker. Representative data are shown from at least three experiments for each construct, performed in duplicate. (c) DJ-1 is not imported into mitochondria. Cells transfected with WT DJ-1 were exposed to PQ²⁺ and mitochondrial fractions prepared. Duplicate samples were either untreated (-, lanes 1 and 2) or subjected to limited proteolysis with trypsin (+, lanes 3 and 4) and blotted for V5 (Top), the outer mitochondrial membrane protein VDAC1 (Middle), or the inner mitochondrial membrane protein Tim23 (Bottom).