Figure 2. PINK1 phosphorylation on damaged mitochondria is inhibited by most pathogenic mutations.

(a) HeLa cells expressing PINK1-Flag with various pathogenic mutations were treated with CCCP, subjected to SDS–PAGE ± phos-tag and immunoblotted using an anti-PINK1 antibody. Red asterisks show phosphorylated PINK1. (b) PINK1^−/− MEFs co-expressing GFP-Parkin and various pathogenic PINK1 mutants were subjected to non phos-tag PAGE and immunoblotting with anti-Parkin and anti-PINK1 antibodies. Blue asterisks show autoubiquitylation of GFP-Parkin, which is taken as evidence of its mitochondrial localization. (c) The subcellular localization of GFP-Parkin in PINK1^−/− MEFs co-expressing various PINK1 mutants. Example figures indicative of colocalization and no colocalization are shown (bars, 10 μm). The number of cells with Parkin-positive mitochondria in the Parkin-expressing cells was counted in >100 cells. Error bars represent the mean±s.d. values of three experiments. Statistical significance was calculated using analysis of variance with a Tukey–Kramer post hoc test; *P<0.01; ns, not significant. (d) PINK1-GFP undergoes auto-phosphorylation following CCCP treatment. Red asterisks show the phosphorylated PINK1-GFP. (e) Various pathogenic PINK1 mutants were co-transfected with PINK1(WT)-GFP, treated with CCCP and subjected to immunoblotting using an anti-PINK1 antibody. Red asterisks show the phosphorylated bands. (f) HeLa cells expressing PINK1(G409V)-Flag with PINK1-GFP or PINK1(KD)-GFP were treated with CCCP and subjected to conventional PAGE. Red asterisks show phosphorylated PINK1(G409V)-Flag. FL, Δ1 and KD represent full-length PINK1, N-terminal processed PINK1 and KD mutant, respectively.