Figure 4. PINK1F104M mutant has the ability to recruit PARKIN to depolarized mitochondria followed by mitophagy. (A) Immunoblotting of HeLa cells expressing PINK1-YFP variants (F104F, F104M and 104Δ) treated with or without valinomycin (Val) for 3 h. Blue and red arrowheads represent the full-length and the cleaved forms of PINK1-YFP, respectively. (B and C) pink1−/− MEF cells were cotransfected with PINK1-YFP variants and mCherry-PARKIN. After 12 h of transfection, cells were treated with DMSO or valinomycin for 4 h. (B) Cells were then immunostained with anti-TOMM20. Scale bars: 10 μm. (C) Cells with mCherry-PARKIN on mitochondria were quantified. Error bars were calculated from three independent experiments. (D and E) pink1 KO HeLa cells stably expressing mCherry-PARKIN were cotransfected with YFP-LC3B and untagged either PINK1 WT(F104F) or PINK1(F104M) or pcDNA3.1(+) (vector). Cells were treated with valinomycin for 3 h, and immunostained with anti-TOMM20. (D) Scale bars: 10 μm. (E) Cells with YFP-LC3B on or near mitochondria were quantified. Error bars represent the results from three independent experiments. (F–I) pink1 KO HeLa cells stably expressing mCherry-PARKIN were transfected with WT PINK1-YFP (F104F), PINK1F104M-YFP or pEYFP-C1 (vector). Cells were treated with valinomycin for 12 h, and then immunostained with anti-TOMM20 or anti-HSPA9 antibodies. (F and H) YFP-positive cells are shown in white outlines. Scale bars: 10 μm. (G and I) Cells with complete degradation of TOMM20 or HSPA9 were quantified. Error bars represent the results from three independent experiments.