Ectopic lysosomal expression of phosphorylated ubiquitin chains recruit phosphomimetic Parkin. (A and B) Lysosomes were ubiquitylated via a K63-linked ubiquitin chain after LLOMe treatment. WT HeLa cells without Parkin expression were treated with LLOMe or CCCP, and were subjected to immunocytochemistry using anti-Lamp1 (lysosomal marker), anti-ubiquitin, and anti-K63–linked ubiquitin chain (Apu3) antibodies. (C) HeLa cells stably expressing PINK1 were transfected with WT Flag-ubiquitin or the phosphorylation-deficient Flag-ubiquitin(S65A) mutant. Cells were treated with CCCP and LLOMe before immunocytochemical staining using anti-Flag and α-pUb antibodies. The phosphorylated ubiquitin signal merged well with that of unphosphorylatable Flag-ubiquitin(S65A), suggesting that phosphorylated ubiquitin is passively incorporated into the ubiquitin chain on ruptured lysosomes. However, cells stably expressing PINK1-3×Flag, 2H8 antibody did not detect PINK1-3×Flag (3) because 2H8 reacts with only N-terminally fused mono Flag tag. Higher magnification views (2×) of the boxed areas are shown in the insets. (D) HeLa cells stably expressing PINK1 were transfected with the indicated Parkin mutants, treated with CCCP, LLOMe, or both, and then immunostained with the indicated antibodies. (E) The number of cells with Parkin-positive lysosomes under the indicated experimental conditions. Bars represent the mean ± SD (error bars) values of 100 cells in three independent experiments. Bars, 10 µm.