Parkin translocation to depolarized mitochondria was abrogated in U2OS-shUb cells in which the endogenous ubiquitin was replaced with S65A phosphorylation-deficient mutants. (A) All genomic ubiquitin genes in U2OS-shUb cells were knocked down and complemented with either HA-tagged WT ubiquitin (shUb-Ub(WT)) or a phosphorylation-deficient ubiquitin(S65A) mutant (shUb-Ub(S65A)) as the tetracycline (Dox)-inducible system. Both complemented ubiquitin proteins are fused with L40 and S27a (essential ribosomal proteins). To quantify the total and complementary ubiquitin levels, shUb-Ub(WT) and shUb-Ub(S65A) cells were subjected to immunoblotting using anti-ubiquitin or anti-HA antibodies. (B) Subcellular localization of GFP-Parkin in shUb-Ub(WT) or shUb-Ub(S65A) cells. Translocation of GFP-Parkin to depolarized mitochondria was observed in shUb-Ub(WT) cells, but was completely depressed in shUb-Ub(S65A) cells. Bars, 10 µm. Insets show 4× magnified views of the boxed regions. (C) The rate of Parkin mitochondrial localization in 100 cells was determined. Bars represent the mean ± SD (error bars) of three independent experiments. (D) Autoubiquitylation of GFP-Parkin and VDAC ubiquitylation in shUb-Ub(WT) or shUb-Ub(S65A) cells. Both ubiquitylation types observed after CCCP treatment in shUb-Ub(WT) cells (red vertical bar and asterisk) were substantially suppressed in shUb-Ub(S65A) cells.