Physical interaction between phosphorylated polyubiquitin chains and Parkin. (A and B) Immunoprecipitation shows that phosphomimetic Parkin and phosphomimetic linear ubiquitin chains interact in cells. GFP-Parkin(S65E/C431S) immunoprecipitated from cell lysates was immunoblotted using an anti-V5 antibody that detects Mt-4×Ub. The interaction depends on phosphomimetic mutation of both the ubiquitin chain (A) and Parkin (B). (C and D) A mitochondria-localized linear ubiquitin chain (Mt-4×Ub) recruits Parkin in a CCCP- and PINK1-dependent manner. PINK1 KO HeLa cells were transfected and treated as described in the text, and the GFP-Parkin(S65E/C431S)-derived signal was observed with a fluorescence microscope (C). Bars, 10 µm. The rate of Parkin mitochondrial localization was determined (D). KD, kinase dead; ΔN155, lacking the first 155 N-terminal amino acids. Bars represent the mean ± SD (error bars) values of 100 cells in three independent experiments. (E) Phosphorylation of the linear ubiquitin chain was confirmed by immunoblotting using α-pUb. (F) Interaction between phosphomimetic Parkin and a phosphorylated polyubiquitin chain. GFP-Parkin(S65E/C431S) was immunoprecipitated from PINK1 KO HeLa cells transfected and treated as described in the text, and immunoblotted with an anti-V5 antibody for Mt-4×Ub and α-pUb. (G) Direct interaction between recombinant phosphomimetic Parkin and a phosphorylated ubiquitin chain. Polyubiquitin chain–conjugated agarose beads were phosphorylated by TcPINK1. Recombinant GST-Parkin(WT) or the phosphomimetic GST-Parkin(S65E) mutant were incubated with the agarose beads and examined for GST-Parkin capture by the phosphorylated ubiquitin-conjugated agarose. (H) Model for phosphorylated polyubiquitin chain recruitment of Parkin to damaged mitochondria. See the text for details.