Phosphomimetic ubiquitin chains specifically recruit phosphomimetic Parkin to mitochondria even in the absence of PINK1. (A) WT and PINK1 KO HeLa cells expressing GFP-Parkin(S65E/C431S) and Mt-4×Ub(WT) were treated with MG-132 (10 µM, 3 h), stained with TMRE, and observed with a fluorescence microscope. Accumulation of linear ubiquitin causes mitochondrial aggregation as reported previously (Okatsu et al., 2010). Bars, 10 µm. (B) The number of cells with GFP-Parkin(S65E/C431S) localized to the mitochondria. Bars represent the mean ± SD (error bars) values of 100 cells in three independent experiments. (C) PINK1 is essential for Mt-4×Ub(WT) recruitment of phosphomimetic Parkin. Mt-4×Ub and MG-132-dependent mitochondrial localization of GFP-Parkin(S65E/C431S) in PINK1 KO HeLa cells with or without PINK1 reintroduction is shown. Cell counting was performed as in B. Error bars represent mean ± SD. (D) PINK1 KO HeLa cells coexpressing GFP-Parkin(S65E/C431S) with WT, phosphorylation-deficient (S65A), or phosphomimetic (S65D) Mt-4×Ub with or without MG-132. Bars, 10 µm. (E) Statistical analysis of D. SA, S65A; SD, S65D. Error bars represent mean ± SD. (F) PINK1 KO HeLa cells expressing the indicated GFP-Parkin mutants and Mt-4×Ub after MG-132 treatment were observed with a fluorescence microscope, and the number of cells with Parkin-positive mitochondria was determined as in B. Error bars represent mean ± SD. SA, S65A; SD, S65D.