Mutation of conserved serine/threonine residues of Parkin does not completely inhibit Parkin translocation or activity. (A) YFP-Parkin is normally cytosolic (left panels), but upon mitochondrial damage (10 µM CCCP for 2.5 h), YFP-Parkin translocates to mitochondria and causes the ubiquitination proteins (right panels). Cells were stained for Tom20 (mitochondria, blue) and Ub (red). (B) ParkinΔUBL, as well as alanine mutants of Ser/Thr residues previously reported to be phosphorylated, were all capable of translocating to damaged mitochondria (10 µM CCCP for 2.5 h). Fewer cells expressing ParkinS65A displayed mitochondrial translocation than any other mutant (see Table S1). Cells were stained for Parkin (green) and Tom20 (mitochondria, red). For quantification and references of observed phosphorylation, see Table S1. (C) CCCP-treated (10 µM CCCP for 3 h) PINK1 KO cells expressing the indicated Parkin mutants showed that Parkin translocation is PINK1 dependent. (D) Phos-tag and SDS-PAGE gels revealed a shift of WT Parkin on Phos-tag gels after CCCP treatment (arrow), indicating it is phosphorylated (lane 2 vs. lane 3), and this phosphorylation was removed by phosphatase (CIP, lane 1). ParkinΔUBL, S65A, and S65E displayed no observable shift. (E) Parkin in vitro ubiquitination assay revealed that Parkin mutants are capable of ubiquitinating Mfn1 (bottom arrow). Cytosolic extracts from cells expressing the indicated Parkin mutants were incubated with mitochondria from cells not expressing Parkin (±CCCP). Ubiquitination of Mfn1 was observed (arrow + Ub and polyUb) only in the presence of mitochondria from CCCP-treated cells. Bars: (A–C) 10 µm.