Detection of a PINK1 phosphorylated ubiquitin chain in cells after a decrease in ΔΨm. (A) Mass-spectrometric (MS) analysis identified peptides with a phosphorylated S65 and a K63-GlyGly branch in the middle (14,000–55,000) and high (>55,000), but not low (<14,000), molecular weight fractions of cell lysates after CCCP treatment. The data shown are from a single MS analysis of three independently prepared samples. (B) The extracted m/z 574.29719 ion chromatogram corresponds to the doubly charged ubiquitin phosphopeptide EpSTLHLVLR, which was identified in immunoprecipitates using an Apu2 anti-K48–linked polyubiquitin chain antibody but not control IgG. This experiment was completed once (n = 1). (C) Retarded-mobility bands corresponding to K48-linked, K63-linked, and linear tetra-ubiquitin chains (red vertical lines) were observed in Phos-tag PAGE only after incubation with mitochondria isolated from CCCP-treated cells. P4D1, anti-ubiquitin antibody. IB, immunoblotting. (D) Incorporation of ³²P in the recombinant K48-linked and K63-linked tetra-ubiquitin chains was specifically detected when incubated with immunoprecipitated PINK1 (IP-PINK1) from digitonin-solubilized depolarized mitochondria (+ CCCP Mt). (E and F) TcPINK1 (indicated by the arrowhead in E) purified from E. coli was incubated with mono-ubiquitin or linear, K48-linked, and K63-linked tetra-ubiquitin chains. Retarded-mobility bands for all of the ubiquitin chains (F; red vertical lines) were observed in Phos-tag PAGE. Note that mobility does not reflect the molecular weight of proteins in Phos-tag PAGE (Kinoshita et al., 2012), and thus molecular weight markers are not shown. Black lines indicate that intervening lanes have been spliced out.