(A) Mapping of phosphopeptide on ubiquitin after phosphorylation by TcPINK1 in vitro. Ubiquitin (10 μg) was incubated with 10 μg of either wild-type TcPINK1 or kinase-inactive TcPINK1 (D359A) in the presence of Mg²⁺-[γ-³²P]ATP for 80 min. Assays were terminated by the addition of SDS loading buffer and products were separated by SDS/PAGE. Proteins were detected by Colloidal Coomassie Blue staining and phosphorylated ubiquitin was digested with trypsin. Peptides were chromatographed on a reverse-phase HPLC Vydac C₁₈ column equilibrated in 0.1% trifluoroacetic acid and the column developed with a linear acetonitrile gradient at a flow rate of 0.2 ml/min and fractions (0.1 ml each) were collected and analysed for ³²P radioactivity by Cerenkov counting. One major ³²P-labelled peak was identified following incubation with wild-type TcPINK1, whereas no peaks were identified following incubation with kinase-inactive TcPINK1 (results not shown). (B) The phosphopeptide identified in (A) was analysed by solid-phase Edman sequencing and MS. The amino acid sequence deduced from the single phosphopeptide seen in the LC–MS/MS analysis is shown using the single-letter amino acid code. (C) The S65A mutation abolishes ubiquitin phosphorylation by TcPINK1. Wild-type or S65A mutant ubiquitin (1 μg) was incubated in the presence of wild-type or kinase-inactive TcPINK1 (1 μg) and Mg²⁺-[γ-³²P]ATP for the times indicated and assays were terminated by the addition of SDS loading buffer. Samples were subjected to SDS/PAGE and proteins detected by Colloidal Coomassie Blue staining (bottom panels) and incorporation of [γ-³²P]ATP was detected by autoradiography (top panels). Cerenkov counting was used to calculate the stoichiometry of ubiquitin phosphorylation indicated above autoradiographs as mol of [γ-³²P]ATP incorporated/mol of ubiquitin. KI, kinase-inactive; WT, wild-type.