Figure 2. Ser65 in the Ubl domain of Parkin is phosphorylated upon depolarisation of ΔΨm.

(a) Phos-tag Western blotting detected phosphorylation of Ser65. HeLa cells were transiently transfected with Parkin WT and a series of alanine mutants for the candidate phospho-residues followed by treatment with or without 20 µM CCCP for 1 hr. Cell lysates were analysed by Phos-tag Western blotting. An asterisk indicates degraded Parkin. (b) Alignment of the amino acid sequences surrounding Ser65 (marked by a black dot) from a variety of animal species. The numbers on the left correspond to the residue numbers of Parkin proteins. (c) Introduction of the S65A mutation delayed Parkin translocation to the depolarised mitochondria in PINK1 WT/GFP-Parkin/PINK1^−/− MEFs. Cells retrovirally introduced with GFP-Parkin WT or its phospho-mutants (S65A and S65E) were treated with or without 30 µM CCCP for the indicated periods of time. GFP-Parkin and mitochondria were visualised with anti-GFP (green) and anti-Tom20 (red), respectively. Parkin signals are also shown as monochrome images. Scale bar = 10 µm. (d) Mitochondrial translocation efficiency of Parkin mutants. PINK1 WT/PINK1^−/− MEFs stably expressing GFP-Parkin WT, S65A or S65E were treated as in (c). Cells expressing GFP-Parkin perfectly overlapped (Complete, examples are shown on the right), partially overlapped (Partial) or non-overlapped (No) with the Tom20 signal were counted. The data represent means ± SE from three experiments (n = 99–143 cells in each). ** p < 0.01, * p < 0.05 vs. WT at each time point.