Figure 4. Ser65 phosphorylation affects the subsequent autophagy reaction.

(a) CCCP-dependent degradation of mitochondrial outer membrane proteins in PINK1 WT/PINK1^−/− MEFs expressing WT or mutant forms of GFP-Parkin. Mfn1, VDAC1 and Tom20 were used as markers of mitochondrial outer membrane proteins. Actin: a loading control. Dots: ubiquitinated Mfn1. (b) Long-term time-course analysis of CCCP-dependent mitochondrial protein degradation. The degradation of outer membrane proteins was impaired in cells expressing GFP-Parkin S65A or S65E mutations. Hsp60 was used as a marker of mitochondrial matrix proteins. (c) S65A and S65E mutations do not affect proteasome recruitment to the mitochondria during mitophagy. PINK1 WT/PINK1^−/− MEFs expressing WT or mutant forms of GFP-Parkin (green) were treated with 30 µM CCCP for 3 or 6 hr. Cells were stained with anti-proteasome subunit alpha type 7 (α7, red). α7-immunoreactivity was enriched in the nuclei of all three cell genotypes under normal conditions, as displayed in the representative image of S65E (CCCP 0 hr), and overlapped with the aggregated mitochondria (arrowheads) 6 hr after CCCP treatment irrespective of genotype. Similar results were obtained 3 hr after CCCP treatment. Scale bar = 10 µm. (d) Model for Parkin translocation and activation. The Parkin Ubl domain masks C-terminal RING-IBR-RING (RBR) domains for E3 activity⁴⁶. A Parkin phosphorylation event at Ser65 (P), combined with unknown factor(s) (?), stimulates the mitochondrial translocation of Parkin, releasing the RBR domains from autoinhibition by the Ubl domain.