The molecular mechanism underlying PINK1-madiated mitochondrial maintenance

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We have reported that PGAM5 acts between PINK1 and Parkin. To understand the molecular roles of PAGM5 in the PINK1-Parkin pathway further, we searched for PGAM5-binding proteins using mass spectrometric analysis and screened genes encoding these binding proteins using Drosophila PINK1 models. We found that Rictor modulates the phenotypes caused by PINK1 inactivation in Drosophila. However, PINK1 did not modify the phosphorylation status of Rictor in cultured cells, suggesting that Rictor is not a direct substrate of PINK1. To isolate physiological substrates of PINK1, we performed phospho-proteomics analysis and subsequent in vitro kinase assay using normal and PINK1 mutant cells. We then found that Ser65 in the ubiquitin-like domain (Ubl) of Parkin is phosphorylated in a PINK1-dependent manner upon depolarization of mitochondrial membrane potential. We further showed that the phosphorylation of Parkin is required for the activation of its ubiquitin-ligase activity.