Defining roles of PARKIN and ubiquitin phosphorylation by PINK1 in mitochondrial quality control using a ubiquitin replacement strategy

The PTEN-induced putative kinase protein 1 ( PINK1) and ubiquitin (UB) ligase PARKINdirect damaged mitochondria for mitophagy. PINK1promotes PARKINrecruitment to the mitochondrial outer membrane (MOM) for ubiquitylation of MOM proteins with canonical and noncanonical UB chains. PINK1phosphorylates both Ser65 (S65) in the UB-like domain of PARKINand the conserved Ser in UB itself, but the temporal sequence and relative importance of these events during PARKINactivation and mitochondria quality control remain poorly understood. Using “UB S65A -replacement,” we find that PARKINphosphorylation and activation, and ubiquitylation of Lys residues on a cohort of MOM proteins, occur similarly irrespective of the ability of the UB-replacement to be phosphorylated on S65. In contrast, polyubiquitin (poly-UB) chain synthesis, PARKINretention on the MOM, and mitophagyare reduced in UB S65A -replacement cells. Analogous experiments examining roles of individual UB chain linkage types revealed the importance of K6 and K63 chain linkages in mitophagy, but phosphorylation of K63 chains by PINK1did not enhance binding to candidate mitophagyreceptors optineurin(OPTN), sequestosome-1(p62), and nuclear dotprotein 52 (NDP52) in vitro. Parallel reaction monitoring proteomics of total mitochondria revealed the absence of p-S65-UB when PARKINcannot build UB chains, and