Defining the landscape of the PARKIN- and PINK1-dependent ubiquitin-modified proteome in response to mitochondrial dysfunction

Parkinson's disease (PD) is a progressive neurological disorder resulting from loss of dopaminergic neurons of the substantia nigra, in part due to mitochondrial dysfunction. The E3 ubiquitin ligase, PARKIN, and mitochondrial kinase, PINK1, found mutated in familial early onset recessive forms of PD play central roles in mitochondrial homeostasis, thus maintaining control of a diversity of cellular processes, including energy metabolism, calcium buffering, and cell death. Together, PARKIN and PINK1 control mitochondrial homeostasis via a signaling cascade in which depolarization&ndashinduced PINK1 stabilization and activation on the mitochondrial outer membrane (MOM) promotes recruitment of PARKIN. Consequently, the outer mitochondrial membrane is extensively decorated with ubiquitin, ultimately resulting in removal of the damaged organelles via mitophagy, the selective autophagic removal of mitochondria. While PARKIN has been demonstrated to ubiquitylate Porin, Mitofusin, and Miro proteins on the MOM, the full repertoire of PARKIN substrates — the PARKIN&ndashdependent ubiquitylome — remains poorly defined. Here, large–scale quantitative diGlycine (diGly) capture proteomics was used to identify PARKIN–dependent ubiquitylation on lysine residues in proteins modified upon mitochondrial depolarization. Hundreds of ubiquitylation sites in dozens of proteins were identified, with strong enrichment for MOM proteins, indicating that PARKIN activity has the capacity to dramatically alter the ubiquitylation status of the mitochondrial proteome. Complementary interaction proteomics identified physical association of PARKIN with a cohort of MOM ubiquitylation targets, autophagy receptors, and the proteasome, interactions which were completely dependent upon mitochondrial damage and drastically reduced upon mutation of the active site residue, C431, found mutated in PD patients. Furthermore, structural and evolutionary analysis of PARKIN&ndashdependent ubiquitylation events revealed extensive conservation of target sites on cytoplasmic domains in vertebrate and D. melanogaster MOM proteins. Parallel PINK1 interaction proteomics identified numerous subunits of the translocase of the outer mitochondrial membrane (TOMM) and a novel interactor, CLU1, shown to regulate mitochondrial morphology in lower eukaryotes. Positive genetic interaction between CLU1, PINK1, and PARKIN suggests the potential of a newly identified node of regulation for the PINK1/PARKIN pathway. These studies define how PARKIN and PINK1 re&ndashsculpt the proteome to support mitochondrial homeostasis, ultimately contributing toward an improved understanding of their role in the progression of disease.