This study investigates the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization. PARKIN, a RING-HECT E3 ubiquitin ligase, plays a critical role in mitochondrial homeostasis and mitophagy. When mitochondrial depolarization occurs, PINK1 recruits PARKIN to the mitochondrial outer membrane (MOM), where it ubiquitylates various proteins, including Porin, Mitofusin, and Miro. The study uses quantitative diGLY capture proteomics to identify hundreds of dynamically regulated ubiquitylation sites in dozens of proteins, with a strong enrichment for MOM proteins, indicating that PARKIN significantly alters the ubiquitylation status of the mitochondrial proteome. Complementary interaction proteomics reveals that depolarization-dependent PARKIN associates with numerous MOM targets, autophagy receptors, and the proteasome. Mutation of PARKIN's active site residue C431, which is mutated in Parkinson's disease patients, disrupts these associations. Structural and topological analysis shows that PARKIN-dependent ubiquitylation sites are conserved in cytoplasmic domains of MOM proteins in vertebrates and D. melanogaster. The study identifies 4772 non-redundant ubiquitylation sites in 1654 proteins, with 443 diGLY sites identified in 261 proteins. These sites are enriched in mitochondrial proteins, including 36 MOM proteins. The study also identifies 582 PARKIN-dependent diGLY peptides in 303 proteins, with significant overlap across biological triplicates. Class 1 and Class 2 targets are defined based on their ubiquitylation sites and protein overlap. The study further identifies 838 diGLY sites in HCT116 and 337 diGLY sites in SH-SY5Y cells, with extensive overlap across data sets. The study also identifies 124 Tier 3 sites in 29 Class 1 or 2 proteins that are distinct from Class 1 sites. Many PARKIN-dependent diGLY targets are confirmed at endogenous PARKIN levels, and ubiquitylation of several candidate substrates is demonstrated by immunoblotting. The study also identifies 4 classes of high confidence candidate HA-PARKIN-interacting proteins (HCIPs) in response to depolarization, including 20 MOM proteins, autophagy adaptors, proteasome subunits, and the VCP/p97 ATPase. These findings provide a resource for understanding how the PINK1-PARKIN pathway resculpts the proteome to support mitochondrial homeostasis. The study also highlights the dynamic nature of PARKIN-dependent ubiquitylation and its role in mitochondrial homeostasis. The results suggest that PARKIN promotes rapid turnover of some targets but not others, and that its ubiquitylation activity is regulated by its N-terminal ubiquitin-like domainThis study investigates the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization. PARKIN, a RING-HECT E3 ubiquitin ligase, plays a critical role in mitochondrial homeostasis and mitophagy. When mitochondrial depolarization occurs, PINK1 recruits PARKIN to the mitochondrial outer membrane (MOM), where it ubiquitylates various proteins, including Porin, Mitofusin, and Miro. The study uses quantitative diGLY capture proteomics to identify hundreds of dynamically regulated ubiquitylation sites in dozens of proteins, with a strong enrichment for MOM proteins, indicating that PARKIN significantly alters the ubiquitylation status of the mitochondrial proteome. Complementary interaction proteomics reveals that depolarization-dependent PARKIN associates with numerous MOM targets, autophagy receptors, and the proteasome. Mutation of PARKIN's active site residue C431, which is mutated in Parkinson's disease patients, disrupts these associations. Structural and topological analysis shows that PARKIN-dependent ubiquitylation sites are conserved in cytoplasmic domains of MOM proteins in vertebrates and D. melanogaster. The study identifies 4772 non-redundant ubiquitylation sites in 1654 proteins, with 443 diGLY sites identified in 261 proteins. These sites are enriched in mitochondrial proteins, including 36 MOM proteins. The study also identifies 582 PARKIN-dependent diGLY peptides in 303 proteins, with significant overlap across biological triplicates. Class 1 and Class 2 targets are defined based on their ubiquitylation sites and protein overlap. The study further identifies 838 diGLY sites in HCT116 and 337 diGLY sites in SH-SY5Y cells, with extensive overlap across data sets. The study also identifies 124 Tier 3 sites in 29 Class 1 or 2 proteins that are distinct from Class 1 sites. Many PARKIN-dependent diGLY targets are confirmed at endogenous PARKIN levels, and ubiquitylation of several candidate substrates is demonstrated by immunoblotting. The study also identifies 4 classes of high confidence candidate HA-PARKIN-interacting proteins (HCIPs) in response to depolarization, including 20 MOM proteins, autophagy adaptors, proteasome subunits, and the VCP/p97 ATPase. These findings provide a resource for understanding how the PINK1-PARKIN pathway resculpts the proteome to support mitochondrial homeostasis. The study also highlights the dynamic nature of PARKIN-dependent ubiquitylation and its role in mitochondrial homeostasis. The results suggest that PARKIN promotes rapid turnover of some targets but not others, and that its ubiquitylation activity is regulated by its N-terminal ubiquitin-like domain