The study investigates the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization, a process crucial for mitophagy and mitochondrial homeostasis. Using quantitative diGLY capture proteomics, the researchers identified hundreds of dynamically regulated ubiquitylation sites in dozens of proteins, with a strong enrichment for mitochondrial outer membrane (MOM) proteins. This indicates that PARKIN significantly alters the ubiquitylation status of the mitochondrial proteome. Complementary interaction proteomics revealed that PARKIN associates with numerous MOM targets, autophagy receptors, and the proteasome. The mutation of PARKIN's active site residue C431, which is often mutated in Parkinson's Disease, disrupts these associations. Structural and topological analysis showed extensive conservation of PARKIN-dependent ubiquitylation sites on cytoplasmic domains in vertebrate and Drosophila MOM proteins. These findings provide insights into how the PINK1-PARKIN pathway re-sculpts the proteome to support mitochondrial homeostasis.The study investigates the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization, a process crucial for mitophagy and mitochondrial homeostasis. Using quantitative diGLY capture proteomics, the researchers identified hundreds of dynamically regulated ubiquitylation sites in dozens of proteins, with a strong enrichment for mitochondrial outer membrane (MOM) proteins. This indicates that PARKIN significantly alters the ubiquitylation status of the mitochondrial proteome. Complementary interaction proteomics revealed that PARKIN associates with numerous MOM targets, autophagy receptors, and the proteasome. The mutation of PARKIN's active site residue C431, which is often mutated in Parkinson's Disease, disrupts these associations. Structural and topological analysis showed extensive conservation of PARKIN-dependent ubiquitylation sites on cytoplasmic domains in vertebrate and Drosophila MOM proteins. These findings provide insights into how the PINK1-PARKIN pathway re-sculpts the proteome to support mitochondrial homeostasis.