The PARKIN (PARK2) ubiquitin ligase and its regulatory kinase PINK1 (PARK6), often mutated in familial early onset Parkinson’s Disease (PD), play central roles in mitochondrial homeostasis and mitophagy. While PARKIN is recruited to the mitochondrial outer membrane (MOM) upon depolarization via PINK1 action and can ubiquitylate Porin, Mitofusin, and Miro proteins on the MOM, the full repertoire of PARKIN substrates – the PARKIN-dependent ubiquitylome – remains poorly defined. Graduate student Shireen Sarraf (Harper Lab) and colleagues used a method called "quantitative diGLY proteomics" to elucidate the ubiquitylation site-specificity and topology of PARKIN-dependent target modification in response to mitochondrial depolarization (Sarraf et al, Nature, 2013). This method was developed previously by postdocs Woong Kim (Gygi Lab) and Eric Bennett (Harper Lab) (Molecular Cell, 2011). Sarraf et al. identified hundreds of dynamically regulated ubiquitylation sites in dozens of proteins, with strong enrichment for MOM proteins, indicating that PARKIN dramatically alters the ubiquitylation status of the mitochondrial proteome. Using complementary interaction proteomics, Sarraf et al. found depolarization-dependent PARKIN association with numerous MOM targets, autophagy receptors, and the proteasome. Mutation of PARKIN’s active site residue C431, which has been found mutated in PD patients, largely disrupts these associations. Structural and topological analysis revealed extensive conservation of PARKIN-dependent ubiquitylation sites on cytoplasmic domains in vertebrate and D. melanogaster MOM proteins. These studies provide a resource for understanding how the PINK1-PARKIN pathway re-sculpts the proteome to support mitochondrial homeostasis, and can be interrogated via a website constructed by Mat Sowa (Harper Lab) that allows the dynamics and structural biology of modification sites to be mined. This paper provides the first global view of the site specificity for target modification by any E3 ubiquitin ligase.