Digital snapshot proteomics of mitochondrial ubiquitylation events during Parkin-mediated mitophagy in model systems and neurons Alban Ordureau1, Joao A Paulo1, Jiuchun Zhang1, Wade Harper1 1Harvard Medical School, Department of Cell Biology, Boston, MA
Flux through kinase and ubiquitin (Ub)-driven signaling systems depends upon the modification kinetics, stoichiometry, primary site-specificity, and target abundance within the pathway, yet we rarely understand these parameters and their spatial organization within cells. Recent work indicates that a major role for the kinase PINK1 and E3 Ub ligase Parkin, two proteins mutated in Parkinson's disease, is in mitochondrial outer membrane (MOM) ubiquitylation, which promotes mitophagy. We have previously found that Parkin can build K6, K11, K48, and K63 Ub chains in response to mitochondrial depolarization. The ability of Parkin to build these chains is greatly stimulated by PINK1-dependent phosphorylation of not only Parkin, but also Ub itself. Importantly, phosphorylation of Ub chains by PINK1 as they are assembled by Parkin leads to further recruitment of Parkin through its phospho-Ub binding site. Here, we've developed temporal digital snapshots of Ub signaling on the MOM in WT or gene-edited embryonic stem cell-derived neurons and model HeLa cell systems upon activation of PINK1 and Parkin by proteomic counting of ubiquitylation and phosphorylation events. This includes over 150 peptides representative of 15 proteins to examine primary ubiquitylation and phosphorylation sites on MOM proteins. We have defined the kinetics and site-specificity of Parkin-dependent target ubiquitylation in neurons and demonstrate the power of this approach to quantify pathway modulators endogenously, and to also examine mechanisms related to the role of Ub and Parkin phosphorylation in mitophagic flux. The ability to accurately monitor Parkin activity at the primary substrate level is critical for multiple purposes, including: a) measuring PINK1/Parkin pathway activity precisely in cells, b) measuring the effect of small molecule activators or inhibitors of the pathway on mitochondrial ubiquitylation, c) examining if there is selectivity in Parkin substrate targeting in various experimental settings. In summary, we demonstrate that we can now quantitatively monitor primary ubiquitylation of sites in a cohort of substrates simultaneously using this novel assay and advance more generally our understanding of Ub signaling cascades.
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