title: Defining roles of PARKIN and ubiquitin phosphorylation by PINK1 in mitochondrial quality control using a ubiquitin replacement strategy.
abstract: The PTEN-induced putative kinase protein 1 (PINK1) and ubiquitin (UB) ligase PARKIN direct damaged mitochondria for mitophagy. PINK1 promotes PARKIN recruitment to the mitochondrial outer membrane (MOM) for ubiquitylation of MOM proteins with canonical and noncanonical UB chains. PINK1 phosphorylates both Ser65 (S65) in the UB-like domain of PARKIN and the conserved Ser in UB itself, but the temporal sequence and relative importance of these events during PARKIN activation and mitochondria quality control remain poorly understood. Using "UB(S65A)-replacement," we find that PARKIN phosphorylation and activation, and ubiquitylation of Lys residues on a cohort of MOM proteins, occur similarly irrespective of the ability of the UB-replacement to be phosphorylated on S65. In contrast, polyubiquitin (poly-UB) chain synthesis, PARKIN retention on the MOM, and mitophagy are reduced in UB(S65A)-replacement cells. Analogous experiments examining roles of individual UB chain linkage types revealed the importance of K6 and K63 chain linkages in mitophagy, but phosphorylation of K63 chains by PINK1 did not enhance binding to candidate mitophagy receptors optineurin (OPTN), sequestosome-1 (p62), and nuclear dot protein 52 (NDP52) in vitro. Parallel reaction monitoring proteomics of total mitochondria revealed the absence of p-S65-UB when PARKIN cannot build UB chains, and <0.16% of the monomeric UB pool underwent S65 phosphorylation upon mitochondrial damage. Combining p-S65-UB and p-S65-PARKIN in vitro showed accelerated transfer of nonphosphorylated UB to PARKIN itself, its substrate mitochondrial Rho GTPase (MIRO), and UB. Our data further define a feed-forward mitochondrial ubiquitylation pathway involving PARKIN activation upon phosphorylation, UB chain synthesis on the MOM, UB chain phosphorylation, and further PARKIN recruitment and enzymatic amplification via binding to phosphorylated UB chains.
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115;
authors.forename: Alban
authors.initials: A
authors.lastname: Ordureau
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115;
authors.forename: Jin-Mi
authors.initials: JM
authors.lastname: Heo
authors.affiliation: Department of Structural Biology, Howard Hughes Medical Institute, St. Jude Children's Research Hospital, Memphis, TN 38105; and.
authors.forename: David M
authors.initials: DM
authors.lastname: Duda
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115;
authors.forename: Joao A
authors.initials: JA
authors.lastname: Paulo
authors.affiliation: Department of Structural Biology.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Olszewski
authors.affiliation: Department of Structural Biology.
authors.forename: David
authors.initials: D
authors.lastname: Yanishevski
authors.affiliation: Department of Cellular & Molecular Physiology, Systems Biology Institute, Yale University School of Medicine, New Haven, CT 06520.
authors.forename: Jesse
authors.initials: J
authors.lastname: Rinehart
authors.affiliation: Department of Structural Biology, Howard Hughes Medical Institute, St. Jude Children's Research Hospital, Memphis, TN 38105; and brenda.schulman@stjude.org wade_harper@hms.harvard.edu.
authors.forename: Brenda A
authors.initials: BA
authors.lastname: Schulman
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115; brenda.schulman@stjude.org wade_harper@hms.harvard.edu.
authors.forename: J Wade
authors.initials: JW
authors.lastname: Harper
mesh_terms: D002460:Cell Line; D006367:HeLa Cells; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000378:metabolism; D063306:Mitophagy; D008954:Models, Biological; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*; D054875:Ubiquitination; D000099202:PTEN-Induced Putative Kinase
chemical_list: D024101:Mitochondrial Proteins; D011494:Protein Kinases; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D000099202:PTEN-Induced Putative Kinase; C111567:parkin protein
keywords: PARKIN; PINK1; mitochondria; phosphorylation; ubiquitin