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Proc Natl Acad Sci U S A. 2015 May 26;112(21):6637-42. doi: 10.1073/pnas.1506593112. Epub 2015 May 12.

Defining roles of PARKIN and ubiquitin phosphorylation by PINK1 in mitochondrial quality control using a ubiquitin replacement strategy.

Author information

  • 1Department of Cell Biology, Harvard Medical School, Boston, MA 02115;
  • 2Department of Structural Biology, Howard Hughes Medical Institute, St. Jude Children's Research Hospital, Memphis, TN 38105; and.
  • 3Department of Structural Biology.
  • 4Department of Cellular & Molecular Physiology, Systems Biology Institute, Yale University School of Medicine, New Haven, CT 06520.
  • 5Department 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.
  • 6Department of Cell Biology, Harvard Medical School, Boston, MA 02115; brenda.schulman@stjude.org wade_harper@hms.harvard.edu.

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.

KEYWORDS:

PARKIN; PINK1; mitochondria; phosphorylation; ubiquitin

PMID:
25969509
PMCID:
PMC4450373
DOI:
10.1073/pnas.1506593112
[PubMed - indexed for MEDLINE]
Free PMC Article
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