Defining roles of PARKIN and ubiquitin phosphorylation by PINK1 in mitochondrial quality control using a ubiquitin replacement strategy
Open accessAbstract
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.
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The most important and better understood pathway to mitophagy is the canonical PINK1/PARKIN pathway.
“There are apparently several pathways to mitophagy [169,170], but the most important and the better understood one is the canonical PINK1/PARKIN pathway [171,172,173,174,175,176].”
From The mitochondrial permeability transition: Nexus of aging, disease and longevity (2021) · cites this paper as [172]
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- Authors
- Ordureau, A., Heo, J.-M., Duda, D. M., Paulo, J. A., Olszewski, J. L., Yanishevski, D., Rinehart, J., Schulman, B. A., & Harper, J. W.
- Affiliations
- Harvard University (US)
- St. Jude Children's Research Hospital (US)
- Department of Cell Biology, Harvard Medical School, Boston, MA 02115;
- Department of Structural Biology,
- Yale University (US)
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- National Academy of Sciences
- Published
- 2015-05-12
- Volume
- 112
- Issue
- 21
- Pages
- 6637-6642
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC4450373
- OpenAlex
- W2102313940
- Cited by
- 310
- References
- 26
- Citations by year
- 2026: 16 · 2025: 28 · 2024: 24 · 2023: 21 · 2022: 28 · 2021: 25 · 2020: 27 · 2019: 26 · 2018: 38 · 2017: 37 · 2016: 28 · 2015: 12
- FWCI
- 27.03
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- HHS | NIH | National Institute of General Medical Sciences (R37GM069530)
- HHS | NIH | National Institute of Neurological Disorders and Stroke (R37NS083524)
- Howard Hughes Medical Institute (045102)
- Topics
- Health Sciences › Medicine › Neurology › Parkinson's Disease Mechanisms and Treatments
- Health Sciences › Medicine › Epidemiology › Autophagy in Disease and Therapy
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
- Keywords
- PINK1, Parkin, mitophagy, mitochondrial quality control, ubiquitin phosphorylation, mitochondrial outer membrane, ubiquitination, optineurin, p62, NDP52, Miro
- MeSH terms
- PTEN-Induced Putative Kinase, Cell Line, HeLa Cells, Humans, Mitochondria, Models, Biological, Phosphorylation, Protein Kinases, Mitochondrial Proteins, Ubiquitin, Ubiquitin-Protein Ligases, Ubiquitination, Mitophagy
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