Article | Published:

The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy

Nature volume 524, pages 309314 (20 August 2015) | Download Citation

Abstract

Protein aggregates and damaged organelles are tagged with ubiquitin chains to trigger selective autophagy. To initiate mitophagy, the ubiquitin kinase PINK1 phosphorylates ubiquitin to activate the ubiquitin ligase parkin, which builds ubiquitin chains on mitochondrial outer membrane proteins, where they act to recruit autophagy receptors. Using genome editing to knockout five autophagy receptors in HeLa cells, here we show that two receptors previously linked to xenophagy, NDP52 and optineurin, are the primary receptors for PINK1- and parkin-mediated mitophagy. PINK1 recruits NDP52 and optineurin, but not p62, to mitochondria to activate mitophagy directly, independently of parkin. Once recruited to mitochondria, NDP52 and optineurin recruit the autophagy factors ULK1, DFCP1 and WIPI1 to focal spots proximal to mitochondria, revealing a function for these autophagy receptors upstream of LC3. This supports a new model in which PINK1-generated phospho-ubiquitin serves as the autophagy signal on mitochondria, and parkin then acts to amplify this signal. This work also suggests direct and broader roles for ubiquitin phosphorylation in other autophagy pathways.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    & Selective autophagy. Essays Biochem. 55, 79–92 (2013)

  2. 2.

    , & Cargo recognition and trafficking in selective autophagy. Nature Cell Biol. 16, 495–501 (2014)

  3. 3.

    et al. PINK1 is selectively stabilized on impaired mitochondria to activate parkin. PLoS Biol. 8, e1000298 (2010)

  4. 4.

    , , & Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J. Cell Biol. 183, 795–803 (2008)

  5. 5.

    et al. PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity. J. Cell Biol. 205, 143–153 (2014)

  6. 6.

    et al. Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65. Biochem. J. 460, 127–139 (2014)

  7. 7.

    et al. Ubiquitin is phosphorylated by PINK1 to activate parkin. Nature 510, 162–166 (2014)

  8. 8.

    et al. PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nature Cell Biol. 12, 119–131 (2010)

  9. 9.

    & Optineurin is an autophagy receptor for damaged mitochondria in parkin-mediated mitophagy that is disrupted by an ALS-linked mutation. Proc. Natl Acad. Sci. USA 111, E4439–E4448 (2014)

  10. 10.

    , , , & p62/SQSTM1 is required for Parkin-induced mitochondrial clustering but not mitophagy; VDAC1 is dispensable for both. Autophagy 6, 1090–1106 (2010)

  11. 11.

    et al. p62/SQSTM1 cooperates with Parkin for perinuclear clustering of depolarized mitochondria. Genes Cells 15, 887–900 (2010)

  12. 12.

    , & Autophagic clearance of polyQ proteins mediated by ubiquitin-Atg8 adaptors of the conserved CUET protein family. Cell 158, 549–563 (2014)

  13. 13.

    et al. Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth. Science 333, 228–233 (2011)

  14. 14.

    et al. Adult-onset primary open-angle glaucoma caused by mutations in optineurin. Science 295, 1077–1079 (2002)

  15. 15.

    et al. Mutations of optineurin in amyotrophic lateral sclerosis. Nature 465, 223–226 (2010)

  16. 16.

    et al. Association between variants of PRDM1 and NDP52 and Crohn's disease, based on exome sequencing and functional studies. Gastroenterology 145, 339–347 (2013)

  17. 17.

    et al. Optineurin negatively regulates the induction of IFNβ in response to RNA virus infection. PLoS Pathog. 6, e1000778 (2010)

  18. 18.

    , , , & The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria. Nature Immunol. 10, 1215–1221 (2009)

  19. 19.

    , , & Enhanced binding of TBK1 by an optineurin mutant that causes a familial form of primary open angle glaucoma. FEBS Lett. 582, 997–1002 (2008)

  20. 20.

    et al. Crystal structure and mechanism of activation of TANK-binding kinase 1. Cell Rep. 3, 734–746 (2013)

  21. 21.

    et al. Quantitative proteomics reveal a feedforward mechanism for mitochondrial PARKIN translocation and ubiquitin chain synthesis. Mol. Cell 56, 360–375 (2014)

  22. 22.

    et al. Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis. EMBO J. 34, 307–325 (2015)

  23. 23.

    et al. PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65. Open Biol. 2, 120080 (2012)

  24. 24.

    et al. Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization. Nature 496, 372–376 (2013)

  25. 25.

    et al. Phosphorylation of mitochondrial polyubiquitin by PINK1 promotes Parkin mitochondrial tethering. PLoS Genet. 10, e1004861 (2014)

  26. 26.

    et al. Phosphorylated ubiquitin chain is the genuine Parkin receptor. J. Cell Biol. 209, 111–128 (2015)

  27. 27.

    et al. Defining roles of PARKIN and ubiquitin phosphorylation by PINK1 in mitochondrial quality control using a ubiquitin replacement strategy. Proc. Natl Acad. Sci. USA 112, 6637–6642 (2015)

  28. 28.

    et al. LC3C, bound selectively by a noncanonical LIR motif in NDP52, is required for antibacterial autophagy. Mol. Cell 48, 329–342 (2012)

  29. 29.

    , & The LC3 interactome at a glance. J. Cell Sci. 127, 3–9 (2014)

  30. 30.

    , & The autophagosome: origins unknown, biogenesis complex. Nature Rev. Mol. Cell Biol. 14, 759–774 (2013)

  31. 31.

    et al. Role of membrane association and Atg14-dependent phosphorylation in beclin-1-mediated autophagy. Mol. Cell. Biol. 33, 3675–3688 (2013)

  32. 32.

    , , & Temporal analysis of recruitment of mammalian ATG proteins to the autophagosome formation site. Autophagy 9, 1491–1499 (2013)

  33. 33.

    , , & AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nature Cell Biol. 13, 132–141 (2011)

  34. 34.

    , , & Structures containing Atg9A and the ULK1 complex independently target depolarized mitochondria at initial stages of Parkin-mediated mitophagy. J. Cell Sci. 125, 1488–1499 (2012)

  35. 35.

    , , & MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5. J. Cell Biol. 210, 435–450 (2015)

  36. 36.

    et al. Mitofusin-1 protein is a generally expressed mediator of mitochondrial fusion in mammalian cells. J. Cell Sci. 116, 2763–2774 (2003)

  37. 37.

    et al. Heritable gene targeting in zebrafish using customized TALENs. Nature Biotechnol. 29, 699–700 (2011)

  38. 38.

    et al. High-content genome-wide RNAi screens identify regulators of parkin upstream of mitophagy. Nature 504, 291–295 (2013)

  39. 39.

    et al. A TALE nuclease architecture for efficient genome editing. Nature Biotechnol. 29, 143–148 (2011)

  40. 40.

    et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013)

  41. 41.

    , , & Role of PINK1 binding to the TOM complex and alternate intracellular membranes in recruitment and activation of the E3 ligase Parkin. Dev. Cell 22, 320–333 (2012)

  42. 42.

    , , , & A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery. Chem. Biol. 18, 1042–1052 (2011)

Download references

Acknowledgements

We thank C. Nezich and S. Banerjee in the Youle laboratory, C. Smith and the NINDS and NHLBI Flow Cytometry Core Facilities. This work was supported by the Intramural Research Program of the NIH, NINDS and the National Health and Medical Research Council (GNT1063781).

Author information

Author notes

    • Michael Lazarou
    • , Danielle A. Sliter
    •  & Lesley A. Kane

    These authors contributed equally to this work.

    • Michael Lazarou

    Present address: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne 3800, Australia

Affiliations

  1. Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA

    • Michael Lazarou
    • , Danielle A. Sliter
    • , Lesley A. Kane
    • , Shireen A. Sarraf
    • , Chunxin Wang
    • , Jonathon L. Burman
    • , Dionisia P. Sideris
    • , Adam I. Fogel
    •  & Richard J. Youle

Authors

  1. Search for Michael Lazarou in:

  2. Search for Danielle A. Sliter in:

  3. Search for Lesley A. Kane in:

  4. Search for Shireen A. Sarraf in:

  5. Search for Chunxin Wang in:

  6. Search for Jonathon L. Burman in:

  7. Search for Dionisia P. Sideris in:

  8. Search for Adam I. Fogel in:

  9. Search for Richard J. Youle in:

Contributions

M.L., D.A.S., L.A.K. and R.J.Y. conceived the projects; M.L., D.A.S., L.A.K., S.A.S., C.W., D.P.S., A.I.F. and R.J.Y. designed experiments; M.L., D.A.S., L.A.K., S.A.S., C.W., J.L.B., D.P.S. and A.I.F. performed experiments; M.L., D.A.S., L.A.K. and R.J.Y. wrote the manuscript, and all authors contributed to editing the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Richard J. Youle.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary information

    This file contains figures showing the uncropped immunoblots present in the main text and extended data section of the manuscript.

Excel files

  1. 1.

    Supplementary Tables

    This file contains Supplementary Table 1, which shows the genotyping results.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature14893

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.