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Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization


This article has been updated


The PARKIN ubiquitin ligase (also known as PARK2) and its regulatory kinase PINK1 (also known as PARK6), often mutated in familial early-onset Parkinson’s disease, have central roles in mitochondrial homeostasis and mitophagy1,2,3. Whereas PARKIN is recruited to the mitochondrial outer membrane (MOM) upon depolarization via PINK1 action and can ubiquitylate porin, mitofusin and Miro proteins on the MOM1,4,5,6,7,8,9,10,11, the full repertoire of PARKIN substrates—the PARKIN-dependent ubiquitylome—remains poorly defined. Here we use quantitative diGly capture proteomics (diGly)12,13 to elucidate the ubiquitylation site specificity and topology of PARKIN-dependent target modification in response to mitochondrial depolarization. Hundreds of dynamically regulated ubiquitylation sites in dozens of proteins were identified, with strong enrichment for MOM proteins, indicating that PARKIN dramatically alters the ubiquitylation status of the mitochondrial proteome. Using complementary interaction proteomics, we found depolarization-dependent PARKIN association with numerous MOM targets, autophagy receptors, and the proteasome. Mutation of the PARKIN active site residue C431, which has been found mutated in Parkinson’s disease patients, largely disrupts these associations. Structural and topological analysis revealed extensive conservation of PARKIN-dependent ubiquitylation sites on cytoplasmic domains in vertebrate and Drosophila melanogaster MOM proteins. These studies provide a resource for understanding how the PINK1–PARKIN pathway re-sculpts the proteome to support mitochondrial homeostasis.

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Figure 1: QdiGly proteomics for PARKIN-dependent ubiquitylation.
Figure 2: PARKIN-dependent ubiquitylation sites revealed by QdiGly proteomics.
Figure 3: PARKIN associates with mitochondrial proteins and the proteasome in response to depolarization.
Figure 4: Structural anatomy and conservation of PARKIN-dependent diGly sites.

Change history

  • 17 April 2013

    Two gene names were corrected (PDCD6IP and IER3IP1) and an indicated site on CLTC and MAOB was removed.


  1. Narendra, D., Walker, J. E. & Youle, R. Mitochondrial quality control mediated by PINK1 and Parkin: links to parkinsonism. Cold Spring Harb. Perspect. Biol. 4, a011338 (2012)

    Article  Google Scholar 

  2. Youle, R. J. & Narendra, D. P. Mechanisms of mitophagy. Nature Rev. Mol. Cell Biol. 12, 9–14 (2011)

    CAS  Article  Google Scholar 

  3. Dawson, T. M. & Dawson, V. L. The role of parkin in familial and sporadic Parkinson’s disease. Mov. Disord. 25 (Suppl 1). S32–S39 (2010)

    Article  Google Scholar 

  4. Chan, N. C. et al. Broad activation of the ubiquitin-proteasome system by Parkin is critical for mitophagy. Hum. Mol. Genet. 20, 1726–1737 (2011)

    CAS  Article  Google Scholar 

  5. Glauser, L., Sonnay, S., Stafa, K. & Moore, D. J. Parkin promotes the ubiquitination and degradation of the mitochondrial fusion factor mitofusin 1. J. Neurochem. 118, 636–645 (2011)

    CAS  Article  Google Scholar 

  6. Ziviani, E., Tao, R. N. & Whitworth, A. J. Drosophila parkin requires PINK1 for mitochondrial translocation and ubiquitinates mitofusin. Proc. Natl Acad. Sci. USA 107, 5018–5023 (2010)

    ADS  CAS  Article  Google Scholar 

  7. Tanaka, A. et al. Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin. J. Cell Biol. 191, 1367–1380 (2010)

    CAS  Article  Google Scholar 

  8. Poole, A. C., Thomas, R. E., Yu, S., Vincow, E. S. & Pallanck, L. The mitochondrial fusion-promoting factor mitofusin is a substrate of the PINK1/parkin pathway. PLoS ONE 5, e10054 (2010)

    ADS  Article  Google Scholar 

  9. Sun, Y., Vashisht, A. A., Tchieu, J., Wohlschlegel, J. A. & Dreier, L. VDACs recruit Parkin to defective mitochondria to promote mitochondrial autophagy. J. Biol. Chem. 287, 40652–40660 (2012)

    CAS  Article  Google Scholar 

  10. Wang, X. et al. PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility. Cell 147, 893–906 (2011)

    CAS  Article  Google Scholar 

  11. Liu, S. et al. Parkinson’s disease-associated kinase PINK1 regulates Miro protein level and axonal transport of mitochondria. PLoS Genet. 8, e1002537 (2012)

    CAS  Article  Google Scholar 

  12. Kim, W. et al. Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol. Cell 44, 325–340 (2011)

    CAS  Article  Google Scholar 

  13. Wagner, S. A. et al. A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles. Mol. Cell Proteomics 10, M111.013284 (2011)

    Article  Google Scholar 

  14. Wenzel, D. M., Lissounov, A., Brzovic, P. S. & Klevit, R. E. UbcH7-reactivity profile reveals Parkin and HHARI to be RING/HECT hybrids. Nature 474, 105–108 (2011)

    CAS  Article  Google Scholar 

  15. Joselin, A. P. et al. ROS-dependent regulation of Parkin and DJ-1 localization during oxidative stress in neurons. Hum. Mol. Genet. 21, 4888–4903 (2012)

    CAS  Article  Google Scholar 

  16. Narendra, D., Tanaka, A., Suen, D. F. & Youle, R. J. Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J. Cell Biol. 183, 795–803 (2008)

    CAS  Article  Google Scholar 

  17. Lazarou, M., Jin, S. M., Kane, L. A. & Youle, R. J. 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)

    CAS  Article  Google Scholar 

  18. Kondapalli, C. 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)

    Article  Google Scholar 

  19. Sha, D., Chin, L. S. & Li, L. Phosphorylation of parkin by Parkinson disease-linked kinase PINK1 activates parkin E3 ligase function and NF-κB signaling. Hum. Mol. Genet. 19, 352–363 (2010)

    CAS  Article  Google Scholar 

  20. Lazarou, M. et al. PINK1 drives Parkin self-association and HECT-like E3 activity upstream of mitochondrial binding. J. Cell Biol. 200, 163–172 (2013)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  22. Sowa, M. E., Bennett, E. J., Gygi, S. P. & Harper, J. W. Defining the human deubiquitinating enzyme interaction landscape. Cell 138, 389–403 (2009)

    CAS  Article  Google Scholar 

  23. Chaugule, V. K. et al. Autoregulation of Parkin activity through its ubiquitin-like domain. EMBO J. 30, 2853–2867 (2011)

    CAS  Article  Google Scholar 

  24. Narendra, D., Kane, L. A., Hauser, D. N., Fearnley, I. M. & Youle, R. J. p62/SQSTM1 is required for Parkin-induced mitochondrial clustering but not mitophagy; VDAC1 is dispensable for both. Autophagy 6, 1090–1106 (2010)

    CAS  Article  Google Scholar 

  25. Onoue, K. et al. Fis1 acts as mitochondrial recruitment factor for TBC1D15 that is involved in regulation of mitochondrial morphology. J. Cell Sci.. (2012)

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

    CAS  Article  Google Scholar 

  27. Huang, P., Galloway, C. A. & Yoon, Y. Control of mitochondrial morphology through differential interactions of mitochondrial fusion and fission proteins. PLoS ONE 6, e20655 (2011)

    ADS  CAS  Article  Google Scholar 

  28. Villén, J. & Gygi, S. P. The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry. Nature Protocols 3, 1630–1638 (2008)

    Article  Google Scholar 

  29. Haas, W. et al. Optimization and use of peptide mass measurement accuracy in shotgun proteomics. Mol. Cell. Proteomics 5, 1326–1337 (2006)

    CAS  Article  Google Scholar 

  30. Huttlin, E. L. et al. A tissue-specific atlas of mouse protein phosphorylation and expression. Cell 143, 1174–1189 (2010)

    CAS  Article  Google Scholar 

  31. Eng, J. K., McCormack, A. L. & Yates, J. R., III An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989 (1994)

    CAS  Article  Google Scholar 

  32. Beausoleil, S. A., Villen, J., Gerber, S. A., Rush, J. & Gygi, S. P. A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nature Biotechnol. 24, 1285–1292 (2006)

    CAS  Article  Google Scholar 

  33. Huang, D. W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature Protocols 4, 44–57 (2009)

    CAS  Article  Google Scholar 

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We thank W. Kim for LC-MS and for development of QdiGly profiling, R. Kunz for peptide purification, J. Lydeard, S. Hayes and A. White for proteomics, M. Comb and S. Beausoleil (Cell Signaling Technologies) for antibodies, Nikon Imaging Center (Harvard Medical School) for microscopy, and D. Finley and B. Schulman for discussions. Supported by NIH grants GM070565 and GM095567 to J.W.H., GM067945 to S.P.G., CA139885 to M.R., and the Michael J. Fox Foundation for Parkinson’s Research to J.W.H.

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Authors and Affiliations



S.A.S. and J.W.H. conceived the experiments. S.A.S. performed QdiGly profiling, biochemical, and interaction experiments and analysis. S.A.S., M.R. and V.G.-P. performed cell biological experiments. M.E.S. designed site visualization software. E.L.H. and S.P.G. provided proteomics software and analysis. S.A.S. and J.W.H. wrote the manuscript. All authors assisted in editing the manuscript.

Corresponding author

Correspondence to J. Wade Harper.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-7, Supplementary Text and Supplementary References. (PDF 11288 kb)

Supplementary Table 1

This file contains experimental parameters of QdiGLY proteomic experiments reported in this study, It shows the experiment numbers in relation to cell lines used, treatments employed, and the number of sequential immunoprecipitations. (XLSX 44 kb)

Supplementary Table 2

This file contains a complete list of all proteins and sites identified and quantified from all experiments from this study, as well as Tier1, 2, and 3 and Class1 and Class 2 site lists. (XLSX 15491 kb)

Supplementary Table 3

This file contains proteomic analysis of HA-FLAG-PARKIN associated proteins in 293T cells in response to depolarization using CompPASS. It shows all the WDN-scores, Z-scores, and APSMs for the PARKIN immunoprecipitation data from 293T cells. (XLSX 5435 kb)

Supplementary Table 4

This file contains proteomic analysis of HA-FLAG-PARKIN associated proteins in HeLa cells in response to depolarization using CompPASS. It shows all the WDN-scores, Z-scores, and APSMs for the PARKIN immunoprecipitation data from HeLa cells. (XLSX 212 kb)

Supplementary Table 5

This file shows conservation and structural analysis of selected candidate PARKIN substrates. This file contains Protein DataBase (PDB) identifiers, the identity of ubiquitylation sites that change in response to depolarization, and the conservation of sites in M. musculus, D. rerio, and D. melanogaster. (XLSX 40 kb)

Supplementary Table 6

This file contains quantification of the PARKIN-mitochondrial overlap for the imaging experiments in Supplementary Figure 6. (XLSX 12 kb)

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Sarraf, S., Raman, M., Guarani-Pereira, V. et al. Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization. Nature 496, 372–376 (2013).

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