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

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Abstract

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.

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Change history

  • 17 April 2013

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

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Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

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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Competing interests

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). https://doi.org/10.1038/nature12043

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