Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization - PubMed (original) (raw)

. 2013 Apr 18;496(7445):372-6.

doi: 10.1038/nature12043. Epub 2013 Mar 17.

Affiliations

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

Shireen A Sarraf et al. Nature. 2013.

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 mitophagy. 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 MOM, the full repertoire of PARKIN substrates--the PARKIN-dependent ubiquitylome--remains poorly defined. Here we use quantitative diGly capture proteomics (diGly) 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.

PubMed Disclaimer

Figures

Figure 1

Figure 1. QdiGLY proteomics for PARKIN-dependent ubiquitylation

a, Proteomics work flow. b, diGLY sites identified and quantified across 73 experiments. FDR, false discovery rate. c, log2(H:L) plots for quantified diGLY peptides for HCT116PARKIN (experiment 17) or HeLaPARKIN (experiment 57) cells (Table S2). d, Overlap of ubiquitylation sites in HeLaPARKIN biological triplicates (1h CCCP + Btz) (Table S1, S2). e, Ubiquitylation site and protein overlap between all HCT116PARKIN and HeLaPARKIN experiments treated with CCCP and Btz for 1h. f, log2(H:L) ratios for selected diGLY sites from HCT116PARKIN (Ex 17) and HeLaPARKIN (Ex 57) (1h CCCP + Btz).

Figure 2

Figure 2. PARKIN-dependent ubiquitylation sites revealed by QdiGLY proteomics

Class 1 sites are in black font. Additional sites found in Class 1 proteins are in red (HCT116PARKIN) or blue font (HeLaPARKIN). Site overlap in HCT116 and/or SH-SY5Y: magenta, orange, and green octagons. Dotted lines: interacting proteins. Rectangles represent Class 2 substrates. Red or blue boxes refer to additional sites identified in either HCT116PARKIN and HeLaPARKIN cells (Table S2). * and ^, protein levels decrease or increase, respectively, upon depolarization.

Figure 3

Figure 3. PARKIN associates with mitochondrial proteins and the proteasome in response to depolarization

a, Heat map of HCIPs (represented by APSMs) for HA-FLAG-PARKIN and mutants in response to depolarization in 293T cells, with or without Btz or BafA. Proteins indicated had WDN-scores ≥ 1.0, Z-score ≥ 5, and APSMs ≥ 2 unless otherwise noted (see METHODS).b, Summary of PARKIN-interacting proteins in 293T and HeLa and integration with diGLY sites.

Figure 4

Figure 4. Structural anatomy and conservation of PARKIN-dependent diGLY sites

Structures or domain schematics are shown for Class 1 depolarization and PARKIN-dependent diGLY sites (PDB codes, Table S5). Color-coded circles (a) indicate the conservation of Lys at the homologous position in M. musculus, D. rerio, and D. melanogaster (Table S5). For structures, regulated sites are shown in red space-filled models. b, Cytoplasmic proteins. RPN10, red circle. c, MOM proteins with structures. d, MOM proteins without structures.

Similar articles

Cited by

References

    1. Narendra DP, Walker JE, Youle R. Mitochondrial Quality Control Mediated by PINK1 and Parkin: Links to Parkinsonism. Cold Spring Harb Perspect Biol. 2012;4:a011338. - PMC - PubMed
    1. Youle RJ, Narendra DP. Mechanisms of mitophagy. Nat Rev Mol Cell Biol. 2011;12:9–14. - PMC - PubMed
    1. Dawson TM, Dawson VL. The role of parkin in familial and sporadic Parkinson's disease. Mov Disord. 2010;25(Suppl 1):S32–S39. - PMC - PubMed
    1. Chan NC, et al. Broad activation of the ubiquitin-proteasome system by Parkin is critical for mitophagy. Hum Mol Genet. 2011;20:1726–1737. - PMC - PubMed
    1. Glauser L, Sonnay S, Stafa K, Moore DJ. Parkin promotes the ubiquitination and degradation of the mitochondrial fusion factor mitofusin 1. J Neurochem. 2011;118:636–645. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources