Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

The mitochondrial deubiquitinase USP30 opposes parkin-mediated mitophagy

Abstract

Cells maintain healthy mitochondria by degrading damaged mitochondria through mitophagy; defective mitophagy is linked to Parkinson’s disease. Here we report that USP30, a deubiquitinase localized to mitochondria, antagonizes mitophagy driven by the ubiquitin ligase parkin (also known as PARK2) and protein kinase PINK1, which are encoded by two genes associated with Parkinson’s disease. Parkin ubiquitinates and tags damaged mitochondria for clearance. Overexpression of USP30 removes ubiquitin attached by parkin onto damaged mitochondria and blocks parkin’s ability to drive mitophagy, whereas reducing USP30 activity enhances mitochondrial degradation in neurons. Global ubiquitination site profiling identified multiple mitochondrial substrates oppositely regulated by parkin and USP30. Knockdown of USP30 rescues the defective mitophagy caused by pathogenic mutations in parkin and improves mitochondrial integrity in parkin- or PINK1-deficient flies. Knockdown of USP30 in dopaminergic neurons protects flies against paraquat toxicity in vivo, ameliorating defects in dopamine levels, motor function and organismal survival. Thus USP30 inhibition is potentially beneficial for Parkinson’s disease by promoting mitochondrial clearance and quality control.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: USP30 antagonizes parkin-mediated mitophagy.
Figure 2: USP30 antagonizes mitophagy in neurons.
Figure 3: USP30 and parkin act antagonistically on common substrates.
Figure 4: USP30 knockdown rescues mitophagy defects associated with mutant parkin.
Figure 5: USP30 knockdown provides protection in vivo.

References

  1. 1

    Narendra, D. P. & Youle, R. J. Targeting mitochondrial dysfunction: role for PINK1 and Parkin in mitochondrial quality control. Antioxid. Redox Signal. 14, 1929–1938 (2011)

    CAS  Article  Google Scholar 

  2. 2

    Hauser, D. N. & Hastings, T. G. Mitochondrial dysfunction and oxidative stress in Parkinson’s disease and monogenic parkinsonism. Neurobiol. Dis. 51, 35–42 (2013)

    CAS  Article  Google Scholar 

  3. 3

    Kitada, T. et al. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 392, 605–608 (1998)

    CAS  Article  ADS  Google Scholar 

  4. 4

    Valente, E. M. et al. Hereditary early-onset Parkinson’s disease caused by mutations in PINK1. Science 304, 1158–1160 (2004)

    CAS  Article  ADS  Google Scholar 

  5. 5

    Yang, Y. et al. Mitochondrial pathology and muscle and dopaminergic neuron degeneration caused by inactivation of Drosophila Pink1 is rescued by Parkin. Proc. Natl Acad. Sci. USA 103, 10793–10798 (2006)

    CAS  Article  ADS  Google Scholar 

  6. 6

    Clark, I. E. et al. Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin. Nature 441, 1162–1166 (2006)

    CAS  Article  ADS  Google Scholar 

  7. 7

    Park, J. et al. Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin. Nature 441, 1157–1161 (2006)

    CAS  Article  ADS  Google Scholar 

  8. 8

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

    Article  Google Scholar 

  9. 9

    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 

  10. 10

    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 

  11. 11

    Matsuda, N. et al. PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy. J. Cell Biol. 189, 211–221 (2010)

    CAS  Article  Google Scholar 

  12. 12

    Vives-Bauza, C. et al. PINK1-dependent recruitment of Parkin to mitochondria in mitophagy. Proc. Natl Acad. Sci. USA 107, 378–383 (2010)

    CAS  Article  ADS  Google Scholar 

  13. 13

    Guzman, J. N. et al. Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1. Nature 468, 696–700 (2010)

    CAS  Article  ADS  Google Scholar 

  14. 14

    Nakamura, N. & Hirose, S. Regulation of mitochondrial morphology by USP30, a deubiquitinating enzyme present in the mitochondrial outer membrane. Mol. Biol. Cell 19, 1903–1911 (2008)

    CAS  Article  Google Scholar 

  15. 15

    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 

  16. 16

    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 

  17. 17

    Lee, J.-Y., Nagano, Y., Taylor, J., Lim, K. & Yao, T.-P. Disease-causing mutations in parkin impair mitochondrial ubiquitination, aggregation, and HDAC6-dependent mitophagy. J. Cell Biol. 189, 671–679 (2010)

    CAS  Article  Google Scholar 

  18. 18

    Katayama, H., Kogure, T., Mizushima, N., Yoshimori, T. & Miyawaki, A. A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery. Chem. Biol. 18, 1042–1052 (2011)

    CAS  Article  Google Scholar 

  19. 19

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

    CAS  Article  ADS  Google Scholar 

  20. 20

    Yoshii, S. R., Kishi, C., Ishihara, N. & Mizushima, N. Parkin mediates proteasome-dependent protein degradation and rupture of the outer mitochondrial membrane. J. Biol. Chem. 286, 19630–19640 (2011)

    CAS  Article  Google Scholar 

  21. 21

    Guo, M. Drosophila as a model to study mitochondrial dysfunction in Parkinson’s disease. Cold Spring Harb. Perspect. Med. http://dx.doi.org/10.1101/cshperspect.a009944 (2012)

  22. 22

    Brand, A. H. & Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415 (1993)

    CAS  PubMed  Google Scholar 

  23. 23

    Greene, J. C. et al. Mitochondrial pathology and apoptotic muscle degeneration in Drosophila parkin mutants. Proc. Natl Acad. Sci. USA 100, 4078–4083 (2003)

    CAS  Article  ADS  Google Scholar 

  24. 24

    Whitworth, A. J. et al. Increased glutathione S-transferase activity rescues dopaminergic neuron loss in a Drosophila model of Parkinson’s disease. Proc. Natl Acad. Sci. USA 102, 8024–8029 (2005)

    CAS  Article  ADS  Google Scholar 

  25. 25

    Wang, C. et al. Drosophila overexpressing parkin R275W mutant exhibits dopaminergic neuron degeneration and mitochondrial abnormalities. J. Neurosci. 27, 8563–8570 (2007)

    CAS  Article  Google Scholar 

  26. 26

    Sang, T. K. et al. A Drosophila model of mutant human parkin-induced toxicity demonstrates selective loss of dopaminergic neurons and dependence on cellular dopamine. J. Neurosci. 27, 981–992 (2007)

    CAS  Article  Google Scholar 

  27. 27

    Cha, G. H. et al. Parkin negatively regulates JNK pathway in the dopaminergic neurons of Drosophila. Proc. Natl Acad. Sci. USA 102, 10345–10350 (2005)

    CAS  Article  ADS  Google Scholar 

  28. 28

    Cochemé, H. M. & Murphy, M. P. Complex I is the major site of mitochondrial superoxide production by paraquat. J. Biol. Chem. 283, 1786–1798 (2008)

    Article  Google Scholar 

  29. 29

    Tanner, C. M. et al. Rotenone, paraquat, and Parkinson’s disease. Environ. Health Perspect. 119, 866–872 (2011)

    CAS  Article  Google Scholar 

  30. 30

    Seeburg, D. P. & Sheng, M. Activity-induced Polo-like kinase 2 is required for homeostatic plasticity of hippocampal neurons during epileptiform activity. J. Neurosci. 28, 6583–6591 (2008)

    CAS  Article  Google Scholar 

  31. 31

    Dooley, C. T. et al. Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators. J. Biol. Chem. 279, 22284–22293 (2004)

    CAS  Article  Google Scholar 

  32. 32

    Burchell, V. S. et al. The Parkinson’s disease-linked proteins Fbxo7 and Parkin interact to mediate mitophagy. Nature Neurosci. 16, 1257–1265 (2013)

    CAS  Article  Google Scholar 

  33. 33

    Shiba-Fukushima, K. et al. PINK1-mediated phosphorylation of the Parkin ubiquitin-like domain primes mitochondrial translocation of Parkin and regulates mitophagy. Sci. Rep. 2, 1002 (2012)

    Article  Google Scholar 

  34. 34

    Xu, G., Paige, J. S. & Jaffrey, S. R. Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling. Nature Biotechnol. 28, 868–873 (2010)

    CAS  Article  Google Scholar 

  35. 35

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

    CAS  Article  Google Scholar 

  36. 36

    Rappsilber, J., Ishihama, Y. & Mann, M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663–670 (2003)

    CAS  Article  Google Scholar 

  37. 37

    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 

  38. 38

    Bustos, D., Bakalarski, C. E., Yang, Y., Peng, J. & Kirkpatrick, D. S. Characterizing ubiquitination sites by peptide based immunoaffinity enrichment. Mol. Cell. Proteomics 11, 1529–1540 (2012)

    Article  Google Scholar 

  39. 39

    Seyfried, N. T. et al. Systematic approach for validating the ubiquitinated proteome. Anal. Chem. 80, 4161–4169 (2008)

    CAS  Article  Google Scholar 

  40. 40

    Pinheiro, J., Bates, D., DebRoy, S. & Sarkar, D. the R Core Team. nlme: linear and nonlinear mixed effects models. R package version 3. (2011)

  41. 41

    Wu, J. S. & Luo, L. A protocol for dissecting Drosophila melanogaster brains for live imaging or immunostaining. Nature Protocols 1, 2110–2115 (2006)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank J. Corn and C. Cunningham for discussions. The park25 line was a gift of L. Pallanck. PTMscan studies are performed at Genentech under a limited use license from Cell Signaling Technology.

Author information

Affiliations

Authors

Contributions

B.B. designed the study and executed and analysed the imaging and biochemistry experiments. J.S.T. executed and analysed the biochemistry and fly experiments. M.R. and O.F. gathered the electron microscopy data. L.P. executed, and C.E.B., Q.S. and D.S.K. analysed the mass spectrometry experiments. B.B. and M.S. wrote the manuscript.

Corresponding author

Correspondence to Baris Bingol.

Ethics declarations

Competing interests

All authors are employees of Genentech, Inc.

Extended data figures and tables

Extended Data Figure 1 USP30 is a mitochondrial protein.

a, Immunostaining of transfected USP30–Flag (red) and mitochondria-targeted GFP (green) in cultured rat hippocampal neurons. Merge is shown in colour; individual channels in greyscale. Scale bar, 5 µm. b, Immunostaining of SH-SY5Y cells transfected with control or USP30 siRNA. 3 days after transfection, cells were fixed and immunostained for endogenous USP30 and HSP60. USP30 siRNA primarily decreases mitochondrial USP30 antibody staining (scale bar, 5 µm). Higher magnification images of the boxed regions are shown in the right panels (scale bar, 2 µm). c, Immunoblots of cytoplasm- and mitochondria-enriched fractions from rat brain with USP30, HSP60 and GAPDH antibodies. d, Immunoblots of cell lysates from HEK-293 cells stably expressing GFP–parkin, transfected with the indicated control (β-Gal) and USP30 constructs. 24 h after transfection, cells were treated with CCCP (5 µM, 2 h) and lysed. e, Quantification of immunoblot signal for GFP–parkin from d, normalized to actin. *P < 0.05 by Kruskal–Wallis test and Dunn’s multiple comparison test. n = 6 experiments. Error bars represent s.e.m.

Extended Data Figure 2 USP30 counteracts mitochondrial ubiquitination and recruitment of p62 and LC3–GFP in CCCP-treated parkin-expressing cells.

a, Immunostaining of SH-SY5Y cells co-transfected with GFP–parkin and the indicated control (β-Gal) and Flag-tagged USP30 constructs. 24 h after transfection, cells were treated with CCCP (20 µM, 4 h) and immunostained for GFP, Flag, endogenous TOM20, and polyubiquitin chains (detected with FK2 antibody). Co-localization of GFP–parkin (shown in red) and polyubiquitin (shown in green) is shown in the right panel. Scale bars, 5 µm. b, Quantification of GFP–parkin-associated polyubiquitin staining intensity from a, normalized by GFP–parkin area (integrated fluorescence intensity of FK2 staining colocalizing with GFP–parkin/area of GFP–parkin staining). ***P < 0.001 by Kruskal–Wallis test and Dunn’s multiple comparison test. n = 6 experiments. Error bars represent s.e.m. c, Immunostaining of HeLa cells co-transfected with GFP–parkin and the indicated control (β-Gal) and Flag-tagged USP30 constructs. Cells were treated as in a and immunostained for GFP, Flag, endogenous p62, and HSP60. Co-localization of GFP–parkin (shown in red) and p62 (shown in green) is shown in the right panel. Scale bars, 10 µm. d, Quantification of GFP–parkin-associated p62 staining intensity from c, normalized by GFP–parkin area (integrated fluorescence intensity of p62 staining colocalizing with GFP–parkin/area of GFP–parkin staining). *P < 0.05 by Kruskal–Wallis test and Dunn’s multiple comparison test. n = 5 experiments. Error bars represent s.e.m. e, Immunostaining of HeLa cells co-transfected with RFP-parkin, LC3–GFP and the indicated control (β-Gal) and Flag-tagged USP30 constructs. Cells were treated as in a and immunostained for GFP, Flag and endogenous HSP60. Co-localization of RFP-parkin (shown in red) and LC3–GFP (shown in green) is shown in the right panel. Scale bars, 10 µm. f, Quantification of RFP-parkin-associated LC3–GFP puncta area from e, normalized by RFP-parkin area (area of LC3–GFP puncta colocalizing with RFP-parkin/area of RFP-parkin staining). *P < 0.05 by Kruskal–Wallis test and Dunn’s multiple comparison test. n = 5 experiments. Error bars represent s.e.m.

Extended Data Figure 3 mt-Keima imaging of mitophagy; PINK1 acts upstream of parkin in the mitophagy pathway.

a, mt-Keima differentially highlights cytoplasmic (green) and lysosomal (red) mitochondria. Cultured hippocampal neurons were transfected with mt-Keima and GFP. Following 2 days of expression, cells were imaged with 458 nm (shown in green) or 543 nm (shown in red) light excitation. GFP signal was used to outline the cell (shown in white). Scale bar, 5 µm. b, mt-Keima imaging in cultured hippocampal neurons before and after NH4Cl treatment (50 mM, 2 min). mt-Keima signal, collected with 543 nm or 458 nm laser excitation, is shown in red and green, respectively. Neutralizing cells with NH4Cl completely reversed the high ratio (543 nm/458 nm) signal to low ratio signal specifically in the round structures without affecting the tubular-reticular mitochondrial signal. Scale bar, 5 µm. c, Imaging of mt-Keima and Lysotracker (Lysotracker green DND-26 shown in grey scale) in hippocampal neurons, showing Lysotracker stained the high ratio mt-Keima structures. Scale bar, 5 µm. d, Post hoc immunostaining for endogenous Lamp1 in neurons imaged for mt-Keima signal, showing the colocalization of high-ratio mt-Keima pixels with Lamp1 staining. Immediately following mt-Keima imaging, cells were fixed and stained with anti-Lamp1 antibody (shown in grey scale). Scale bar, 5 µm. e, Quantification of mitophagy index following 1, 3 and 6–7 days of mt-Keima expression in cultured hippocampal neurons. **P < 0.01 and ***P < 0.001 by Kruskal–Wallis test and Dunn’s multiple comparison test. n = 29–85 cells. n = 2–4 experiments. Error bars represent s.e.m. f, g, Immunoblots of HEK-293 cell lysates transfected with the indicated cDNA and parkin (f) or PINK1 (g) shRNA constructs. PSD-95–Flag was co-transfected as a control. Representative blots from three independent experiments are shown. h, i, Immunoblots of endogenous parkin (h) and PINK1 (i) in cultured hippocampal neurons infected with adeno-associated virus expressing the indicated shRNAs. Representative blots from two independent experiments are shown. j, mt-Keima imaging in neurons transfected with PINK1–GFP and parkin-shRNA 1 (β-Gal and luciferase shRNA as controls). Scale bar, 5 µm. k, Quantification of mitophagy index from j. ***P < 0.001 by Kruskal–Wallis test and Dunn’s multiple comparison test. n = 55–75 cells. n = 3 experiments. Error bars represent s.e.m. l, mt-Keima imaging in neurons transfected with GFP–parkin or GFP control. Scale bar, 5 µm. m, Quantification of mitophagy index from l. (P = 0.22 by Mann–Whitney test. n = 37–43 cells. n = 3 experiments. Error bars represent s.e.m. n, Mitochondria-targeted GFP (mito-GFP) imaging in neurons transfected with luciferase shRNA or USP30 shRNA constructs. Scale bar, 10 μm. Higher magnification images shown in the bottom panel. Scale bar, 5 μm. o, Quantification of fold change in area of individual dendritic mitochondria from n. ***P < 0.001 by Mann–Whitney test. n = 9 experiments. Error bars represent s.e.m.

Extended Data Figure 4 USP30 opposes autophagic flux.

a, Immunoblots of cell lysates from HEK-293 cells, transfected with GFP–parkin and the indicated control (β-Gal) or USP30 constructs. b, Quantification of the LC3-II and p62 immunoblot signal from a, normalized to actin. **P < 0.01 by Kruskal–Wallis test and Dunn’s multiple comparison test. n = 6 experiments. Error bars represent s.e.m. c, Immunoblots of cell lysates from HEK-293 cells, transfected with GFP–parkin and β-Gal or USP30 wild type constructs, as indicated. 24 h after transfection, cells were treated with bafilomycin (100 nM, 0–8 h). d, Quantification of the LC3-II immunoblot signal from c, normalized to actin. P = 0.97 by two-way ANOVA and Bonferroni’s multiple comparison test. n = 5 experiments. Error bars represent s.e.m. e, Immunoblots of cell lysates from HEK-293 cells, transfected with GFP–parkin and control (luciferase) or USP30 shRNA constructs. f, Quantification of the LC3-II and p62 immunoblot signal from e, normalized to actin. g, Immunoblots of cell lysates from HEK-293 cells, transfected with GFP–parkin and control (luciferase) or USP30 shRNA constructs. 6 days after transfection, cells were treated with bafilomycin (100 nM, 0–8 h). h, Quantification of the LC3-II immunoblot signal from g, normalized to actin. **P < 0.01 by two-way ANOVA and Bonferroni’s multiple comparison test. n = 4 experiments. Error bars represent s.e.m. i, j, Immunoblots of cell lysates from HEK-293 cells, transfected with GFP–parkin and the indicated control (β-Gal) or USP30 constructs. 24 h after transfection, cells were treated with CCCP (20 µM, 0–6 h). β-Gal transfected cells were also treated with bafilomycin (100 nM, 0–6 h) as a control (shown in j). k, Quantification of the p62 immunoblot signal from i, j, normalized to actin. *P < 0.05 and **P < 0.01 by two-way ANOVA and Bonferroni’s multiple comparison test. n = 6 experiments. Error bars represent s.e.m. l, Immunoblots of cell lysates from HEK-293 cells, transfected with GFP–parkin and control (luciferase) or USP30 knockdown constructs. 24 h after transfection, cells were treated with CCCP (20 µM, 0-8 h) and bafilomycin (100 nM), as indicated. m, Quantification of the p62 immuoblot signal from l, normalized to actin. *P < 0.05 by two-way ANOVA and Bonferroni’s multiple comparison test. n = 6 experiments. Error bars represent s.e.m.

Extended Data Figure 5 USP30 deubiquitinates multiple mitochondrial proteins.

a, Proteins whose ubiquitination is regulated by both USP30 and parkin. Asymmetric ‘volcano plot’ showing the subset of 41 proteins whose ubiquitination significantly increased (P < 0.05) by both GFP–parkin overexpression (right side) and USP30 knockdown (left side) in ‘combo’ treatments compared to ‘CCCP-treatment’ alone. ‘Combo’ refers to cells treated with ‘CCCP + GFP–parkin’ or ‘CCCP + USP30-shRNA’. For this subset of proteins, fold-increase in ubiquitination (x-axis) and the P value (y-axis) are reported. Mitochondrial proteins (as identified by the Human MitoCarta database) are shown in red. Fold-changes and P values for all proteins with quantified K-GG peptides are reported in Supplementary Table 1. b, Immunoblots of anti-HA-immunoprecipitates for endogenous MIRO1 and TOM20 in parental HEK-293 cell line (that lacks GFP–parkin) transfected with HA–ubiquitin and the indicated Flag-tagged USP30 constructs (β-Gal as control). 24 h after transfection, cells were treated with CCCP (5 µM, 2 h) and ubiquitinated proteins were immunoprecipitated with anti-HA beads. Immunoprecipitates and inputs were blotted with the indicated antibodies. n = 2 experiments. c, Immunoblots of total lysates of GFP–parkin HEK-293 stable cells that were transfected with the indicated Flag-tagged USP30 constructs, and then treated with CCCP (5 µM, 0–6 h). d, Quantification of MIRO1 and TOM20 immunoblot signals from c, normalized to actin. Immunoblot signals for all other proteins (VDAC, Mfn-1, Tom70, HSP60, TIMM8a) did not reach significance. *P < 0.05, **P < 0.01, ***P < 0.001 compared to β-Gal control, by two-way ANOVA and Bonferroni’s multiple comparison test. n = 3-5 experiments.) e, Immunoblots of anti-HA-immunoprecipitates for endogenous MIRO1 and TOM20 with USP30 knockdown. HEK-293 cells stably expressing GFP–parkin were transfected as indicated with HA–ubiquitin, human USP30 shRNA and rat USP30–Flag cDNA that is insensitive to the shRNA (luciferase shRNA and β-Gal as controls). 6 days after transfection, cells were processed as in b. n = 2 experiments.

Extended Data Figure 6 TOM20 activates mitophagy through ubiquitination; USP30 is a parkin substrate.

a, Extracted ion chromatograms corresponding to K-GG peptides identified from TOM20 in the USP30 knockdown mass spectrometry. Relative abundance of each ubiquitinated peptide is shown on the y-axis relative to the most abundant analysis, with precursor ion m/z indicated above each peak. The sequence of each K-GG peptide is shown below in green. Asterisks denote modified lysine residues. b, Immunoblots of HA–ubiquitin precipitates from GFP–parkin HEK-293 cells transfected with the indicated constructs. Following transfection and treatment with CCCP (5 µM, 2 h), ubiquitinated proteins were immunoprecipitated with anti-HA beads, and precipitates and inputs were blotted with the indicated antibodies. n = 3 experiments. c, mt-Keima imaging in neurons transfected with the indicated TOM20–MYC and USP30 constructs (β-Gal as control). Scale bar, 5 µm. d, Quantification of mitophagy index from c. ***P < 0.001 by Kruskal–Wallis test and Dunn’s multiple comparison test. n = 67–80 cells for all groups. n = 3 experiments. Error bars represent s.e.m. e, Extracted ion chromatograms corresponding to K-GG peptides identified from USP30 in the parkin overexpression mass spectrometry. Similar to a. f, Immunoblots of anti-HA-immunoprecipitates for endogenous USP30 from cells transfected with wild-type, K161N and G430D GFP–parkin constructs. After 24 h of expression, cells were treated with CCCP (20 µM, 2 h) and ubiquitinated proteins were immunoprecipitated with anti-HA beads. Immunoprecipitates and inputs were blotted with the indicated antibodies. g, Quantification of immunoblot signal for co-immunoprecipitated USP30 from f. Protein levels co-immunoprecipitating with anti-HA beads are normalized to the ‘wild-type GFP–parkin + CCCP’ group. ***P < 0.001 by one-way ANOVA and Dunnett’s multiple comparison test, compared to ‘wild-type GFP–parkin + CCCP’. n = 5 experiments. Error bars represent s.e.m. h, Immunoblots of lysates prepared from HEK-293 cells transfected with the indicated GFP and GFP–parkin constructs and treated with CCCP (20 µM, 0–6 h). i, Quantification of immunoblot signal for USP30 from h, normalized to actin. **P < 0.01, ***P < 0.001 compared to wild-type GFP–parkin, by two-way ANOVA and Bonferroni’s multiple comparison test. n = 4 experiments. Error bars represent s.e.m. j, Immunoblots of lysates prepared from HEK-293 cells transfected with GFP–parkin and treated as indicated (CCCP 20 µM, 6 h; bafilomycin (100 nM), MG132 (20 µM), and epoxomicin (2 µM) were added 15 min before CCCP treatment). k, Quantification of immunoblot signal for USP30 from j, normalized to actin. *P < 0.05 and ***P < 0.001 by one-way ANOVA and Dunnett’s multiple comparison test, compared to ‘DMSO + CCCP’. n = 4 experiments. Error bars represent s.e.m.

Extended Data Figure 7 USP30 knockdown rescues mitophagy defects in cells expressing mutant parkin.

a, Immunoblot for endogenous USP30 in SH-SY5Y cells transfected with USP30 siRNA for 3 days. b, c, Immunostaining in SH-SY5Y cells stably expressing GFP–parkin(G430D), transfected with siRNAs against USP30, USP6 or USP14. 3 days after transfection, cells were treated with CCCP (20 µM, 24 h), then fixed and stained for GFP and endogenous TOM20. Scale bars, 5 µm. d, Quantification of fold change in TOM20 staining intensity from b and c, normalized to control siRNA. **P < 0.01 by Kruskal–Wallis test and Dunn’s multiple comparison test. n = 3 experiments. Error bars represent s.e.m. e, Immunostaining of SH-SY5Y cells expressing GFP–parkin(G430D), transfected as indicated, and treated with CCCP (20 µM, 24 h). Rat USP30 cDNA is insensitive to human USP30 siRNA. f, Quantification of fold change in TOM20 intensity from e. Kruskal–Wallis test, n = 3 experiments. g, Immunostaining of SH-SY5Y cells expressing GFP–parkin(K161N), and transfected with USP30 siRNA. Following 3 days of knockdown, cells were treated with CCCP (20 µM, 24 h), then fixed and stained for GFP and endogenous TOM20 and HSP60. Scale bars, 5 µm. h, Quantification of fold change in TOM20 or HSP60 staining intensity from g, normalized to control siRNA. *P < 0.05 by Mann–Whitney test, n = 4 experiments. Error bars represent s.e.m. i, k, Immunostaining of SH-SY5Y cells expressing GFP–parkin(K161N), and transfected with USP30 siRNA. Following 3 days of knockdown, cells were treated with CCCP (20 µM, 4 h), then fixed and stained for GFP and endogenous p62 (i) or LC3 (k). Co-localization of GFP–parkin (show in green) and p62 or LC3 (shown in red) is shown in the lower panel. Scale bars, 5 µm. j, l, Quantification of GFP–parkin(K161N)-associated p62 (j) or LC3 (l) staining intensity normalized by GFP–parkin(K161N) area, from i, k. **P < 0.01 by Mann–Whitney test, n = 9–10 experiments. Error bars represent s.e.m. m, mt-Keima imaging in neurons transfected with PINK1 shRNA and USP30(C77A)–Flag. Scale bar, 5 µm. n, Quantification of mitophagy index from m. Kruskal–Wallis test. n = 127–166 cells. n = 7 experiments. Error bars represent s.e.m.

Extended Data Figure 8 USP30 knockdown decreases oxidative stress in neurons and rescues mitochondrial morphology defects in PINK1 mutant flies.

a, Ratiometric mito-roGFP imaging in hippocampal neurons transfected with USP30 shRNA. Following measurement of ratiometric mito-roGFP signal in individual cells, the dynamic range of the probe was calibrated by treating cultures sequentially with DTT (1 mM) to fully reduce the probe, and aldrithiol (100 µM) to fully oxidize the probe13. The ‘relative oxidation index’ is shown in a ‘colour scale’ from 0 (mito-roGFP ratio after DTT treatment, shown in black) to 1 (mito-roGFP ratio after aldrithiol treatment, shown in red). b, Quantification of relative oxidation index from a. ***P < 0.001 by Mann–Whitney test. n = 24 cells for luciferase shRNA and 36 cells for USP30 shRNA. n = 3 experiments. Error bars represent s.e.m. c, Quantitative RT–PCR of dUSP30 mRNA. qRT–PCR in Actin-GAL4, UAS-dUSP30RNAi, and Actin-GAL4 > UAS-dUSP30RNAi flies, shown relative to Actin-GAL4. dUSP30 mRNA levels were normalized to internal control Drosophila RpII140 mRNA levels. ***P < 0.001 by one-way ANOVA and Dunnett’s multiple comparison test. n = 3 experiments. Error bars represent s.e.m. d, Transverse sections of Drosophila indirect flight muscles of indicated genotypes. Arrowheads, electron-dense mitochondria; dashed lines, ‘pale’ mitochondria with disorganized cristae. Scale bars, 1 µm (top), 0.2 µm (bottom panels). e, f, Quantification of mitochondrial morphology (e) and size distribution (f) from d. Mann–Whitney test (e). Kolmogorov–Smirnov test, pink1B9 versus ‘pink1B9 + dUSP30 knockdown’ (f). n = 4 flies per genotype. g, Transverse sections of indirect flight muscles (IFMs) from vehicle- or paraquat-treated flies of indicated genotypes. Scale bar, 0.5 µm.

Extended Data Figure 9 Neurodegeneration was not observed in genetic parkin fly models; dUSP30 knockdown protects against paraquat-induced climbing and dopamine deficits.

a, c, e, Representative images of the indicated dopaminergic neuron clusters in flies with indicated genotypes. Scale bars, 10 µm. b, d, f, Blind quantification for panels a, c, e. P values calculated by Student’s t-test (f) and one-way ANOVA and Bonferroni’s multiple comparison test (b, d). n = 4–5 hemibrains per genotype. Similar results were obtained with additional counts performed for the PPL1 cluster, n = 18–40 hemibrains per genotype. Error bars represent s.e.m. g, Dopamine levels in fly brains for the indicated genotypes. n = 16 flies per genotype. P values calculated by one-way ANOVA and Bonferroni’s multiple comparison test. h, Climbing assay in control flies (Actin-GAL4). Flies were treated with vehicle control (5% sucrose) or paraquat (10 mM, 48 h). l-3,4-dihydroxyphenylalanine (1 mM, 48 h) was administered simultaneously with paraquat, as indicated. Graph shows % of flies climbing 15 cm in 30 s. **P < 0.01 by Kruskal–Wallis test and Dunn’s multiple comparison test. n = 6 experiments. Error bars represent s.e.m. i, Serotonin levels per fly head, as assessed by ELISA. Flies were treated with paraquat (10 mM, 48 h) or vehicle control (5% sucrose). P values calculated by Kruskal–Wallis test and Dunn’s multiple comparison test. n = 8 heads, n = 2 experiments. Error bars represent s.e.m. j, Climbing assay of dUSP30 knockdown flies driven by Th-GAL4. Flies were treated with paraquat (10 mM, 48 h) or vehicle control (5% sucrose). Graph shows % of flies climbing 15 cm in 30 s. *P < 0.05 by Kruskal–Wallis test and Dunn’s multiple comparison test. n = 4 experiments. Error bars represent s.e.m. k, Climbing assay of dUSP30 knockdown flies driven by Actin-GAL4. Flies were treated with paraquat (10 mM, 48 h) or vehicle control (5% sucrose). Graph shows % of flies climbing 15 cm in 30 s. **P < 0.01 and ***P < 0.001 by one-way ANOVA and Bonferroni’s multiple comparison test. n = 6–10 experiments. Error bars represent s.e.m. l, m, Normalized dopamine levels per fly head, as assessed by ELISA. Flies of the indicated genotype were treated with paraquat (10 mM, 48 h) or vehicle control (5% sucrose). *P < 0.05, **P < 0.01, and ***P < 0.001 by Mann–Whitney test. n = 8–28 heads. Error bars represent s.e.m.

Extended Data Figure 10 Knockdown of DUBs dYOD1 or dUSP47 in flies does not provide protection against paraquat; hUSP30 overexpression reverses dUSP30 knockdown benefits.

a, b, Quantitative RT–PCR measurement of dUSP47 (a) and dYOD1 (b) mRNA levels in flies of the indicated genotypes, expressed as relative to Actin-GAL4 genotype. **P < 0.01 and ***P < 0.001 by one-way ANOVA and Dunnett’s multiple comparison test. n = 3 technical replicates. Error bars represent s.e.m. c, Survival curves of flies with dUSP47 or dYOD1 knockdown, treated with vehicle (5% sucrose) or paraquat (10 mM). Graph shows percent of flies alive at indicated times. *P < 0.05 and **p<0.01 by two-way ANOVA and Bonferroni’s multiple comparison test. n = 5–7 experiments. Error bars represent s.e.m. d, Survival curves of flies with dUSP30 knockdown driven by Th-GAL4, treated with paraquat (10 mM). Graph shows percent of flies alive at indicated times after feeding with paraquat. **P < 0.01 and ***P < 0.001 by two-way ANOVA and Bonferroni’s multiple comparison test. n = 3 experiments. Error bars represent s.e.m. e, f, Quantitative RT–PCR measurement of hUSP30 and dUSP30 mRNA levels in flies of the indicated genotypes. **P < 0.01 and ***P < 0.001 by one-way ANOVA and Dunnett’s multiple comparison test. n = 4 experiments. Error bars represent s.e.m. g, Climbing assay for flies overexpressing hUSP30. Flies of indicated genotypes were fed with vehicle (5% sucrose) or paraquat (10 mM, 48 h); graph shows percent of flies climbing 15 cm in 30 s. *P < 0.05 by Kruskal–Wallis test and Dunn’s multiple comparison test. n = 4 experiments. Error bars represent s.e.m. h, Survival assay for flies overexpressing hUSP30. Flies were fed paraquat (10 mM); graph shows % live flies at indicated times. *P < 0.05 and ***P < 0.001 by two-way ANOVA and Bonferroni’s multiple comparison test. n = 4–11 experiments. Error bars represent s.e.m.

Supplementary information

Supplementary Information

This file contains Supplementary Results, a Supplementary Discussion, an additional reference and legends for Supplementary Tables 1-3 (see separate files for the Supplementary Tables). (PDF 229 kb)

Supplementary Table 1

This file shows that LiME plots reveal putative substrates of parkin and USP30 (see Supplementary Information file for full legend). (PDF 6218 kb)

Supplementary Table 2

This file contains K-GG Peptides identified by mass spectrometry (see Supplementary Information file for full legend). (XLSX 8194 kb)

Supplementary Table 3

This file shows proteins differentially ubiquitinated by parkin or USP30 (see Supplementary Information file for full legend). (XLSX 274 kb)

dUSP30 knockdown by Ddc-GAL4 rescues paraquat induced climbing impairment in flies.

1-day old adult males from indicated genotypes were treated with paraquat (10 µM, 48 hours) or vehicle control (5% sucrose) on saturated Whatman filter paper. Following treatment, flies were collected in groups of 10 and transferred to fresh vials containing 1% agarose (in water) for 1 hour to recover from the effects of CO2 exposure. The flies were then transferred to new glass test tubes, gently tapped to the bottom, and scored for their ability to climb. Numbers of flies crossing 15 cm mark are shown in the video. (MP4 8973 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Bingol, B., Tea, J., Phu, L. et al. The mitochondrial deubiquitinase USP30 opposes parkin-mediated mitophagy. Nature 510, 370–375 (2014). https://doi.org/10.1038/nature13418

Download citation

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.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing