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USP30 and parkin homeostatically regulate atypical ubiquitin chains on mitochondria

Nature Cell Biology volume 17, pages 160–169 (2015)Cite this article

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Abstract

Multiple lines of evidence indicate that mitochondrial dysfunction is central to Parkinson’s disease. Here we investigate the mechanism by which parkin, an E3 ubiquitin ligase, and USP30, a mitochondrion-localized deubiquitylase, regulate mitophagy. We find that mitochondrial damage stimulates parkin to assemble Lys 6, Lys 11 and Lys 63 chains on mitochondria, and that USP30 is a ubiquitin-specific deubiquitylase with a strong preference for cleaving Lys 6- and Lys 11-linked multimers. Using mass spectrometry, we show that recombinant USP30 preferentially removes these linkage types from intact ubiquitylated mitochondria and counteracts parkin-mediated ubiquitin chain formation in cells. These results, combined with a series of chimaera and localization studies, afford insights into the mechanism by which a balance of ubiquitylation and deubiquitylation regulates mitochondrial homeostasis, and suggest a general mechanism for organelle autophagy.

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Figure 1: Lys 6 Ub chains are enriched in HEK293 and SH-SY5Y whole cell lysates and mitochondria on CCCP treatment.
Figure 2: USP30 is a Ub-specific deubiquitylase that prefers compact chain types.
Figure 3: USP30 preferentially removes Lys 6 and Lys 11 chains from intact mitochondria.
Figure 4: USP30 deubiquitylates outer membrane mitochondrial substrates.
Figure 5: A typical Ub chain linkage signal for mitophagy.
Figure 6: Ectopically expressed USP30 is capable of preventing autophagy of peroxisomes.
Figure 7: Model for the regulation of mitophagy by deubiquitylation of mitochondrial proteins by USP30.

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Acknowledgements

The authors wish to acknowledge C. Balakarski, A. Decker and W. Forrest for assistance with bioinformatic tools and statistical analyses, Y. Franke for assistance with construct design and creation and E. Dueber, J. Lill and D. Vucic for critical reading of the manuscript. PTMScan is carried out at Genentech under limited license from Cell Signaling Technologies.

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Author notes

  1. Jacob E. Corn

    Present address: Present address: Innovative Genomics Initiative, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.,

  2. Joshua M. Baughman and Lilian Phu: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Early Discovery Biochemistry, Genentech, Inc., 1 DNA Way South San Francisco, California 94080, USA,

    Christian N. Cunningham & Jacob E. Corn

  2. Department of Protein Chemistry, Genentech, Inc., 1 DNA Way South San Francisco, California 94080, USA,

    Joshua M. Baughman, Lilian Phu & Donald S. Kirkpatrick

  3. Department of Neuroscience, Genentech, Inc., 1 DNA Way South San Francisco, California 94080, USA,

    Joy S. Tea & Baris Bingol

  4. Department of Structural Biology, Genentech, Inc., 1 DNA Way South San Francisco, California 94080, USA,

    Christine Yu & Mary Coons

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Contributions

C.N.C., J.M.B., L.P., J.S.T., B.B. and J.E.C. were responsible for the experimental work. C.N.C., D.S.K., B.B. and J.E.C. planned the project and designed the experiments. C.N.C., J.M.B., L.P., D.S.K., B.B. and J.E.C. analysed the data. C.Y. and M.C. provided materials and reagents. C.N.C. and J.E.C. wrote the manuscript with help from all authors.

Corresponding authors

Correspondence to Donald S. Kirkpatrick, Baris Bingol or Jacob E. Corn.

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The authors declare competing financial interests. C.N.C., J.M.B., L.P., C.Y., M.C., D.S.K. and B.B. are all employees of Genentech, Inc.

Integrated supplementary information

Supplementary Figure 4 Lys6 ubiquitin chains are enriched in HEK293 and SH-SY5Y whole cell lysates and mitochondria upon CCCP treatment.

(A) SH-SY5Y Western blot confirmation of mitochondrial depolarization upon CCCP treatment over time. MFN1 levels decrease and Opa1 processing increased over the course of 4 h with 10 μM CCCP treatment in SH-SY5Y cells. (B) Base peak chromatograms of immunoaffinity-purified K-GG peptides from SH-SY5Y cells following Parkin overexpression (oe) and/or 10 μM CCCP treatment for 2 h. Chromatographic features showing ubiquitin signature peptides for Lys6, Lys11, Lys48, and Lys63-linked chains are denoted by arrows. Inset shows extracted ion chromatograms from Lys6 peptides in the treated and untreated samples. (C) SDS-PAGE gel regions excised for ubiquitin linkage analysis in Supplementary Fig. 1D. HEK293 cells stably expressing GFP-Parkin were treated with either DMSO or 20 μM CCCP for 3 h, fractionated into mitochondria, and lysed for trypsinization and subsequent ubiquitin linkage analysis. Data represent one out of three independent experiments. (D) Ubiquitin linkage analysis on 4 gel regions ranging from 5–250 kDa. Abundance (fmol) of each ubiquitin linkage is reported for DMSO (black circles) or CCCP (red circles) treatment for each gel region. Data represent one out of three experiments. (E) Representative SDS-PAGE gel showing excised regions for ubiquitin linkage analysis in Supplementary Fig. 1F. SH-SY5Y cells were treated with either DMSO or 10 μM CCCP for 3 h, fractionated into cytosolic and mitochondrial fractions, and lysed for trypsinization and subsequent ubiquitin linkage analysis. (F) Ubiquitin linkage analysis of cytosolic and mitochondrial fractions of 2 gel regions ranging from 40–100 kDa. Abundance (fmol) of each ubiquitin linkage is reported for DMSO (black circles) or CCCP (red circles) treatment for each gel region from either the cytosolic or mitochondrial fractions of two replicate experiments in SH-SY5Y cells.

Supplementary Figure 5 Immunodepletion of GFP-Parkin confirms substrates other than Parkin carry Lys6-linked polyubiquitin chains.

(A) HEK293 cells overexpressing GFP-Parkin were treated with 10 μM CCCP or a DMSO vehicle control for 2 h. Lysates were immunodepleted with anti-GFP beads and input and flow through (FT) fractions were run on Western blot. MFN1 levels decreased and Opa1 processing increased confirming mitochondrial depolarization upon CCCP treatment. This experiment was performed once. (B) Input and flow through (FT) fractions treated with CCCP from (A) were run on SDS-PAGE and excised into 10 gel regions spanning 5–250 kDa. (C) Ubiquitin linkage analysis was used to measure the abundance of Lys6 chains (fmol) for each gel region excised (left) and Parkin peptide abundance (AUC) was measured for each gel region excised (right).

Supplementary Figure 6 USP30 is an ubiquitin specific deubiquitinase that prefers compact chain types.

(A) Michaelis–Menten plot of USP2 (blue), USP30 (orange), and USP7 (red) catalytic domain activity. Data were fit using the equation v = Vmax[S]/KM + [S]. (B) USP30’s preference for compact ubiquitin linkages is not driven by a hyper affinity for dimeric ubiquitin chain types in a biolayer interferometry experiment. Equilibrium response is shown as a function of di-ubiquitin concentration. Compact chains (Lys6, Lys11, and Lys48) are shown in colors. Greater overall signal was observed with Lys6 dimers, but individual kinetic curves displayed some non-specific adherence to the tip and fitting showed no significant (NS) decrease in dissociation constant (all KDs > 5 μM). (C) Gel of Lys6, Lys48, and Lys63 purified tetramers. The lower band of Lys6 (∗) migrates most closely to Lys63 tetramers. (D) Intact mass spectrometry of Lys6 tetramer shows a single molecular weight peak at the expected MW of tetrameric ubiquitin. (E) SDS-PAGE gel of Lys6 tetramers showing regions excised for ubiquitin linkage analysis. (F) Relative amounts of detected chain types for each excised band in (E). Lys63 make up a larger fraction of linkages in the lower band indicating this chain type as an off-target product during the enzymatic formation of K6 tetramers.

Supplementary Figure 7 Mass spectrometry validation and ubiquitin linkage analysis of USP30 knockout cell line.

(A,B) Mass spectrometry confirmation of USP30 CRISPR knockout. Ratios of area under the curve (AUC) between USP30 knockout HEK293 cells expressing GFP-Parkin and a wild type USP30 isogenic cell line for 2 mitochondrial matrix proteins (HSPD1, ATP5B) and three independent USP30 peptides (A; see Supplementary Table 2 for raw data). Ratios are not shown for the three USP30 signature peptides since no signal was detected in the USP30 knockout cell line, as represented in (B). Error bars represent standard deviation from n = 3 independent experiments (Supplementary Table 2). (C) Representative SDS-PAGE gel from three independent experiments showing excision locations used for ubiquitin linkage analysis in Fig. 3a and Supplementary Fig. 3D. USP30 knockout (KO) or WT USP30 (WT) HEK293 cells stably expressing GFP-Parkin were treated with either DMSO or CCCP (10 μM, 3 h), enriched for mitochondria, and lysed for trypsinization and subsequent ubiquitin linkage analysis. (D) Ubiquitin linkage analysis on 4 gel regions ranging from 5–250 kDa. fmol of each ubiquitin linkage is reported for CCCP treated WT (black circles) or USP30 knockout (red circles) mitochondrial enriched fractions for each gel region. Data represent one out of two replicate experiments. (E) Representative SDS-PAGE gel showing regions excised for ubiquitin linkage analysis in Fig. 3c.

Supplementary Figure 8 USP30 deubiquitinates outer membrane mitochondrial substrates.

(A) Western blot confirmation of USP30 induction following doxycycline administration and mitochondrial depolarization upon CCCP treatment. USP30 showed strong induction, while reduced MFN1 levels and increase in Opa1 processing were observed. Representative Western blot shown from experiment 1. (B,C) Volcano plots of ubiquitinated proteins identified in Experiments 1 and 2, respectively. The fold change of ubiquitination for each protein between Combo (USP30 overexpression and CCCP) versus CCCP is denoted on the x-axis with significance reported on the y-axis. Both values were determined based on the aggregate values of individual KGG peptides using LiME analysis. Proteins with p-value < 0.01 are labelled; mitochondria-associated proteins are colored red.

Supplementary Figure 9 Atypical ubiquitin chain linkages signal for mitophagy.

(A) Immunostaining of HeLa cells co-transfected with GFP-Parkin and mitochondrial-localized, FLAG-tagged DUBs. 24 h after transfection cells were immunostained for FLAG (green) and endogenous HSP60 (red). Each DUB co-localizes with HSP60 indicating proper mitochondrial targeting (see merge). In addition, expression levels appear uniform across all DUBs transfected. Representative cells are shown for each transfection. Scale bar, 10 μm. (B) Large field images from Fig. 5a. Solid yellow outlines highlight cells with GFP-Parkin and DUB-Flag expressing cells. Dashed yellow outlines highlight cells expressing only GFP-Parkin. Scale bar, 30 μm. (C) Immunostaining of HeLa cells co-transfected with GFP-Parkin and HA-tagged ubiquitin variants as listed in Fig. 5c. Only GFP-Parkin expressing cells are outlined. Solid lines indicate TOM20 positive staining cells; dashed lines indicate TOM20 negative staining cells. Scale bar, 20 μm. (D) Quantification of TOM20 in HeLa cells treated with CCCP (15 μM, 24 h) and transfected with either beta-Gal or HA-ubiquitin. A significant difference in TOM20 was observed with a 24 h CCCP treatment preventing analysis of individual HA-ubiquitin mutants tested in Fig. 5c. Each point represents a single experiment with a mean intensity from 25–40 cells measured; the line represents the mean from n = 5 experiments and error bars represent s.e.m. Significance is reported as one-Way ANOVA with Holm–Sidak’s multiple comparison test (∗P < 0.05).

Supplementary Table 1 Total peptide counts of selected proteins from isolated cytosolic (orange highlights) and mitochondrial fractions of HEK293T cells stably expressing GFP-Parkin confirm proper enrichment within each fraction.
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Supplementary Table 2 Area under the curve (AUC) measurements for a USP30 knockout HEK293 cell line expressing GFP-Parkin and a wild type USP30 isogenic cell line.
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Supplementary Table 3 Ubiquitin chain type analysis calculations for each chain type in four separate replicate experiments.
Full size table

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Cunningham, C., Baughman, J., Phu, L. et al. USP30 and parkin homeostatically regulate atypical ubiquitin chains on mitochondria. Nat Cell Biol 17, 160–169 (2015). https://doi.org/10.1038/ncb3097

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