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1 SUPPLEMENTARY INFORMATION doi: /nature13418 Supplementary Results: USP30 opposes autophagic flux In HEK-293 cells, USP30 overexpression increased basal LC3-II levels, dependent on enzymatic activity, suggesting USP30 increases LC3-II synthesis and/or reduces LC3-II degradation (Extended Data Fig. 4a, b). To assess LC3-II synthesis, we inhibited lysosomal degradation with bafilomycin (100 nm, 0-8 hours) and measured LC3-II stabilization. USP30 overexpression did not affect accumulation of LC3-II with bafilomycin treatment, implying increased LC3-II levels with USP30 overexpression reflects reduced LC3-II degradation (Extended Data Fig. 4c, d). Conversely, USP30 knockdown did not affect basal LC3-II levels, yet accumulated higher levels of LC3-II upon bafilomycin treatment, implying USP30 suppression enhances basal autophagic flux (Extended Data Fig. 4e-h). USP30 overexpression also reduced lysosomal degradation of p62 in CCCP-treated parkin-expressing cells, dependent on USP30 enzymatic activity (Extended Data Fig. 4i-k). In contrast, USP30 knockdown accelerated p62 degradation in CCCP-treated parkin-expressing cells (Extended Data Fig. 4l, m). These data confirm the mt-keima imaging data in neurons that USP30 counteracts mitophagic activity. USP30 deubiquitinates multiple mitochondrial proteins Since parkin and USP30 antagonistically regulate mitochondrial degradation, we hypothesized that this E3 ligase and DUB act on some common substrates. To identify parkin and USP30 substrates, we analyzed global ubiquitination by mass spectrometry (MS) following immunoaffinity enrichment of ubiquitinated peptides with the ubiquitin branch-specific (K-GG) 1

2 RESEARCH SUPPLEMENTARY INFORMATION immunoaffinity enrichment of ubiquitinated peptides with the ubiquitin branch-specific (K-GG) antibodies (See Methods). Global ubiquitination was analyzed and quantified by MS in HEK- 293 cells under two different sets of conditions: 1) inducible parkin overexpression, or 2) USP30 knockdown (USP30 knockdown efficiency was 85 ± 5% (see Fig. 3d)). In each set, cells were treated with CCCP (5 µm, 2 hours) or vehicle control (DMSO). In aggregate, MS analysis revealed >15,000 unique ubiquitination sites on ~3200 proteins (Supplementary Table 1, 2). Among these proteins, ubiquitination of 245 and 319 proteins were increased by parkin overexpression or by USP30 knockdown, respectively; i.e. these proteins exhibited significantly more ubiquitination (fold change > 1.3; p < 0.05) in parkin overexpression + CCCP or USP30 knockdown + CCCP vs. CCCP-alone (Supplementary Table 3). Of these proteins, 41 were regulated by both parkin overexpression and USP30 knockdown (Extended Data Fig. 5a). 12 of these 41 proteins were mitochondrial or associated with mitochondria (Tom20, Miro1, Fkbp8, Pth2, Mul1, Mat2b, Tom70, Prdx3, Ide, and VDAC1-3), based on the Human MitoCarta database 42. Others included nuclear import proteins (e.g. Ipo5), demethylases (e.g. Kdm3b), and components of the ubiquitin-proteasome system (e.g. Psd13, UBP13) (Extended Data Fig. 5a). 2

3 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Discussion In this study, we identified USP30 as a DUB that biochemically and functionally counteracts parkin. Through its enzymatic activity, USP30 opposes parkin-mediated mitophagy and deubiquitinates some, but not all, mitochondrial parkin substrates. Different ubiquitin chain types that accumulate on parkin substrates may dictate which parkin substrates are deubiquitinated by USP30, if USP30 prefers to cleave certain forms of ubiquitin chains over others. Since USP30 is physically attached on the mitochondrial outer membrane, USP30 most likely reverses ubiquitination of parkin substrates in a direct manner. However, the possibility remains that USP30 antagonizes parkin through an indirect mechanism. Interestingly, a subset of USP30 substrates -- exemplified by Tom20, but not Miro -- was regulated by USP30 even under basal conditions i.e. the ubiquitination of these substrates increased with USP30 knockdown even without CCCP treatment. Thus, USP30 basally deubiquitinates this set of proteins, presumably counterbalancing a mitochondrial E3 ligase that is active in the absence of CCCP and that acts on Tom20 but not Miro. Following CCCP-induced mitochondrial damage, USP30 also counteracts parkin--dependent ubiquitination of this same set of substrates. On the other hand, a different set of proteins exemplified by Miro showed enhanced ubiquitination with USP30 knockdown only following CCCP. This observation suggests that this set of proteins undergoes a low degree of basal ubiquitination in the absence of recruited parkin (i.e. parkin is their major E3 ligase), or that USP30 is inactive toward those proteins under basal conditions. 3

4 RESEARCH SUPPLEMENTARY INFORMATION Supplementary References: 42 Pagliarini, D. J. et al. A mitochondrial protein compendium elucidates complex I disease biology. Cell 134, (2008) 4

5 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Table 1 LiME plots reveal putative substrates of parkin and USP30 Linear mixed effects (LiME) plots used to display the results of global ubiquitination site profiling. Each page displays the LiME plots of K-GG peptides (i.e. ubiquitin modified) from two experiments: GFP-parkin overexpression (left) and USP30 knockdown (right). In each set of experiments, cells were treated with vehicle control (DMSO) or CCCP (5 µm, 2 hours). Gray lines report the area under the curve (log10) for a single non-overlapping, chromatographic peak stemming from direct peptide spectral match and/or cross quantification event. The red line denotes the fitted values of treatments from the linear mixed effect for each protein in each experiment, with fold-change and P-values reported relative to Control for each treatment. Control groups are DMSO + β-gal for GFP-parkin overexpression experiment, and DMSO + luciferase shrna for USP30 knockdown experiment. Proteins from the decoy database are denoted as ##REF_HUMAN. Supplementary Table 2 K-GG Peptides identified by mass spectrometry Aggregated list of high confidence peptide spectral matches (PSM) obtained from LC- MS/MS analysis of anti-k-gg enriched samples. Raw MS/MS spectra were searched using Mascot (+/-50 ppm) against a concatenated target-decoy database comprised of human protein sequences (Uniprot v2011_12) and common contaminants. Proteins in the decoy database are denoted as ##REF_HUMAN. Linear discriminant analysis was used for systematic filtering at the peptide and protein levels. For the final list, filtered PSMs were further curated to include only PSMs with mass errors within +/- 10 ppm. PSMs bearing a C-terminal K-GG and no 5

6 RESEARCH SUPPLEMENTARY INFORMATION available lysines were similarly discarded. PSMs passing through the systematic filtering but discarded during manual curation are provided as a separate tab. The final list contains total K-GG PSMs stemming from 3217 proteins at peptide and protein level FDRs of 0.11% and 1.12%, respectively. The 'Peptide' column refers to the modified peptide sequence returned by the Mascot algorithm. The observed m/z, precursor ion mass difference (PPM), and Mascot Ion Score (IonScore) are provided for each peptide spectral match. The localization score generated by the AScore algorithm and the corresponding site number (from the Uniprot reference sequence) are provided for each modification site (e.g. AScore 1, Site 1, AScore 2, Site 2 ). Where the score is ambiguous, the region of uncertainty is provided (e.g. Region 1, Region 2 ). For PSMs bearing a C-terminal K-GG modification and an available lysine within the internal portion of the sequence, the position of the modified lysine is reported in the Relocalized Seq as the internal lysine. In the Peptide, AScore Seq, and Relocalized Seq columns, variable lysine modifications of Da (corresponding to K-GG) are denoted by K*. Oxidized methionine residues are denoted by M#. Supplementary Table 3 Proteins differentially ubiquitinated by parkin or USP30 List of proteins exhibiting a significant increase in ubiquitination (>1.3 fold increase and p < 0.05) with GFP-parkin overexpression or USP30 knockdown in CCCP-treated groups (i.e. CCCP-alone versus Combo where Combo refers to either CCCP + GFP-parkin overexpression or CCCP + USP30 knockdown ). For each protein, fold change and p-values are reported relative to Control group (left side) and relative to Combo group (right side). Control groups are DMSO + β-gal for GFP-parkin overexpression experiment, and DMSO + luciferase shrna for USP30 knockdown experiment. 6