Article12 September 2014free access Source Data USP8 regulates mitophagy by removing K6-linked ubiquitin conjugates from parkin Thomas M Durcan Thomas M Durcan McGill Parkinson Program, Department of Neurology & Neurosurgery, Montreal Neurological Institute, McGill University, Montréal, QC, Canada Search for more papers by this author Matthew Y Tang Matthew Y Tang McGill Parkinson Program, Department of Neurology & Neurosurgery, Montreal Neurological Institute, McGill University, Montréal, QC, Canada Search for more papers by this author Joëlle R Pérusse Joëlle R Pérusse Institut de Recherches Cliniques de Montréal (IRCM), Montréal, QC, Canada Search for more papers by this author Eman A Dashti Eman A Dashti McGill Parkinson Program, Department of Neurology & Neurosurgery, Montreal Neurological Institute, McGill University, Montréal, QC, Canada Search for more papers by this author Miguel A Aguileta Miguel A Aguileta McGill Parkinson Program, Department of Neurology & Neurosurgery, Montreal Neurological Institute, McGill University, Montréal, QC, Canada Search for more papers by this author Gian-Luca McLelland Gian-Luca McLelland McGill Parkinson Program, Department of Neurology & Neurosurgery, Montreal Neurological Institute, McGill University, Montréal, QC, Canada Search for more papers by this author Priti Gros Priti Gros McGill Parkinson Program, Department of Neurology & Neurosurgery, Montreal Neurological Institute, McGill University, Montréal, QC, Canada Search for more papers by this author Thomas A Shaler Thomas A Shaler Physics Laboratory, SRI International, Menlo Park, CA, USA Search for more papers by this author Denis Faubert Denis Faubert Institut de Recherches Cliniques de Montréal (IRCM), Montréal, QC, Canada Search for more papers by this author Benoit Coulombe Benoit Coulombe Institut de Recherches Cliniques de Montréal (IRCM), Montréal, QC, Canada Department of Biochemistry, Université de Montréal, Montréal, QC, Canada Search for more papers by this author Edward A Fon Corresponding Author Edward A Fon McGill Parkinson Program, Department of Neurology & Neurosurgery, Montreal Neurological Institute, McGill University, Montréal, QC, Canada Search for more papers by this author Thomas M Durcan Thomas M Durcan McGill Parkinson Program, Department of Neurology & Neurosurgery, Montreal Neurological Institute, McGill University, Montréal, QC, Canada Search for more papers by this author Matthew Y Tang Matthew Y Tang McGill Parkinson Program, Department of Neurology & Neurosurgery, Montreal Neurological Institute, McGill University, Montréal, QC, Canada Search for more papers by this author Joëlle R Pérusse Joëlle R Pérusse Institut de Recherches Cliniques de Montréal (IRCM), Montréal, QC, Canada Search for more papers by this author Eman A Dashti Eman A Dashti McGill Parkinson Program, Department of Neurology & Neurosurgery, Montreal Neurological Institute, McGill University, Montréal, QC, Canada Search for more papers by this author Miguel A Aguileta Miguel A Aguileta McGill Parkinson Program, Department of Neurology & Neurosurgery, Montreal Neurological Institute, McGill University, Montréal, QC, Canada Search for more papers by this author Gian-Luca McLelland Gian-Luca McLelland McGill Parkinson Program, Department of Neurology & Neurosurgery, Montreal Neurological Institute, McGill University, Montréal, QC, Canada Search for more papers by this author Priti Gros Priti Gros McGill Parkinson Program, Department of Neurology & Neurosurgery, Montreal Neurological Institute, McGill University, Montréal, QC, Canada Search for more papers by this author Thomas A Shaler Thomas A Shaler Physics Laboratory, SRI International, Menlo Park, CA, USA Search for more papers by this author Denis Faubert Denis Faubert Institut de Recherches Cliniques de Montréal (IRCM), Montréal, QC, Canada Search for more papers by this author Benoit Coulombe Benoit Coulombe Institut de Recherches Cliniques de Montréal (IRCM), Montréal, QC, Canada Department of Biochemistry, Université de Montréal, Montréal, QC, Canada Search for more papers by this author Edward A Fon Corresponding Author Edward A Fon McGill Parkinson Program, Department of Neurology & Neurosurgery, Montreal Neurological Institute, McGill University, Montréal, QC, Canada Search for more papers by this author Author Information Thomas M Durcan1, Matthew Y Tang1, Joëlle R Pérusse2, Eman A Dashti1, Miguel A Aguileta1, Gian-Luca McLelland1, Priti Gros1, Thomas A Shaler3, Denis Faubert2, Benoit Coulombe2,4 and Edward A Fon 1 1McGill Parkinson Program, Department of Neurology & Neurosurgery, Montreal Neurological Institute, McGill University, Montréal, QC, Canada 2Institut de Recherches Cliniques de Montréal (IRCM), Montréal, QC, Canada 3Physics Laboratory, SRI International, Menlo Park, CA, USA 4Department of Biochemistry, Université de Montréal, Montréal, QC, Canada *Corresponding author. Tel: +1 514 398 8398; Fax: +1 514 398 5214; E-mail: [email protected] The EMBO Journal (2014)33:2473-2491https://doi.org/10.15252/embj.201489729 See also: I Dikic & A Bremm (November 2014) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mutations in the Park2 gene, encoding the E3 ubiquitin-ligase parkin, are responsible for a familial form of Parkinson's disease (PD). Parkin-mediated ubiquitination is critical for the efficient elimination of depolarized dysfunctional mitochondria by autophagy (mitophagy). As damaged mitochondria are a major source of toxic reactive oxygen species within the cell, this pathway is believed to be highly relevant to the pathogenesis of PD. Little is known about how parkin-mediated ubiquitination is regulated during mitophagy or about the nature of the ubiquitin conjugates involved. We report here that USP8/UBPY, a deubiquitinating enzyme not previously implicated in mitochondrial quality control, is critical for parkin-mediated mitophagy. USP8 preferentially removes non-canonical K6-linked ubiquitin chains from parkin, a process required for the efficient recruitment of parkin to depolarized mitochondria and for their subsequent elimination by mitophagy. This work uncovers a novel role for USP8-mediated deubiquitination of K6-linked ubiquitin conjugates from parkin in mitochondrial quality control. Synopsis The deubiquitinase USP8 is required for parkin recruitment to mitochondria and thus mitophagy by removing K6-linked ubiquitin conjugates from parkin hence opposing its auto-ubiquitination. USP8 is a deubiquitinating enzyme that is required for parkin to efficiently mediate the autophagic clearance of damaged mitochondria. Knockdown of USP8 delays the mitochondrial recruitment of parkin and parkin-mediated mitophagy. Parkin has a propensity to form ubiquitin conjugates on itself predominantly linked via K6. USP8 directly deubiquitinates parkin, by preferentially hydrolyzing K6 linkages within parkin-Ub conjugates. Increased levels of K6-linked Ub conjugates impede the normal function of parkin in mitophagy. Introduction Mutations in the Park2 gene are associated with a familial form of Parkinson's disease (PD) and are believed to impair the normal function of the parkin protein as an E3 ubiquitin (Ub)-ligase (Shimura et al, 2000). These loss-of-function mutations impede the function of parkin in a variety of cellular pathways, including mitochondrial quality control, a process believed to be central to the pathogenesis of PD (Narendra et al, 2008; Geisler et al, 2010; McLelland et al, 2014). In this pathway, parkin collaborates with another PD gene, PTEN-Induced Kinase I (PINK1), to promote the removal of dysfunctional mitochondria by autophagy (mitophagy) (Narendra et al, 2010b). The PINK1 protein is a mitochondrial kinase that is rapidly degraded upon import into healthy mitochondria (Jin et al, 2010; Greene et al, 2012). When the mitochondrial membrane potential is abolished, PINK1 import is stalled leading to its accumulation on the mitochondrial surface (Lazarou et al, 2012), where it phosphorylates the parkin Ub-like domain (Ubl) and Ub at serine 65 (S65), a residue conserved in both proteins (Kondapalli et al, 2012; Kane et al, 2014; Kazlauskaite et al, 2014; Koyano et al, 2014). S65 phosphorylation, in turn, promotes the activation and translocation of parkin from the cytosol to mitochondria (Kondapalli et al, 2012; Kane et al, 2014; Kazlauskaite et al, 2014; Koyano et al, 2014), where it ubiquitinates a number of mitochondrial proteins (Chan et al, 2011; Sarraf et al, 2013), in addition to ubiquitinating itself (Matsuda et al, 2010; Lazarou et al, 2013). Parkin E3 ubiquitin-ligase activity is critical for the efficient elimination of dysfunctional mitochondria by mitophagy (Geisler et al, 2010; Matsuda et al, 2010). However, parkin activity is auto-inhibited at baseline (Trempe et al, 2013; Wauer & Komander, 2013), and understanding how PINK1-mediated phosphorylation of Ub and parkin triggers its activation and recruitment to mitochondria is incomplete (Kondapalli et al, 2012; Kane et al, 2014; Kazlauskaite et al, 2014; Koyano et al, 2014). A common mode of regulation used by E3 ubiquitin ligases involves auto-ubiquitination. Depending on which of the seven lysines within Ub is used for conjugation, E3 Ub-ligases can assemble poly-Ub chains on themselves and on substrates, with different topologies, to mediate distinct biological functions (Komander & Rape, 2012; Kulathu & Komander, 2012). Chains linked via Lys48 (K48) in Ub, the best-characterized type of Ub conjugation, typically lead to degradation of the substrate by the proteasome (Voges et al, 1999). In contrast, chains linked via one of the other six lysines in Ub can protect modified substrates from proteasomal degradation and divert them toward functions in a variety of cellular pathways including trafficking, signaling, and autophagy (Komander & Rape, 2012; Kulathu & Komander, 2012). Thus, regulation of parkin by auto-ubiquitination has the potential to profoundly affect its function. Auto-ubiquitination can be antagonized by deubiquitinating enzymes (DUBs), which remove Ub from the E3 (de Bie & Ciechanover, 2011). This is typically believed to protect the E3 from proteasomal degradation (Wu et al, 2004; Nathan et al, 2008; Mei et al, 2011). However, certain DUBs have been shown to exhibit specificity in hydrolyzing non-K48-linked Ub chains (Komander et al, 2009a; Reyes-Turcu et al, 2009; Mevissen et al, 2013). Whether or how this could affect the stability and function of a cognate E3 partner is only beginning to be explored (Marfany & Denuc, 2008; Ventii & Wilkinson, 2008; de Bie et al, 2010). We recently reported that ataxin-3, a DUB responsible for Machado-Joseph's disease, can deubiquitinate parkin (Durcan et al, 2011, 2012). However, the effects of ataxin-3 on parkin-mediated mitophagy were not investigated. Moreover, as E3s can be regulated by multiple DUBs (Daviet & Colland, 2008; Nathan et al, 2008), we used an unbiased siRNA-based approach to ask whether deubiquitination of parkin regulates its function in mitophagy. We report here that USP8, a DUB previously associated with endosomal trafficking (Mizuno et al, 2005; Row et al, 2006), but not mitochondrial quality control, is required for the efficient recruitment of parkin to depolarized mitochondria and subsequent mitophagy. Moreover, we find that USP8 preferentially removes K6-linked Ub conjugates from parkin but has little effect on the ubiquitination or stability of other known mitochondrial substrates of parkin. Whereas K6-linked Ub conjugates on parkin appear to protect it from proteasomal degradation and to impede mitophagy, USP8-mediated removal of K6-linked Ub from parkin promotes parkin turnover and is required for mitophagy to proceed efficiently. Taken together, our work identifies USP8 and K6-linked Ub conjugates as key players in the regulation of parkin-mediated mitophagy. Results Reducing USP8 levels impairs recruitment of parkin to depolarized mitochondria Given that DUBs can regulate the function of E3 Ub-ligases, we surmised that parkin might also collaborate with a DUB during mitophagy. Using an unbiased siRNA-based approach, we knocked down 87 putative DUBs encoded by the human genome (Supplementary Table S1) (Nijman et al, 2005) in U2OS cells stably expressing GFP-parkin and screened for effects on CCCP-induced parkin recruitment to mitochondria. Expression of endogenous parkin in these cells was undetectable relative to other cell lines (Supplementary Fig S1A). Silencing of USP8, but not any of the other DUBs, impaired parkin recruitment to mitochondria after 1 h of treatment with CCCP (Fig 1A–C, Supplementary Fig S1B–D). Immunoblotting confirmed that the USP8 protein was indeed reduced in these cells (Fig 1C, Supplementary Fig S1C). Time-lapse microscopy showed that knockdown of USP8 delayed but did not abolish parkin recruitment to depolarized mitochondria (Fig 1D and E, Supplementary Video S1). Indeed, after 2 h of CCCP treatment, parkin was ultimately recruited in USP8 siRNA-transfected cells (Fig 1D and E). This effect was not cell type-specific as knockdown of USP8 in HeLa cells transiently transfected with GFP-parkin caused a similar delay in parkin recruitment to mitochondria (data not shown). Furthermore, the delay does not appear related to impairment in the endocytic function of USP8, as knockdown of STAM1 and 2; two other core components of this pathway had no effect on parkin recruitment (Supplementary Fig S2). Figure 1. USP8 siRNA delays parkin recruitment onto mitochondria A, B. USP8 siRNA impedes parkin recruitment onto mitochondria following CCCP treatment. U2OS-GFP-parkin cells were transfected with non-targeting or USP8 siRNA (10 nM) for 60 h (A). Untreated cells or cells treated with CCCP for 1 h were fixed, and images were acquired after staining for the mitochondrial protein TOM20. After 1-h CCCP treatment, cells were analyzed for GFP-parkin co-localization onto TOM20-positive mitochondria (B). Experiments were blinded and performed in triplicate with 100 cells analyzed for each condition. The vertical bars represent SEM for three independent experiments. For statistical analysis, a two-way ANOVA with Tukey post-test was performed, **P < 0.01. C. Validation of USP8 siRNA knockdown. U2OS-GFP-parkin cells were transfected with non-targeting or USP8 siRNA oligos (10 nM) for 60 h. Cells were lysed and analyzed by immunoblotting for USP8, parkin (long and short exposure), and actin. D, E. A delay in parkin recruitment onto mitochondria is observed in cells transfected with USP8 siRNA by live-cell microscopy. U2OS-GFP-parkin cells were transfected with non-targeting or USP8 siRNA (10 nM) for 60 h (D). 16 h prior to imaging, cells were infected with CellLight® mitochondria-RFP (Mito-RFP) to visualize mitochondria. Live-cell imaging was initiated 5 min after CCCP treatment, and images were acquired every 5 min over a 140-min period. Parkin recruitment upon membrane depolarization is visualized by the appearance of punctate GFP fluorescence superposed onto mitochondrial RFP fluorescence. Quantification of GFP-parkin recruitment to mitochondria (E) is facilitated by calculating the percentage of cells showing recruitment of GFP-parkin onto mitochondria at 5-min intervals over a period of 145 min. Experiments were performed in triplicate with 350 cells analyzed in each condition. The vertical bars represent the mean ± SEM for three independent experiments (see also Supplementary Video S1). F, G. Expression of FLAG-HA-USP8 rescues the effects of USP8 siRNA. U2OS-GFP-parkin cells were co-transfected with USP8 RNAi (5 nM) and FLAG-USP8 (0.5 μg) for 60 h (F). Cells treated with CCCP for 1 h were fixed, and images were acquired after staining for the mitochondrial protein, TOM20. After 1-h CCCP treatment, cells were analyzed for GFP-parkin co-localization onto TOM20-positive mitochondria in cells either negative or positive for FLAG-USP8 (G). Experiments were blinded and performed in triplicate with 100 cells analyzed for each condition. For statistical analysis, a two-way ANOVA with Tukey post-test was performed, ****P < 0.0001. The vertical bars represent the mean ± SEM for three independent experiments (see also Supplementary Fig S3D and E). Source data are available online for this figure. Source Data for Figure 1C [embj201489729-SourceData-Fig1C.pdf] Download figure Download PowerPoint USP8 knockdown also led to an increase in steady-state parkin levels (Fig 1C, Supplementary Fig S1C), with noticeable cell-to-cell variability based on the intensity of GFP fluorescence (Fig 1D, Supplementary Fig S1D). However, impaired parkin recruitment could not simply be explained by an increase in parkin expression, as cells with both high and low parkin levels displayed a comparable delay in mitochondrial recruitment (Supplementary Fig S1E). Nonetheless, our findings suggest that USP8 is required not only for the efficient recruitment of parkin to mitochondria but also for the efficient turnover of parkin itself. Indeed, we noticed that knocking down USP8 in cell lines and primary neurons increased both transfected and endogenous parkin levels (Fig 1C, Supplementary Figs S1C and S3A–C). Conversely, USP8 overexpression reduced steady-state parkin levels in a dose-dependent manner and rescued the effects of USP8 siRNA on parkin levels (Supplementary Fig S3D and E). Moreover, the delay in parkin recruitment onto mitochondria induced by USP8 siRNA was rescued in cells transduced with a FLAG-USP8 expression plasmid (Fig 1F and G). Thus, knockdown of USP8 increases parkin levels and delays its recruitment onto mitochondria. Silencing USP8 impairs mitophagy Prolonged treatment with CCCP ultimately leads to the elimination of depolarized mitochondria by parkin-dependent mitophagy (Narendra et al, 2008). Given that silencing USP8 delayed parkin recruitment to mitochondria, we asked whether it also affected mitophagy. U2OS-GFP-parkin cells were transfected with either non-targeting or USP8 siRNA and treated with CCCP for 24 h. As expected, with transfection of non-targeting siRNA, the majority of cells showed a loss of TOM20 (Fig 2A upper), TIM23, and COX1 (data not shown) staining, indicating their mitochondria had been efficiently cleared by autophagy. In contrast, fewer cells transfected with USP8 siRNA lost TOM20 staining, indicating that mitophagy was impaired (Fig 2A and B). Prolonged CCCP treatment also led to a marked reduction in parkin staining in cells transfected with non-targeting siRNA, as has been noted previously (Fig 2A upper) (Geisler et al, 2010; Tanaka et al, 2010; Rakovic et al, 2013). In contrast, in USP8 siRNA-transfected cells, GFP-parkin staining persisted after 24 h of CCCP treatment and formed puncta that co-localized with mitochondrial markers (TOM20, COX1, and Tim23), providing a robust readout of parkin-mediated mitophagy (Fig 2A and C, Supplementary Fig S4A and B). However, as with parkin recruitment, the effect on mitophagy appears to be delayed rather than abolished, as no differences were detectable between non-targeting and USP8 siRNA-treated cells by 48 h of CCCP treatment (Fig 2D and E). Figure 2. USP8 siRNA impairs parkin-mediated mitophagy A–E. USP8 siRNA impairs parkin-mediated mitophagy. U2OS-GFP-parkin cells were transfected with non-targeting or USP8 siRNA (10 nM) for 48 h (A). Transfected cells were either left untreated or were treated with CCCP for 24 or 48 h before fixation. Immunofluorescence images of cells were acquired after staining for TOM20. Cells were outlined in white as a result of low GFP-parkin levels. The percentage of all U2OS-GFP-parkin cells after 24 h (B, C) or 48 h (D, E) CCCP treatment lacking TOM20-positive mitochondria or containing GFP-parkin puncta co-localizing with TOM20-positive mitochondria was quantified in cells transfected with non-targeting or USP8 siRNA. Experiments were blinded and performed in triplicate with 100 cells analyzed for each condition. The vertical bars represent SEM. For statistical analysis, a two-way ANOVA with Tukey post-test was performed, *P < 0.05, **P < 0.01; NS, not significant. F. USP8 siRNA has no effect on CCCP-induced mitochondrial depolarization. U2OS-GFP-parkin cells transfected with non-targeting or USP8 siRNA (10 nM) for 60 h were first incubated with the potentiometric dye TMRM (600 nM) 20 min prior to imaging. Prior to CCCP treatment, cells were imaged for 10 min to confirm TMRM staining. Following CCCP treatment, images of cells were acquired every minute for 10 min (see also Supplementary Video S2). The membrane potential in untreated cells was tested by imaging untreated cells for 1 h (see also Supplementary Video S3). G. USP8 siRNA has no discernible effect on CCCP-induced PINK1 accumulation. U2OS-GFP-parkin cells transfected with non-targeting, PINK1 siRNA, or USP8 siRNA (10 nM) for 60 h were either left untreated or were treated with CCCP for 1 or 3 h. Cells were lysed and immunoblotted for parkin, actin, and PINK1. Source data are available online for this figure. Source Data for Figure 2G [embj201489729-SourceData-Fig2G.pdf] Download figure Download PowerPoint CCCP dissipates the proton gradient across the mitochondrial inner membrane, which blocks the import and degradation of PINK1 (Narendra et al, 2010b; Greene et al, 2012). This in turn leads to an accumulation of PINK1 at the mitochondrial outer surface, where it phosphorylates Ub and parkin at serine 65, thereby activating parkin and triggering its translocation from the cytosol (Kondapalli et al, 2012; Kane et al, 2014; Kazlauskaite et al, 2014; Koyano et al, 2014). Thus, we asked whether the effect of USP8 on parkin recruitment was upstream or downstream of mitochondrial depolarization and PINK1 accumulation. Staining with the potentiometric dye TMRM revealed that silencing USP8 affected neither basal mitochondrial membrane potential nor the ability of CCCP to abolish mitochondrial membrane potential (Fig 2F, Supplementary Videos S2 and S3). Similarly, USP8 knockdown affected neither the steady-state levels of endogenous PINK1 nor its accumulation in response to CCCP (Fig 2G). Using time-lapse microscopy, we also demonstrate using mitochondrially targeted RFP that USP8 siRNA neither altered mitochondrial morphology, fusion, or fission at baseline (Supplementary Fig S4C, Supplementary Video S4) nor did it affect mitochondrial fragmentation in response to CCCP (Supplementary Video S5). Finally, steady-state levels of mitochondrial proteins appeared unchanged between non-targeting and USP8 knockdown cells (Supplementary Fig S4D). Thus, the effect of USP8 on parkin recruitment is downstream of mitochondrial depolarization and PINK1 accumulation and is not the result of off-target siRNA effects on PINK1 or on mitochondrial dynamics. USP8 deubiquitinates parkin We noticed an apparent increase in the levels of Ub conjugates on parkin in the absence of USP8 (Fig 1C). These findings were confirmed in HEK293T cells by overexpressing HA-Ub and FLAG-parkin, a paradigm that stimulates parkin auto-ubiquitination at steady state. Indeed, immunoprecipitation with FLAG followed by immunoblotting with HA showed that parkin Ub conjugates were significantly increased in cells transfected with USP8 siRNA (Fig 3A and B). Conversely, HA-Flag-USP8 overexpression reduced parkin auto-ubiquitination (Supplementary Fig S5A). To address whether USP8 could directly deubiquitinate parkin, we carried out in vitro ubiquitination reactions using recombinant and affinity-purified GST-parkin, Ub, ATP and E1 and E2 enzymes. Ubiquitinated GST-parkin, bound to glutathione sepharose beads, was then washed extensively to remove free Ub and other components of the reaction, followed by incubation with either full-length USP8 or one of several commercially available DUBs. These DUBs all exhibited enzymatic activity in Ub-AMC cleavage assays (Durcan et al, 2012). However, save for USP8 and USP2, none could hydrolyze Ub conjugates on parkin (Fig 3C). The DUB activity of USP8 appeared to be specific toward parkin, as it was much less efficient at deubiquitinating other E3s, including HHARI, an RBR family E3 Ub-ligase structurally very similar to parkin (Fig 3D) (Duda et al, 2013; Trempe et al, 2013). In addition to GST-parkin, USP8 could efficiently deubiquitinate untagged parkin, indicating that it targeted Ub conjugated to parkin per se rather than to the N-terminal GST-tag (Fig 3E). The effect of USP2 on parkin was not surprising, as it had been shown previously to disassemble Ub chains indiscriminately (Komander et al, 2009b). Indeed, USP2 was able to deubiquitinate several E3s non-selectively in addition to parkin (Supplementary Fig S5B) (Komander et al, 2009b). However, USP2 siRNA showed no effect on parkin levels or on parkin recruitment to mitochondria (Fig 3F, Supplementary Fig S6). Similarly, knockdown of ataxin-3, the first identified DUB partner of parkin (Durcan et al, 2011), had no effect on the mitochondrial recruitment of parkin (Fig 3F, Supplementary Fig S6). Recently, USP15 and USP30 were shown to inhibit mitophagy by counteracting parkin-mediated ubiquitination (Bingol et al, 2014; Cornelissen et al, 2014). However, in contrast to USP8, these two DUBs were believed to deubiquitinate substrates of parkin on mitochondria downstream of recruitment rather than deubiquitinating parkin itself. Consistent with this model, we find that USP15 cannot deubiquitinate parkin directly (Fig 3C) and that neither the knockdown of USP15 (not shown) nor USP30 (Fig 3F, Supplementary Fig S6) affects parkin recruitment to mitochondria. Taken together, the findings indicate that USP8 directly and specifically deubiquitinates parkin. Figure 3. USP8 deubiquitinates parkin A, B. USP8 knockdown causes Ub conjugates to accumulate on FLAG-parkin in HEK293T cells. HEK293T cells were co-transfected with FLAG-parkin (0.5 μg), HA-UbWT (0.5 μg) and non-targeting or USP8 siRNA (5 nM). Lysates were immunoprecipitated with FLAG resin and analyzed by immunoblotting for HA and FLAG. Input lysates (5% of total input) were analyzed by immunoblotting for USP8 and actin. The optical densities of the Ub-parkin conjugates were quantified using NIH ImageJ, and the data represent the mean ± SEM for three independent experiments. For statistical analysis, a two-way ANOVA with Tukey post-test was performed, **P < 0.01. C. The activity of USP8 toward parkin relative to other DUBs. GST-parkin bound to glutathione beads was left to ubiquitinate for 2 h alone at 37°C. After 2 h, the beads were washed to remove reaction components and ubiquitinated GST-parkin was then incubated in the presence or absence of the indicated DUB for 1 h at 37°C. Reactions were immunoblotted for Ub and Ponceau stained for GST-parkin. D. USP8 preferentially hydrolyzes preassembled parkin Ub conjugates. For these reactions, GST-parkin, GST-CHIP, GST-HHARI (RBR domain), or GST-cIAP1 bound to glutathione beads were left to ubiquitinate for 2 h alone at 37°C. After 2 h, the beads were washed to remove reaction components. The ubiquitinated E3 was then incubated in the presence or absence of the full-length His-USP8 for 1 h at 37°C. Reactions were analyzed by Ponceau S staining and immunoblotting for Ub. E. USP8 can hydrolyze preassembled Ub conjugates on untagged parkin. For these reactions, untagged parkin was left to ubiquitinate for 2 h alone at 37°C with UbcH7 as the E2. After 2 h, apyrase was added for 20 min to terminate the reaction. Ubiquitinated parkin was now incubated for 1 h at 37°C in the presence or absence of His-tagged full-length USP8. Reactions were immunoblotted for Ub and parkin. F. Knockdown of other DUBs compared to USP8 had no effect on the recruitment of parkin onto mitochondria following CCCP treatment. U2OS-GFP-parkin cells were transfected with either non-targeting, USP8, ataxin-3, USP30, or USP2 siRNA (10 nM) for 60 h. Cells treated with CCCP for 1 h were fixed, and images were acquired after staining for the mitochondrial protein, TOM20 (see also Supplementary Fig S6). Source data are available online for this figure. Source Data for Figure 3 [embj201489729-SourceData-Fig3.pdf] Download figure Download PowerPoint USP8 regulates parkin auto-ubiquitination during mitophagy Next, we examined the effect of USP8 on the ubiquitination of parkin and mitochondrial proteins in cells during mitophagy. In cells transfected with non-targeting siRNA, CCCP treatment led to a transient increase followed by a profound reduction in parkin levels, as described previously (Fig 4A–C) (Chan et al, 2011; Rakovic et al, 2013). Concurrently with the changes in parkin levels, the electrophoretic mobility of parkin was transiently shifted upward, consistent with a CCCP-induced activation of parkin and parkin auto-ubiquitination, as reported previously (Matsuda et al, 2010; Lazarou et al, 2013). Indeed, in control conditions, Ub affinity chromatography using Ub binding Agarose (TUBEs) followed by immunoblotting for parkin confirmed that it was markedly but transiently ubiquitinated in response to CCCP (Fig 4B, upper). In comparison, the increase in parkin levels and ubiquitination after CCCP persisted several hours longer in USP8 knockdown cells (Fig 4A–C). No comparable differences were noted in total Ub levels, suggesting that the effect of USP8 on parkin ubiquitination was specific