Parkin‐mediated ubiquitylation redistributes MITOL/March5 from mitochondria to peroxisomes

Article10 October 2019Open Access Transparent process Parkin-mediated ubiquitylation redistributes MITOL/March5 from mitochondria to peroxisomes Fumika Koyano Fumika Koyano Ubiquitin Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Search for more papers by this author Koji Yamano Koji Yamano Ubiquitin Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Search for more papers by this author Hidetaka Kosako Hidetaka Kosako Division of Cell Signaling, Fujii Memorial Institute of Medical Sciences, Tokushima University, Tokushima, Japan Search for more papers by this author Yoko Kimura Yoko Kimura Department of Agriculture Graduate School of Integrated Science and Technology, Shizuoka University, Shizuoka, Japan Search for more papers by this author Mayumi Kimura Mayumi Kimura Ubiquitin Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Search for more papers by this author Yukio Fujiki Yukio Fujiki Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka, Japan Search for more papers by this author Keiji Tanaka Keiji Tanaka Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Search for more papers by this author Noriyuki Matsuda Corresponding Author Noriyuki Matsuda [email protected] orcid.org/0000-0001-8199-952X Ubiquitin Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Search for more papers by this author Fumika Koyano Fumika Koyano Ubiquitin Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Search for more papers by this author Koji Yamano Koji Yamano Ubiquitin Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Search for more papers by this author Hidetaka Kosako Hidetaka Kosako Division of Cell Signaling, Fujii Memorial Institute of Medical Sciences, Tokushima University, Tokushima, Japan Search for more papers by this author Yoko Kimura Yoko Kimura Department of Agriculture Graduate School of Integrated Science and Technology, Shizuoka University, Shizuoka, Japan Search for more papers by this author Mayumi Kimura Mayumi Kimura Ubiquitin Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Search for more papers by this author Yukio Fujiki Yukio Fujiki Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka, Japan Search for more papers by this author Keiji Tanaka Keiji Tanaka Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Search for more papers by this author Noriyuki Matsuda Corresponding Author Noriyuki Matsuda [email protected] orcid.org/0000-0001-8199-952X Ubiquitin Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Search for more papers by this author Author Information Fumika Koyano1, Koji Yamano1, Hidetaka Kosako2, Yoko Kimura3, Mayumi Kimura1, Yukio Fujiki4, Keiji Tanaka5 and Noriyuki Matsuda *,1 1Ubiquitin Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan 2Division of Cell Signaling, Fujii Memorial Institute of Medical Sciences, Tokushima University, Tokushima, Japan 3Department of Agriculture Graduate School of Integrated Science and Technology, Shizuoka University, Shizuoka, Japan 4Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka, Japan 5Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan *Corresponding author. Tel: +81 3 5316 3123; Fax: +81 3 5316 3152; E-mail: [email protected] EMBO Reports (2019)20:e47728https://doi.org/10.15252/embr.201947728 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 Ubiquitylation of outer mitochondrial membrane (OMM) proteins is closely related to the onset of familial Parkinson's disease. Typically, a reduction in the mitochondrial membrane potential results in Parkin-mediated ubiquitylation of OMM proteins, which are then targeted for proteasomal and mitophagic degradation. The role of ubiquitylation of OMM proteins with non-degradative fates, however, remains poorly understood. In this study, we find that the mitochondrial E3 ubiquitin ligase MITOL/March5 translocates from depolarized mitochondria to peroxisomes following mitophagy stimulation. This unusual redistribution is mediated by peroxins (peroxisomal biogenesis factors) Pex3/16 and requires the E3 ligase activity of Parkin, which ubiquitylates K268 in the MITOL C-terminus, essential for p97/VCP-dependent mitochondrial extraction of MITOL. These findings imply that ubiquitylation directs peroxisomal translocation of MITOL upon mitophagy stimulation and reveal a novel role for ubiquitin as a sorting signal that allows certain specialized proteins to escape from damaged mitochondria. Synopsis Mitochondrial E3 ubiquitin ligase MITOL/March5 translocates from depolarized mitochondria to peroxisomes following mitophagy stimulation. Ubiquitylation of MITOL at K268 by Parkin is required for p97/VCP-dependent extraction from damaged mitochondria. MITOL is ubiquitylated in a PINK1/Parkin-dependent manner in response to mitochondrial depolarization. Ubiquitylated MITOL translocates from mitochondria to peroxisomes instead of undergoing degradation. Specific peroxins (Pex3 and Pex16) and p97/VCP are required for MITOL translocation to peroxisomes. MITOL may affect peroxisomal size and abundance. Introduction The mitochondria are a well-organized organelle. When the mitochondrial membrane potential is decreased, PINK1 and Parkin, which are causal gene products of inherited Parkinson's disease, play pivotal roles in the selective degradation of damaged mitochondria via the proteasome and autophagy pathway (the following is called mitophagy). PINK1 is a Ser/Thr kinase, and Parkin is an RBR-type E3 (ubiquitin ligase) that catalyzes ubiquitin conjugation to protein substrates from a ubiquitin-adducted E2 (ubiquitin-conjugating enzyme) 1-9. PINK1 accumulates on damaged mitochondria and subsequently phosphorylates Ser65 of both ubiquitin and ubiquitin-like (Ubl) domain of Parkin 10-15. Through the interaction with phosphorylated ubiquitin, Parkin also becomes phosphorylated, which triggers auto-activation and subsequent conformational changes 16-18. Ubiquitylation of various mitochondrial proteins by the activated Parkin then leads to selective elimination of damaged mitochondria 19-31. MITOL/March5 is a RING-type E3 with four transmembrane domains that is integrated into the outer mitochondrial membrane 32-34. It has been reported that MITOL regulates mitochondrial morphology via association with and ubiquitylation of the mitochondrial fission factors, Fis1 and Drp1 34. Indeed, siRNA-mediated knockdown of MITOL resulted in mitochondrial fragmentation. MITOL is also known to ubiquitylate mitochondrial mitofusin2 (MFN2), which leads to the tethering of mitochondria to the endoplasmic reticulum (ER) 33, 35, 36. When we investigated whether a mitochondrial E3(s) cooperate with Parkin to eliminate damaged mitochondria 37, we noticed that MITOL showed ectopic localization during Parkin-mediated mitochondrial ubiquitylation. This finding is of interest as the disconnected localization of MITOL and mitochondria had not been previously reported. In this study, we found that both overexpressed and endogenous MITOL translocated from damaged mitochondria to peroxisomes in response to mitochondrial depolarization. Notably, MITOL translocation was completely dependent on PINK1 and the E3 activity of Parkin with ubiquitylation of MITOL at K268 triggering mitochondrial extraction in a p97/VCP-dependent manner. Furthermore, peroxin-dependent translocation of MITOL was also observed, suggesting that the p97/VCP–peroxin axis regulates ubiquitylated MITOL translocation following mitophagy stimulation. Results MITOL redistributes to peroxisomes in response to mitochondrial depolarization in a Parkin-dependent manner To understand the molecular details of MITOL localization when the mitochondrial membrane potential is decreased, C-terminal HA-tagged MITOL and Flag-Parkin were transiently expressed in HeLa cells. Cells treated with or without the uncoupler CCCP (carbonyl cyanide m-chlorophenylhydrazone) were stained with anti-HA and anti-Tom20/TOMM20 antibodies (Fig 1A). Without CCCP treatment, MITOL-HA overlapped with the mitochondrial outer membrane protein Tom20, indicating its mitochondrial localization. Interestingly, after 3 h of CCCP treatment, a large population of MITOL clustered in dot-like structures that did not co-localize with Tom20, and we assumed that MITOL had localized in another organelle rather than the cytosol. To clarify MITOL localization after mitophagy stimulation, we first performed co-immunostaining experiments using various organelle markers. The MITOL-HA-positive dot-like structures were not coincident with the immunostaining patterns of Sec61β (ER marker) or LAMP1 (lysosome marker; Fig 1B and C), but did largely co-localize with catalase (peroxisome marker) after 3 h of CCCP treatment (Fig 1D and F). Next, co-localization between MITOL and each marker protein was assessed as a Pearson correlation coefficient. The quantitative analysis supported our aforementioned findings that co-localization between MITOL and Tom20 was significantly decreased following CCCP treatment for 3 h (Fig 1E, columns 1 and 2). Although a small percentage of MITOL was merged with catalase in the absence of CCCP treatment, the co-localization markedly increased in response to CCCP treatment for 3 h (Fig 1E, columns 7 and 8). Peroxisomal membrane protein PMP34 fused with FusionRed at its C-terminus (PMP34-FusionRed) also co-localized with MITOL-GFP in Parkin-expressing HeLa cells after 3 h of CCCP treatment (Fig 1G), and this co-localization was confirmed by quantitative analysis (Fig 1H). In addition, the peroxisomal localization of MITOL following dissipation of the mitochondrial membrane potential was unaffected by C-terminal addition of the 3Flag epitope (Appendix Fig S1A). Cells exposed to the potassium ionophore valinomycin (another type of mitochondrial uncoupler) for 3 h also exhibited MITOL and catalase co-localization, suggesting that peroxisomal localization of MITOL was not an artifact of CCCP treatment (Appendix Fig S1B). These results indicate that MITOL moves to peroxisomes in response to mitophagy stimulation. Next, we tested the possibility that peroxisomes are the halfway point of a translocation process that leads to another organelle. Following extended CCCP treatment, HeLa cells stably expressing HA-Parkin and 3Flag-MITOL were stained with Pex14 (Appendix Fig S2A), Sec61β (Appendix Fig S2B), and Tom20 (Appendix Fig S2C: because Tom20 was substantially degraded via mitophagy for > 12 h, Tom20-positive cells were selected to be shown). MITOL and Pex14 co-localization peaked within 3 h of CCCP treatment and then dispersed over time. However, even with extended CCCP treatment, we found no evidence of MITOL co-localization with Tom20 or Sec61β. Figure 1. MITOL redistributes to peroxisomes in response to a loss in the mitochondrial membrane potential A–C. MITOL-HA formed small dot-like structures following CCCP treatment. HeLa cells transiently expressing Flag-Parkin and MITOL-HA were treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry. MITOL and Tom20 (mitochondrial marker; A) signals co-localized well without CCCP treatment, whereas the MITOL-positive small dots were not coincident with Tom20, Sec61β (ER marker; B), or LAMP1 (lysosomal marker; C) after CCCP treatment. Scale bars, 10 μm. D. MITOL-HA co-localized with catalase (peroxisome marker) following CCCP treatment. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Arrowheads indicate representative examples of MITOL-HA co-localization with catalase. E. Correlation statistics for the localization of MITOL-HA and Tom20, Sec61β, LAMP1, or catalase. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test. F. The line graph shows a line scan of fluorescence through three MITOL-positive peroxisomes (red bar in D) that clearly indicates co-localization of MITOL (green line) and catalase (magenta line). G. Peroxisomal membrane protein (PMP) 34-FusionRed also co-localized with MITOL-GFP in Parkin-expressing HeLa cells after 3 h of CCCP treatment. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. H. Correlation statistics for the localization of MITOL-GFP and PMP34-FusionRed. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test. Download figure Download PowerPoint PINK1 and the E3 activity of Parkin are required for MITOL redistribution to peroxisomes Next, we investigated whether Parkin is required for the redistribution of MITOL from impaired mitochondria to peroxisomes. For this purpose, MITOL-HA was transfected into HeLa cells that lack a functional PARK2 gene by genomic mutation 4, 38. As expected, MITOL-HA moved to peroxisomes in HeLa cells stably expressing GFP-Parkin after 3 h of CCCP treatment and did not merge with Tom20 (Fig 2A, bottom panel). In contrast, MITOL-HA was retained on mitochondria after CCCP treatment in HeLa cells lacking endogenous Parkin expression (Fig 2A, upper panel). Valinomycin-treated cells showed the same phenomena (Appendix Fig S1C), and quantitative analysis confirmed that in the absence of Parkin, MITOL-HA was retained on depolarized mitochondria (Fig 2B). These results indicate that Parkin is required for MITOL relocation from mitochondria to peroxisomes. Figure 2. Parkin is required for MITOL redistribution to peroxisomes MITOL-HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild-type HeLa cells or HeLa cells stably expressing GFP-Parkin were transfected with MITOL-HA, treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti-HA and anti-Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Correlation statistics for the localization of MITOL-HA and Tom20 in the absence or presence of GFP-Parkin. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test. MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag-MITOL were transfected with a GFP-Parkin plasmid or the pEGFP-C1 vector, treated with 15 μM CCCP ± 10 μM MG132 for 3 h, and then immunoblotted with anti-Flag, anti-ubiquitin, and anti-tubulin antibodies. Black arrowheads indicate 3Flag-MITOL in the upper panel and mono-ubiquitin in the middle panel, respectively. The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that mitofusin2 (MFN2) underwent rapid degradation within 3 h of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria-rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 h 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor Z-VAD-FMK (10 μM) was added to cells along with valinomycin to prevent cell death. Quantification of 3Flag-MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 μM valinomycin + Z-VAD-FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m. relative to untreated samples in three biological replicates. Pre-existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 h to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 h, cells were immunostained using anti-Flag, anti-Pex14 (peroxisomal membrane protein), and anti-Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 μm. Download figure Download PowerPoint Next, we sought to demonstrate that pre-existing mitochondrial MITOL moved to peroxisomes in response to mitochondrial depolarization, rather than direct peroxisomal targeting of newly synthesized MITOL following CCCP treatment. The simplest experiment would suggest the use of cycloheximide (CHX), which blocks protein synthesis. However, we cannot use CHX as Parkin translocation to impaired mitochondria depends on the accumulation of newly synthesized PINK1 on the outer mitochondrial membrane following CCCP treatment, and thus, CHX treatment would block PINK1 synthesis and consequently impede Parkin translocation/activation 39. Instead of CHX, we utilized a doxycycline induction/repression system. HeLa cells stably expressing HA-Parkin were transiently transfected with pTRE3G-3Flag-MITOL and pCMV-Tet3G plasmids. Before doxycycline treatment, MITOL expression was repressed and no signal was observed (Fig 2F, top panel). After 3 h of doxycycline treatment, MITOL expression was induced and the protein localized on mitochondria (Fig 2F). Cells were then repeatedly washed to remove doxycycline induction (i.e., no new MITOL synthesis) and treated with CCCP for an additional 3 h. Examination of these cells revealed co-localization of MITOL and the peroxisomal marker Pex14 (Fig 2F), suggesting that previously synthesized MITOL that had been localized on mitochondria had moved to peroxisomes in response to CCCP treatment. We also wanted to eliminate the trivial possibility that (i) MITOL exists in two distinct organellar states, one that is predominantly localized on mitochondria and a smaller grouping localized on peroxisomes, and (ii) that very rapid degradation (within 3 h) of mitochondria-localized MITOL leaves peroxisome-localized MITOL as the dominant grouping. This possibility could lead to an erroneous conclusion that MITOL translocated to the peroxisomes. If true, then the total amount of MITOL should decrease rapidly; however, the protein level of MITOL was not drastically altered following CCCP or MG132 treatment, suggesting that a large fraction of MITOL is not rapidly degraded during mitophagy stimulation (Fig 2C). Furthermore, we monitored the levels of MITOL over an extended period of valinomycin treatment (6 and 24 h) in conjunction with fractionation analysis. Mitofusin2 (MFN2), which is degraded by the proteasome following mitochondrial depolarization 22, underwent rapid degradation within 3 h of valinomycin treatment (Fig 2D and E), in particular the mitochondria-rich fractions. MTCO2, an inner mitochondrial protein that is degraded via mitophagy following mitochondrial depolarization 40, was significantly decreased following 24 h of valinomycin treatment (Fig 2D and E). In contrast, 3Flag-MITOL persisted throughout the first 6 h and only underwent a slight reduction after 24 h, confirming that the total cellular amount of MITOL is not dramatically decreased. Although MITOL underwent a modest reduction at 24 h, it was not as rapid as that seen with MFN2 and MTCO2 that are destined for Ub- and mitophagy-mediated degradation (Fig 2D and E). Given that the mitochondria-enriched fraction contains some peroxisomes (as shown in Fig 11), the results are consistent with our hypothesis that mitochondrial MITOL is not degraded but rather migrates to peroxisomes. MITOL redistribution from impaired mitochondria to peroxisomes occurs downstream of Parkin. Because ATG5 is indispensable for the progression of autophagy, an ATG5 knockout cell line was used to investigate whether mitophagy inhibition affects the peroxisomal translocation of MITOL. When MITOL localization in ATG5 knockout cells stably expressing HA-Parkin and 3Flag-MITOL was analyzed by immunocytochemistry, MITOL clearly overlapped with PMP70 (peroxisomal marker) following CCCP treatment, which was similar to that seen in wild-type cells (Fig EV1). This indicates that inhibition of mitophagy does not affect the redistribution of MITOL to peroxisomes. These results further refute the possibility that MITOL is localized on both peroxisomes and mitochondria under steady-state conditions and that peroxisomal localization only becomes apparent following the rapid degradation of mitochondrial MITOL via CCCP-induced mitophagy. Click here to expand this figure. Figure EV1. Autophagy activity is not a prerequisite for the translocation of MITOL to peroxisomesThe localization of MITOL in ATG5 knockout cells stably expressing Parkin was analyzed by immunocytochemistry using anti-Flag and anti-PMP70 antibodies. Following 15 μM CCCP treatment, MITOL clearly overlapped with PMP70 in ATG5 knockout cells, suggesting that inhibition of autophagy (including mitophagy) does not affect the translocation of MITOL to peroxisomes. Scale bars, 10 μm. Download figure Download PowerPoint Collectively, it is unlikely that newly synthesized MITOL is directly targeted to peroxisomes in response to CCCP treatment or that mitochondrial MITOL is rapidly degraded within 3 h of CCCP treatment; rather, the data support translocation of pre-existing mitochondrial MITOL to peroxisomes in response to mitochondrial depolarization. To further analyze Parkin dependency, we examined whether Parkin E3 activity is required for MITOL translocation to peroxisomes. In the presence of wild-type Parkin, 3Flag-MITOL localized to peroxisomes after CCCP treatment as described above. In contrast, no MITOL translocation was observed in cells expressing a Parkin (C431S) mutant in which the catalytic Cys critical for E3 activity had been mutated (Fig 3A, middle panels), indicating that the redistribution of MITOL from mitochondria to peroxisomes requires Parkin E3 activity. Furthermore, we also confirmed that PINK1 is required for MITOL redistribution. In PINK1 knockout (KO) HeLa cells 41, the redistribution of MITOL to peroxisomes was not observed when mitochondria were damaged despite the presence of wild-type Parkin (Fig 3A, lower panel). These results were further substantiated by quantitative analysis (Fig 3C). To determine whether endogenous levels of Parkin are sufficient for the transition of MITOL from damaged mitochondria to peroxisomes, we utilized an SH-SY5Y human neuroblastoma cell line that expresses endogenous Parkin. SH-SY5Y cells transiently expressing 3Flag-MITOL were treated with valinomycin + Z-VAD-FMK for 3 or 6 h and then analyzed by immunofluorescence (note that Z-VAD-FMK is an apoptosis inhibitor that was included in the incubation because SH-SY5Y cells are more sensitive to uncoupler treatment and detach easily). As shown in Appendix Fig S3, MITOL once again redistributed to peroxisomes. However, the frequency of MITOL redistribution in SH-SY5Y cells appeared to be lower than that observed following overexpression in HeLa cells. We think this difference arises from the experimental conditions. Typically, only a minor population of mitochondria are damaged, and thus, the physiological levels of Parkin are sufficient to manage them. However, valinomycin or CCCP treatment causes depolarization of all mitochondria, and as a result, the endogenous levels of Parkin are overwhelmed. Given that the endogenous levels of Parkin in SH-SY5Y cell lines are lower than in the overexpression conditions, the endogenous Parkin might be insufficient to assist in relocation of all the MITOL molecules from damaged mitochondria to peroxisomes. However, we do observe peroxisomal localization of MITOL without exogenous Parkin expression. We thus conclude that in addition to mitochondrial membrane dissipation, both PINK1 and the E3 activity of Parkin are essential for the translocation of MITOL to peroxisomes. Figure 3. Both PINK1 and the E3 activity of Parkin are required for MITOL redistribution to peroxisomes Wild-type or PINK1 knockout (KO) HeLa cells were transfected with GFP-Parkin wild-type or the C431S mutant, treated with 15 μM CCCP, and then immunostained with anti-Flag and anti-catalase antibodies. Wild-type Parkin, but not the catalytically inactive Parkin (C431S) mutant, mediated 3Flag-MITOL translocation to peroxisomes following mitophagy stimulation. In PINK1 KO HeLa cells, the redistribution of MITOL to peroxisomes was not observed even in the presence of wild-type Parkin when mitochondria were damaged. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Arrowheads indicate representative examples of MITOL–peroxisome co-localization observed only in the presence of wild-type Parkin. The redistribution of MITOL from mitochondria to peroxisomes does not require its own E3 activity. The E3-inactive MITOL Cys65Ser/Cys68Ser (CS) and H43W mutants were transfected into HeLa cells stably expressing HA-Parkin, treated with 15 μM CCCP, and then subjected to immunocytochemistry with anti-Flag and anti-catalase antibodies. After 3 h of CCCP treatment, both the CS and H43W mutants co-localized with catalase. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Arrowheads indicate representative examples of 3Flag-MITOL co-localization with catalase. Correlation statistics for the localization of 3Flag-MITOL and catalase in the presence of GFP-Parkin wild-type or an inactive C431S mutant. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test. MITOL overlapped with catalase only in the presence of wild-type Parkin in wild-type HeLa cells. In PINK1 knockout (KO) HeLa cells, wild-type Parkin was unable to induce the peroxisomal translocation of MITOL. Correlation statistics for the localization of 3Flag-MITOL wild-type or inactive mutants with catalase. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test. Download figure Download PowerPoint MITOL specifically relocates to peroxisomes Since MITOL is an E3, we next examined whether the E3 activity of MITOL itself is required for translocation to peroxisomes. To impair the MITOL E3 activity, we mutated Cys65/Cys68 within the RING domain of MITOL to Ser (CS) or the conserved Zn-binding His43 to Trp (H43W) 34, 35. Despite higher background peroxisomal signals, MITOL CS and H43W mutants localize on mitochondria. However, following 3 h of CCCP treatment, both mutants clearly co-localized with catalase (Fig 3B). Quantitative analysis confirmed the cytochemical results (Fig 3D), indicating that the redistribution of MITOL from mitochondria to peroxisomes does not require its own E3 activity. Next, to determine whether the Parkin-dependent translocation from depolarized mitochondria to peroxisomes is limited to MITOL, the localization of other mitochondrial proteins was analyzed by immunostaining. The proteins examined included both N-terminal anchored proteins, MitoNEET/CISD1 and Tom70, and C-terminal anchored proteins Fis1 and Miro1/2 (Fig 4A). No peroxisomal translocation following CCCP treatment was observed for either N-terminal anchored or C-terminal anchored proteins (Fig 4B). The matrix protein, Hsp60, likewise did not undergo peroxisomal translocation following CCCP treatment (Fig 4B). It has been reported that Drp1, Fis1, Mitofusin (MFN) 1/2, MiD49, and Mcl1 interact with MITOL as substrates 33, 34, 42-44. We consequently also examined the subcellular localization of these proteins following CCCP treatment. Because antibodies that detect endogenous MFN2, MiD49, and Mcl1 were not av