Stress-induced OMA1 activation and autocatalytic turnover regulate OPA1-dependent mitochondrial dynamics

Article18 February 2014free access Source Data Stress-induced OMA1 activation and autocatalytic turnover regulate OPA1-dependent mitochondrial dynamics Michael J Baker Michael J Baker Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany Search for more papers by this author Philipp A Lampe Philipp A Lampe Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany Search for more papers by this author Diana Stojanovski Diana Stojanovski Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Vic., Australia Search for more papers by this author Anne Korwitz Anne Korwitz Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany Search for more papers by this author Ruchika Anand Ruchika Anand Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany Search for more papers by this author Takashi Tatsuta Takashi Tatsuta Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany Search for more papers by this author Thomas Langer Corresponding Author Thomas Langer Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany Max-Planck-Institute for Biology of Aging, Cologne, Germany Search for more papers by this author Michael J Baker Michael J Baker Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany Search for more papers by this author Philipp A Lampe Philipp A Lampe Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany Search for more papers by this author Diana Stojanovski Diana Stojanovski Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Vic., Australia Search for more papers by this author Anne Korwitz Anne Korwitz Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany Search for more papers by this author Ruchika Anand Ruchika Anand Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany Search for more papers by this author Takashi Tatsuta Takashi Tatsuta Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany Search for more papers by this author Thomas Langer Corresponding Author Thomas Langer Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany Max-Planck-Institute for Biology of Aging, Cologne, Germany Search for more papers by this author Author Information Michael J Baker1,‡, Philipp A Lampe1,‡, Diana Stojanovski2, Anne Korwitz1, Ruchika Anand1, Takashi Tatsuta1 and Thomas Langer 1,3 1Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany 2Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Vic., Australia 3Max-Planck-Institute for Biology of Aging, Cologne, Germany ‡Both authors contributed equally. *Corresponding author. Tel: +49 221 470 4876; Fax: +49 221 470 6749; E-mail: [email protected] The EMBO Journal (2014)33:578-593https://doi.org/10.1002/embj.201386474 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 The dynamic network of mitochondria fragments under stress allowing the segregation of damaged mitochondria and, in case of persistent damage, their selective removal by mitophagy. Mitochondrial fragmentation upon depolarisation of mitochondria is brought about by the degradation of central components of the mitochondrial fusion machinery. The OMA1 peptidase mediates the degradation of long isoforms of the dynamin-like GTPase OPA1 in the inner membrane. Here, we demonstrate that OMA1-mediated degradation of OPA1 is a general cellular stress response. OMA1 is constitutively active but displays strongly enhanced activity in response to various stress insults. We identify an amino terminal stress-sensor domain of OMA1, which is only present in homologues of higher eukaryotes and which modulates OMA1 proteolysis and activation. OMA1 activation is associated with its autocatalyic degradation, which initiates from both termini of OMA1 and results in complete OMA1 turnover. Autocatalytic proteolysis of OMA1 ensures the reversibility of the response and allows OPA1-mediated mitochondrial fusion to resume upon alleviation of stress. This differentiated stress response maintains the functional integrity of mitochondria and contributes to cell survival. Synopsis A sensor domain for mitochondrial stress and a novel self-degradation mechanism enables the mitochondrial protease OMA1 to reversibly control mitochondrial fragmentation in response to various cellular stresses. Various cellular stress conditions activate OMA1 and induce OPA1 processing and mitochondrial fragmentation. A stress-sensor domain is present outside the conserved metallopeptidase domain in the amino terminal region of mammalian OMA1. OMA1 activation is accompanied by its autocatalytic turnover ensuring the reversibility of the response. Introduction Mitochondria continuously divide and fuse. This dynamic behaviour is required for the inheritance of mitochondria, ensures mitochondrial trafficking and the distribution of mitochondria-derived metabolites throughout the cell and contributes to mitochondrial quality control (Chen & Chan, 2010; Westermann, 2010; Nunnari & Suomalainen, 2012; Youle & van der Bliek, 2012; Anand et al, 2013). Fusion and fission of mitochondrial membranes are mediated by dynamin-like GTPases that act on both the mitochondrial inner (IM) and outer membrane (OM; Hoppins et al, 2007; Ishihara et al, 2013). Mutations in these GTPases are associated with various neurodegenerative disorders highlighting the importance of mitochondrial dynamics for neuronal survival (Knott & Bossy-Wetzel, 2008; Chan, 2012). Both mitochondrial fusion and fission are highly regulated in response to various physiological cues. Starvation and stress conditions inhibit mitochondrial fission and induce the fusion of mitochondrial membranes to form a highly interconnected network (Tondera et al, 2009; Gomes et al, 2011; Rambold et al, 2011). On the other hand, severe mitochondrial damage and depolarisation impair fusion (Duvezin-Caubet et al, 2006; Ishihara et al, 2006; Cereghetti et al, 2008). Unopposed fission leads to fragmentation of the mitochondrial network under these conditions, which allows the selective autophagic removal of damaged mitochondria and which is associated with necrotic and apoptotic cell death (Twig & Shirihai, 2011; Youle & Narendra, 2011; Youle & van der Bliek, 2012). The dynamin-like GTPase OPA1 mediates the fusion of the mitochondrial IM and maintains cristae morphology (Olichon et al, 2003; Cipolat et al, 2004; Griparic et al, 2004; Frezza et al, 2006; Merkwirth et al, 2008; Song et al, 2009). Mounting evidence revealed that its activity is regulated in response to mitochondrial stress (Duvezin-Caubet et al, 2006; Ishihara et al, 2006). Under normal conditions, proteolytic cleavage of long isoforms of OPA1 (L-OPA1) results in the balanced accumulation of long and short OPA1 (S-OPA1) forms (Ishihara et al, 2006; Griparic et al, 2007; Song et al, 2007). Constitutive processing of OPA1 is required to maintain a normal morphology of mitochondria. However, mitochondrial depolarisation induces the complete conversion of L-OPA1 into S-OPA1 (Duvezin-Caubet et al, 2006; Ishihara et al, 2006; Song et al, 2007; Guillery et al, 2008). Stress-induced OPA1 processing inhibits fusion and causes mitochondrial fragmentation and the segregation of damaged mitochondria (Twig et al, 2008). A tubular mitochondrial network is restored upon removal of stress stimuli (Ishihara et al, 2006; Griparic et al, 2007; Guillery et al, 2008; Head et al, 2009), whereas irreversibly damaged mitochondria are selectively degraded by mitophagy if stress persists (Narendra et al, 2008; Twig et al, 2008). The plasticity of the mitochondrial network thus allows a differentiated response to mitochondrial dysfunction and is central to maintain the functional integrity of mitochondria. A number of proteases have been linked to constitutive and stress-induced OPA1 processing. These include the i-AAA protease YME1L that constitutively cleaves L-OPA1 isoforms at the proteolytic site S2 (Griparic et al, 2007; Song et al, 2007; Stiburek et al, 2012), while the metallopeptidase OMA1 processes OPA1 at site S1 (Ehses et al, 2009; Head et al, 2009; Quiros et al, 2012). OMA1 was first identified in yeast as a peptidase with overlapping activities with the m-AAA protease and was shown to degrade misfolded membrane proteins (Käser et al, 2003; Khalimonchuk et al, 2012). Members of the OMA1 family of membrane-embedded peptidases contain a conserved M48 metallopeptidase domain [according to the MEROPS database, (Rawlings et al, 2012)] and are found in eukaryotes as well as eubacteria. The analysis of OMA1-deficient mice revealed an unexpected metabolic phenotype characterised by hepatic steatosis, decreased energy expenditure and impaired thermogenic regulation, pointing to important functions of stress-induced OPA1 processing for metabolic homeostasis (Quiros et al, 2012). It is presently not understood how the activity of OMA1 is regulated to allow an immediate and reversible response to mitochondrial stress. Various stress stimuli were found to trigger mitochondrial fragmentation affecting the membrane potential across the inner membrane (Δψ) and intramitochondrial ATP levels (Baricault et al, 2007), but the role of OMA1 under these conditions remained speculative. Here, we demonstrate a general role of OMA1 for stress-induced OPA1 processing and identify a critical sensory region outside the metallopeptidase domain of OMA1, which is required for OMA1 activation. Activation of OMA1 is accompanied by its autocatalytic turnover ensuring the reversibility of the stress response. Results OMA1 mediates constitutive and stress-induced OPA1 processing At least five different forms of OPA1 accumulate in mouse embryonic fibroblasts (MEFs; Griparic et al, 2007; Guillery et al, 2008; Ishihara et al, 2006; Fig 1A): long (L) forms a and b derived from OPA1 splice variants 1 and 7, respectively, and short (S) forms c, d and e that are generated upon proteolytic processing of L-OPA1 forms at sites S1 and S2. Cleavage of L-OPA1 forms a and b at S1 results in the formation of c and e, respectively, while processing at S2 generates d. To unambiguously establish the role that OMA1 plays in the processing of OPA1, we monitored the accumulation of OPA1 forms in MEFs isolated from Oma1−/− mice by immunoblotting. Both L- and S-OPA1 forms accumulated in Oma1−/− cells; however, the proteolytic forms c and e were absent (Fig 1B). Thus, OMA1 mediates constitutive OPA1 processing of both splice variants 1 and 7 at S1. The morphology of the mitochondrial network appears largely normal in MEFs lacking OMA1 (Ehses et al, 2009; Head et al, 2009; Quiros et al, 2012; see also Fig 1G), suggesting that OPA1 forms c and e are dispensable for mitochondrial fusion. Figure 1. OMA1-dependent processing of OPA1 A. Linear depiction of the OPA1 splice variants 1 and 7. L-OPA1 splice variant 1 (b) and splice variant 7 (a) are shown with cleavage sites for OMA1 (S1) or YME1L (S2). Processing generates S-OPA1 form e (OMA1) or S-OPA1 forms c (OMA1) and d (YME1L). MTS, mitochondrial targeting sequence; TM, transmembrane domain. B. OMA1 is required for inducible and constitutive OPA1 processing. Oma1+/+ and Oma1−/− MEFs expressing OMA1-myc or OMA1E324Q-myc as indicated were incubated in the absence or presence of CCCP for 2 h. C. Stress-induced OPA1 processing by OMA1. Oma1+/+ and Oma1−/− MEFs were incubated with CCCP, valinomycin, oligomycin (Oligo) and/or antimycin A (Anti) for 1 h. D. Determination of ∆Ψ in Oma1+/+ MEFs under various stress conditions by JC-1 staining. Untreated MEFs were used as a control. E. Heat stress-induced OPA1 processing depends on OMA1. Oma1+/+ and Oma1−/− MEFs were cultured at 42°C for the times indicated. F. H2O2 induces OMA1-mediated OPA1 processing. Oma1+/+ and Oma1−/− MEFs were incubated in the presence of H2O2 (0.5 mM) for the times indicated. In all panels, cellular proteins were extracted and analysed by SDS–PAGE and immunoblotting. a-e, OPA1 forms. G, H. The mitochondrial network in Oma1−/− MEFs is protected against H2O2-induced fragmentation. Oma1+/+ and Oma1−/− MEFs were incubated for 4 h in the presence or absence of H2O2 as indicated. Mitochondrial morphology was visualised by immunofluorescence microscopy using cytochrome c-specific antibodies. Scale bar, 15 μm (inset 5 μm). A quantification of mitochondrial morphology in 100 cells is shown in (H). Source data are available online for this figure. Source Data for Figure 1B, C, E, F, G [embj201386474-SourceData-Fig1B-C-E-F-G.pdf] Download figure Download PowerPoint Depolarisation of the mitochondrial membrane potential (∆Ψ) using the ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) results in loss of L-OPA1 in an OMA1-dependent manner (Fig 1B; Ehses et al, 2009; Head et al, 2009; Quiros et al, 2012), suggesting that OMA1 regulates L-OPA1 stability in response to the bioenergetic status of mitochondria in the cell. To further define conditions for OMA1 activation, we first examined the role of the electrical component (∆V) of ∆Ψ. Treatment of cells with valinomycin, a potassium ionophore dissipating ∆Ψ similar to CCCP, triggered proteolysis of L-OPA1 in an OMA1-dependent fashion (Fig 1C). Addition of antimycin A, which blocks transfer of electrons from cytochrome b to cytochrome c, did not efficiently stimulate OPA1 processing by OMA1, while treatment of the cells with oligomycin, which inhibits the ATP synthase, stimulated OMA1-mediated OPA1 processing at least to some extent under the conditions used (Fig 1C). However, addition of antimycin A and oligomycin in combination destabilised L-OPA1 and resulted in a more efficient degradation of L-OPA1 by OMA1 (Fig 1C). These results indicate that the loss of the proton motive force and the collapse of ∆Ψ activate OMA1 and lead to the degradation of L-OPA1. However, OMA1 activation does not strictly correlate with dissipation of ∆Ψ (Fig 1D). In agreement with previous findings (Baricault et al, 2007), oligomycin treatment stimulated proteolysis of L-OPA1, but does not significantly affect ∆Ψ. Conversely, ∆Ψ was lost, but L-OPA1 remained stable upon short incubation with antimycin A (Fig 1D). Similar effects on ∆Ψ were observed in Oma1−/− MEFs (Supplementary Fig S1A). To ascertain whether OMA1 is activated following other types of stress, we analysed L-OPA1 stability under heat stress. In agreement with recent observations (Sanjuan Szklarz & Scorrano, 2012), incubation of MEFs at 42°C accelerated processing of L-OPA1 (Fig 1E). However, OPA1 processing was completely abolished in Oma1−/− MEFs (Fig 1E), demonstrating that heat stress activates OMA1. Heat stress did not dissipate ∆Ψ as revealed by TMRM staining of mitochondria (Supplementary Fig S1B). We also addressed whether reactive oxygen species (ROS) affect OMA1 activity. Following the addition of H2O2 to induce ROS, we observed loss of ∆Ψ (Fig 1D), OMA1-dependent proteolysis of L-OPA1 (Fig 1F) and mitochondrial fragmentation (Fig 1G and H). OMA1 activation under oxidative stress was further substantiated incubating wild-type and OMA1-deficient MEFs with paraquat or rotenone (Supplementary Fig S2). We conclude from these experiments that OMA1 mediates both constitutive and stress-induced OPA1 processing. Multiple stress conditions that impinge on mitochondria activate OMA1, which degrades L-OPA1 and causes fragmentation of the mitochondrial network. These findings identify stress sensing by OMA1 as a critical cellular response to dysfunction of mitochondria. OPA1 processing by OMA1 in the intermembrane space of mitochondria To define the localisation of OMA1 within mitochondria, we exploited a lentiviral system to express OMA1 or C-terminally myc-tagged variants of OMA1 in Oma1−/− MEFs (OMA1-myc). In parallel, we expressed OMA1E324Q-myc harbouring a point mutation in its proteolytic centre. The OPA1 forms c and e were absent in cells lacking OMA1 but accumulated upon expression of OMA1-myc in these cells (Fig 1B). In contrast, OMA1E324Q-myc did not promote formation of c and e, demonstrating that OMA1-myc is active and that OPA1 processing at S1 depends on the proteolytic activity of OMA1 (Fig 1B). Mitochondria were isolated from Oma1−/− MEFs expressing OMA1-myc and subfractionated by osmotic disruption of the OM or solubilisation of mitochondrial membranes (Fig 2A). OMA1 remained largely resistant to externally added protease in intact mitochondria (Fig 2A). However, it became protease-accessible following osmotic disruption of the OM, while matrix-localised Hsp60 remained protected against proteolytic attack (Fig 2A). In contrast, solubilisation of mitochondrial membranes allowed degradation of OMA1-myc and Hsp60 by externally added protease (Fig 2A). Figure 2. OPA1 cleavage by OMA1 occurs in the IMSMitochondria were isolated from Oma1−/− MEFs expressing OMA1-myc. Submitochondrial localisation of OMA1. Mitochondria and mitoplasts, which were generated by hypotonic disruption of the OM (swelling), were incubated with or without proteinase K (PK; 50 μg/ml) and analysed by SDS–PAGE and immunoblotting. The IMS protein Smac and the matrix protein Hsp60 served as controls. Mitochondrial membranes were solubilised with Triton X-100 where indicated. The slightly reduced level of Smac upon PK treatment of mitochondria indicates partial disruption of the OM upon purification of mitochondria. OMA1 is an integral membrane protein. Alkaline extracts of mitochondria in Na2CO3 (pH 11.5) were separated into soluble (S) and membrane (P) fractions by centrifugation and analysed by SDS–PAGE and immunoblotting. The integral inner membrane protein PHB2 and the soluble intermembrane space protein Smac served as controls. T, total. Topological model of mature OMA1 in the IM. The C-terminal M48 metallopeptidase domain is exposed to the IMS, whereas the N-terminal domain is present at the matrix side of the IM. FLAG-tagged OPA1 Sp1 or OPA1 Sp1-TCS, which contains a TEV-cleavage site (TCS) instead of S1, were transfected into cell lines allowing tetracycline-inducible expression of HA-tagged Su9-TEV (targeted to the matrix) or Smac-TEV (targeted to the IMS). Cells were treated with tetracycline when indicated and analysed by SDS–PAGE and immunoblotting. Source data are available online for this figure. Source Data for Figure 2A, B, D [embj201386474-SourceData-Fig2A-B-D.pdf] Download figure Download PowerPoint Hydrophobicity plots reveal the presence of several hydrophobic regions within the amino acid sequence of OMA1 that may form transmembrane helices. We therefore examined the solubility of OMA1 and other mitochondrial marker proteins upon alkaline extraction of mitochondrial membranes isolated from OMA1-myc-expressing MEFs (Fig 2B). While Smac/DIABLO, a soluble protein of the intermembrane space (IMS), accumulated in the supernatant fraction, OMA1 was recovered from the pellet fraction, similar to the integral IM protein PHB2 (Fig 2B). Collectively, these results reveal that OMA1 is integral part of the IM, exposing its C-terminal domain to the IMS (Fig 2C). Previous experiments revealed that the S1 cleavage site of OPA1 is localised in the matrix (Ishihara et al, 2006), that is, in a different subcompartment of mitochondria than the catalytic domain of OMA1. To address these apparently conflicting data, we revisited the previous experiments and replaced S1 in OPA1 splice variant 1 (Sp1; Fig 1A) with the cleavage site for the tobacco etch virus (TEV) protease (Sp1-TCS). Sp1 or Sp1-TCS was co-expressed in HEK293 cells with TEV protease, which was expressed under the control of a tetracycline-inducible promoter and directed either to the mitochondrial matrix (Su9-TEV) or to the IMS (Smac-TEV; Fig 2D). Expression of Sp1 containing the S1 site allowed the formation of S-OPA1, independent of the presence of TEV (Fig 2D). In contrast, Sp1-TCS lacking the S1 site accumulated in the long form only (Fig 2D). While tetracycline-induced expression of matrix-localised TEV in these cells did not allow OPA1 processing, we observed cleavage of Sp1-TCS in the presence of TEV in the IMS (Fig 2D). These findings strongly suggest that the S1 processing site of OPA1 resides in the IMS and therefore is accessible for proteolytic attack by OMA1. OMA1 is unstable in depolarised mitochondria In agreement with previous findings (Head et al, 2009), we observed that OMA1-myc accumulated at a molecular mass of approximately 45 kDa within mitochondria. However, bioinformatic analysis of the amino acid sequence of OMA1 revealed potential targeting sequences of 28 or 86 amino acids, which would result in the formation of a significant larger mature form of OMA1 (55 or 49 kDa, respectively). To unambiguously identify the proteolytically active form of OMA1, we first assessed the import of nuclear-encoded OMA1 into mitochondria. Synthesis of OMA1 in a cell-free system in the presence of 35S-methionine generated a product of approximately 60 kDa, consistent with the predicted molecular mass of OMA1 (Fig 3A). Incubation of 35S-labelled OMA1 with mitochondria isolated from MEFs was accompanied by a time- and ∆Ψ-dependent accumulation of shorter forms of OMA1, indicating proteolytic processing upon import into mitochondria (Fig 3A). The molecular mass of these proteolytic products was approximately 45 kDa and thus of a similar size as OMA1-myc expressed in vivo, suggesting that they represent the mature form of OMA1. Therefore, we sought to determine the N-terminal sequence of mature OMA1 accumulating in vivo. We employed a HEK293 cell line to overexpress proteolytically inactive OMA1E324Q-myc in a tetracycline-inducible manner and purified OMA1E324Q-myc by immunoprecipitation using myc-specific antibodies (Fig 3B). A single protein of the expected molecular weight was detected in the eluate and subjected to N-terminal sequencing (Fig 3B). In contrast to in silico predictions for processing sites in OMA1, this analysis identified alanine at position 140 as the N-terminal amino acid of mature OMA1 (Fig 3C), which thus has a molecular mass of approximately 43 kDa. Figure 3. Turnover of mature OMA1 upon mitochondrial depolarisation In vitro import of OMA1 into mitochondria. Mitochondria were isolated from MEFs and incubated with [35S]-labelled OMA1 that was synthesised in a cell-free system for the time points indicated in the presence or absence of membrane potential (∆Ψ). Mitochondrial proteins were analysed by SDS–PAGE and autoradiography. p, precursor form; m, mature form. N-terminal sequencing of mature OMA1. Mitochondria isolated from HEK293 cells expressing OMA1E324Q-myc were solubilised in 1% (v/v) NP-40 and subjected to immunoprecipitation using myc-specific antibodies. Total (T), pellet (P), supernatant (S) and unbound (U) fractions (0.25%) and the eluate (E; 95%) were analysed by SDS–PAGE and Coomassie staining. The arrow indicates OMA1E324Q-myc in the eluate. N-terminal amino acid sequence of OMA1. Mature OMA1 is generated upon proteolytic cleavage at the site indicated with an arrow. The bold, underlined sequence represents the N-terminal sequence of mature OMA1 determined by Edman degradation. Degradation of pre-existing OMA1 following mitochondrial stress. MEFs stably expressing OMA1-myc were treated with CCCP, with cyclohexamide (CHX) or with ethanol and DMSO (control) as indicated. Samples were isolated and analysed via SDS–PAGE and immunoblotting. #, non-specific cross reaction; a-e, OPA1 forms. Overexpression of OMA1-myc in HEK293 cells. OMA1-myc expression was induced by addition of tetracycline (Tet) for the time points shown. Cell extracts were analysed by SDS–PAGE and immunoblotting. *, proteolytic fragments of OMA1. Overexpressed OMA1 is degraded following mitochondrial dysfunction. After tetracycline-induced expression of OMA1-myc, HEK293 cells were treated with CCCP or, for control, ethanol (Et) as indicated. Samples were assessed by SDS–PAGE and immunoblotting. *, C-terminal, proteolytic fragments of OMA1. Source data are available online for this figure. Source Data for Figure 3A, B, D, E, F [embj201386474-SourceData-Fig3A-B-D-E-F.pdf] Download figure Download PowerPoint The mechanism of OMA1 activation is presently not understood. However, it was previously reported that dissipation of ∆Ψ caused the accumulation of an approximately 60 kDa form of OMA1 at the mitochondrial surface, while the approximately 45 kDa form disappeared. This has led to the hypothesis that the approximately 60 kDa species is the proteolytically active form of OMA1, which is localised to the OM upon depolarisation of mitochondria and degrades L-OPA1 (Head et al, 2009). Therefore, we used the Oma1−/− cell line stably expressing OMA1-myc to investigate the requirement of newly synthesised OMA1 for OPA1 processing. Mitochondrial depolarisation induced rapid degradation of L-OPA1 and led to the accumulation of OPA1 forms c and e, while the OPA1 pattern was not altered in control cells (Fig 3D). Importantly, inhibition of cytosolic protein synthesis by cyclohexamide did not inhibit CCCP-induced OPA1 processing (Fig 3D). Thus, OMA1 pre-existing within mitochondria is activated under mitochondrial stress, demonstrating that the approximately 43 kDa form of OMA1 is proteolytically active. In the course of these experiments, we noted that mitochondrial depolarisation was accompanied by the loss of OMA1-myc (Fig 3D). This suggested that a dysfunction of mitochondria induces proteolysis of OMA1-myc, which is either completely degraded or C-terminally cleaved under these conditions. To further explore this proteolytic process and exclude indirect effects of the C-terminal protein tag, we generated antibodies directed against the proteolytic domain of mouse OMA1. However, we were unable to detect endogenous OMA1 in MEFs or HEK293 cells. We therefore turned to HEK293 cells, which allow tetracycline-inducible expression of OMA1-myc. Following addition of tetracycline, OMA1-myc accumulated in these cells as monitored using myc-specific antibodies. Increased expression of OMA1-myc resulted in processing of L-OPA1 (Fig 3E), consistent with proteolytic activity of OMA1 in polarised mitochondria. The overexpressed protein was detected using OMA1-specific antibodies in cells grown in the presence of tetracycline (Fig 3E), allowing us to monitor proteolysis of OMA1 upon dissipation of ∆Ψ. We induced OMA1-myc expression and then activated OMA1 by adding CCCP (Fig 3F). Two smaller OMA1-derived proteolytic fragments were observed with myc-specific antibodies pointing to degradation of OMA1 from the N-terminus (Fig 3F). Immunoblotting using OMA1-specific antibodies revealed rapid degradation of OMA1 (Fig 3F), indicating that OMA1 is completely degraded upon mitochondrial depolarisation, without the accumulation of stable intermediates. Using OMA1-specific antibodies, we detected a second form of OMA1 that was slightly smaller than mature OMA1 but degraded similarly to mature OMA1 (Fig 3F). This likely reflects clipping of C-terminal amino acids of OMA1-myc, as only the larger band was recognised by myc-specific antibodies. We conclude from these experiments that mitochondrial depolarisation induces the degradation of OMA1 from both termini offering a mechanism to attenuate the stress response. Autocatalytic proteolysis of OMA1 To identify the protease(s) mediating OMA1 turnover, we first tested various protease inhibitors for their ability to block the degradation of OMA1 in depolarised