- Record: found
- Abstract: found
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Metalloprotease OMA1 Fine-tunes Mitochondrial Bioenergetic Function and Respiratory Supercomplex Stability
research-article
Author(s):
Iryna Bohovych
1
,
2 ,
Mario R. Fernandez
4 ,
Jennifer J. Rahn
5 ,
Krista D. Stackley
5 ,
Jennifer E. Bestman
5 ,
Annadurai Anandhan
2
,
3 ,
Rodrigo Franco
2
,
3 ,
Steven M. Claypool
6 ,
Robert E. Lewis
4 ,
Sherine S. L. Chan
5 ,
Oleh Khalimonchuk
a
,
1
,
2
Publication date (Electronic):
14 September 2015
Journal:
Scientific Reports
Publisher:
Nature Publishing Group
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Abstract
Mitochondria are involved in key cellular functions including energy production, metabolic homeostasis, and apoptosis. Normal mitochondrial function is preserved by several interrelated mechanisms. One mechanism – intramitochondrial quality control (IMQC) – is represented by conserved proteases distributed across mitochondrial compartments. Many aspects and physiological roles of IMQC components remain unclear. Here, we show that the IMQC protease Oma1 is required for the stability of the respiratory supercomplexes and thus balanced and tunable bioenergetic function. Loss of Oma1 activity leads to a specific destabilization of respiratory supercomplexes and consequently to unbalanced respiration and progressive respiratory decline in yeast. Similarly, experiments in cultured Oma1-deficient mouse embryonic fibroblasts link together impeded supercomplex stability and inability to maintain proper respiration under conditions that require maximal bioenergetic output. Finally, transient knockdown of OMA1 in zebrafish leads to impeded bioenergetics and morphological defects of the heart and eyes. Together, our biochemical and genetic studies in yeast, zebrafish and mammalian cells identify a novel and conserved physiological role for Oma1 protease in fine-tuning of respiratory function. We suggest that this unexpected physiological role is important for cellular bioenergetic plasticity and may contribute to Oma1-associated disease phenotypes in humans.
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Most cited references39
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- Abstract: found
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Mitochondrial diseases in man and mouse.
D C Wallace (1999)
Over the past 10 years, mitochondrial defects have been implicated in a wide variety of degenerative diseases, aging, and cancer. Studies on patients with these diseases have revealed much about the complexities of mitochondrial genetics, which involves an interplay between mutations in the mitochondrial and nuclear genomes. However, the pathophysiology of mitochondrial diseases has remained perplexing. The essential role of mitochondrial oxidative phosphorylation in cellular energy production, the generation of reactive oxygen species, and the initiation of apoptosis has suggested a number of novel mechanisms for mitochondrial pathology. The importance and interrelationship of these functions are now being studied in mouse models of mitochondrial disease.
- Record: found
- Abstract: found
- Article: not found
Supercomplex assembly determines electron flux in the mitochondrial electron transport chain.
The textbook description of mitochondrial respiratory complexes (RCs) views them as free-moving entities linked by the mobile carriers coenzyme Q (CoQ) and cytochrome c (cyt c). This model (known as the fluid model) is challenged by the proposal that all RCs except complex II can associate in supercomplexes (SCs). The proposed SCs are the respirasome (complexes I, III, and IV), complexes I and III, and complexes III and IV. The role of SCs is unclear, and their existence is debated. By genetic modulation of interactions between complexes I and III and III and IV, we show that these associations define dedicated CoQ and cyt c pools and that SC assembly is dynamic and organizes electron flux to optimize the use of available substrates.
- Record: found
- Abstract: found
- Article: found
Regulation of OPA1 processing and mitochondrial fusion by m-AAA protease isoenzymes and OMA1
Sarah Ehses, Ines Raschke, Giuseppe Mancuso … (2009)
Introduction Mitochondria are dynamic organelles that undergo continuous remodeling through fusion and fission events. Conserved protein machineries mediate fusion and fission of mitochondrial membranes (Cerveny et al., 2007; Hoppins et al., 2007; Westermann, 2008). The dynamin-related GTPase OPA1, which resides in the mitochondrial intermembrane space, is involved in inner membrane fusion, in the regulation of mitochondrial cristae morphology, and in protecting cells from apoptosis (Olichon et al., 2003; Griparic et al., 2004; Frezza et al., 2006; Meeusen et al., 2006; Lenaers et al., 2009). Mutations in OPA1 cause dominant optic atrophy, a progressive neurological disease characterized by degeneration of the retinal ganglion cells and atrophy of the optic nerve (Amati-Bonneau et al., 2009). Mitochondria contain various OPA1 isoforms that arise from alternative splicing and proteolytic processing events at two sites, S1 and S2, generating shorter isoforms (Delettre et al., 2001; Ishihara et al., 2006). A combination of long and short OPA1 isoforms is required for mitochondrial fusion (Song et al., 2007), pointing to a crucial regulatory role of OPA1 processing. Roughly equimolar concentrations of long and short isoforms of OPA1 are formed under normal conditions. However, low mitochondrial ATP levels, the dissipation of the membrane potential across the inner membrane (Δψm), or apoptotic stimuli induce OPA1 cleavage, resulting in the loss of long isoforms (Duvezin-Caubet et al., 2006; Ishihara et al., 2006; Baricault et al., 2007). Various proteases in the inner membrane have been linked to the processing of OPA1. The intermembrane space AAA (ATPase associated with diverse cellular activities) protease YME1L1 regulates OPA1 cleavage at S2, which is only present in a subset of OPA1 variants (Griparic et al., 2007; Song et al., 2007). Debate exists about the protease cleaving OPA1 at S1, as both the rhomboid protease PARL (presenilin-associated rhomboid-like protein) and the matrix AAA (m-AAA) protease, an oligomeric ATP-dependent metallopeptidase in the inner membrane, have been proposed to be involved in OPA1 processing (Cipolat et al., 2006; Ishihara et al., 2006). However, normal OPA1 processing in mouse embryonic fibroblasts (MEFs) lacking PARL or the m-AAA protease subunit paraplegin raised doubts about the role of either protease for OPA1 processing in vivo (Duvezin-Caubet et al., 2007). Notably, human and murine mitochondria possess different m-AAA protease isoenzymes, which differ in their subunit composition but exert at least partially redundant activities (Koppen et al., 2007). Three homologous subunits are expressed in the mouse, AFG3L1 and -2 and paraplegin. Whereas paraplegin was exclusively detected in heterooligomeric complexes with AFG3L1 and -2, homooligomeric complexes composed of AFG3L1 or -2 do exist. Therefore, functionally redundant isoenzymes might substitute for the loss of paraplegin and maintain OPA1 processing in the absence of paraplegin. A complementation study in yeast indeed demonstrated functional conservation of different mammalian m-AAA protease isoenzymes and revealed their ability to cleave OPA1 expressed heterologously in yeast (Duvezin-Caubet et al., 2007). This notwithstanding, they have been linked to different neurodegenerative diseases in human: mutations in Spg7 encoding paraplegin cause a recessive form of hereditary spastic paraplegia, whereas heterozygous mutations in Afg3l2 are responsible for a dominant form of spinocerebellar ataxia, SCA28 (Cagnoli et al. 2008. Annual meeting of the American Society of Human Genetics. Abstr. 1501; DiBella et al. 2008. Annual meeting of the American Society of Human Genetics. Abstr. 216). It is presently unclear whether this reflects functional differences of m-AAA isoenzymes or results from a tissue-specific expression of m-AAA protease subunits (Koppen et al., 2007). In this study, we have assessed the function of different m-AAA isoenzymes in vivo using RNAi and demonstrate that OPA1 processing and mitochondrial fusion depend on AFG3L1 and -2. Moreover, we identify a novel peptidase in the inner membrane, OMA1, which mediates OPA1 processing if m-AAA proteases are absent or mitochondrial activities are impaired. Results AFG3L1 and -2 are required for mitochondrial fusion We used RNAi in MEFs to down-regulate m-AAA protease subunits individually or in combination. Immunoblotting revealed effective depletion of AFG3L1 and -2 and paraplegin (Fig. 1 A). Mitochondrial morphology was monitored by fluorescence microscopy in cells cotransfected with a mitochondrially targeted DsRed variant. Neither paraplegin-depleted mitochondria nor mitochondria from Spg7−/− MEFs showed morphological abnormalities (Fig. S1 and not depicted). Similarly, mitochondria of cells transfected with either Afg3l1- or Afg3l2-specific siRNA appeared largely normal in morphology (Fig. 1, B and C). However, concomitant down-regulation of both subunits caused fragmentation of the mitochondrial network in ∼80% of the cells (Fig. 1, B and C). Examination of these cells by electron microscopy revealed a severe disturbance of the mitochondrial ultrastructure (Fig. 1 D). Lamellar cristae were either completely absent or severely reduced in number in cells lacking AFG3L1 and -2, whereas the structure of cristae was not significantly affected upon down-regulation of AFG3L1 or -2 (Fig. 1 D). These findings reveal overlapping activities of AFG3L1 and -2 and demonstrate an essential role of the m-AAA protease for the ultrastructure of mitochondria. Figure 1. Fragmentation of mitochondria in MEFs depleted of AFG3L1 and -2. (A) Immunoblot analysis of MEFs transfected with siRNAs directed against AFG3L1 and -2 and paraplegin or scrambled siRNA (Scr) using specific antisera recognizing paraplegin (Para), AFG3L1 (L1), AFG3L2 (L2), and the 70-kD subunit of complex II (CII). (B and C) Mitochondrial morphology in AFG3L1- and AFG3L2-deficient MEFs was visualized by expression of mito-DsRed, and nuclear DNA was stained with DAPI. >150 MEFs were scored in each experiment. Bars represent means ± SD of three independent experiments (**, P 200 cells were scored per experiment. (C and D) SIMH depends on AFG3L1 and -2. After depletion of AFG3L1 and -2, MEFs were incubated for 3 (C and D) and 6 h (D) in the presence of 1 µM CHX (+CHX) as indicated. >150 cells were scored for each experiment. Bars represent means ± SD of three independent experiments (**, P 150 cells were scored. Bars represent means ± SD of three independent experiments (*, P 100 cells were scored. WT, AFG3L2; PS, AFG3L2E575Q; WB, AFG3L2E408Q. (E) Membrane potential in cells expressing AFG3L2 (WT) or AFG3L2E408Q (WB). (F) Oxygen consumption in cells expressing AFG3L2 (WT) or AFG3L2E408Q (WB) measured as in Fig. 3 F. Error bars represent the means ± SD of a minimum of three independent experiments (***, P 150 MEFs were scored in each immunofluorescence experiment. Bars represent means ± SD of three independent experiments (**, P 80% (Fig. S4 A). Depletion of OMA1 slightly reduced the level of OPA1 isoforms c and e, which are generated by S1 cleavage and accumulate at low levels under these conditions in MEFs (Fig. 5 C). However, we observed a significant stabilization of L-OPA1 in AFG3L1/AFG3L2-deficient MEFs upon down-regulation of OMA1, whereas transfection of the cells with scrambled oligonucleotides had no effect (Fig. 5 C). We monitored OPA1 processing directly after transient expression of the Flag-tagged OPA1 variant v1 in MEFs (Fig. 5 D). In the presence of AFG3L1 and -2, Flag-tagged L-OPA1 accumulated in OMA1-depleted cells (Fig. 5 D), indicating that OMA1 is able to cleave overexpressed OPA1. In contrast, in the absence of AFG3L1 and -2, down-regulation of OMA1 stabilized L-OPA1, demonstrating that OMA1 mediates turnover and increased OPA1 processing in the absence of the m-AAA protease (Fig. 5 D). OPA1 isoforms accumulated virtually as in wild-type (WT) cells in MEFs depleted of both OMA1 and the m-AAA protease. Therefore, we examined the morphology of the mitochondrial network after expression of a mitochondrially targeted DsRed variant in these cells (Fig. 5, E and F). Depletion of OMA1 did not significantly affect mitochondrial morphology in the presence of the m-AAA protease but restored the tubular network in AFG3L1/AFG3L2-deficient MEFs (Fig. 5, E and F). Electron micrographs revealed a normal, lamellar cristae morphology of mitochondria lacking both the m-AAA protease and OMA1 (Fig. 5 E). These findings substantiate our conclusion that the m-AAA protease regulates mitochondrial fusion via OPA1 and demonstrate that OMA1 is epistatic to the m-AAA protease. To further define the functional interrelationship of OMA1 and the m-AAA protease, we assessed the assembly of m-AAA proteases in OMA1-depleted mitochondria by blue-native gel electrophoresis (Fig. S4 B). Down-regulation of OMA1 did not affect the formation of the proteolytic complexes containing AFG3L2 and/or -1. Moreover, depletion of OMA1 did not interfere with the maturation of AFG3L1 or -2 (Fig. 5 C and Fig. S5), which occurs autocatalytically upon import into mitochondria (Koppen et al., 2009). Similarly, processing of an OMA1 variant carrying a C-terminal myc tag was not impaired in mitochondria depleted of AFG3L1 and -2 (Fig. S4 C). Tissue-specific destabilization of L-OPA1 in Afg3l2−/− mice Mutations in the m-AAA protease subunits paraplegin and AFG3L2 cause neurodegeneration in human, but molecular details of the pathogenesis are currently not understood. Our experiments revealed an at least partially redundant function of AFG3L1 and -2 for mitochondrial fusion in MEFs (Fig. 1). However, as AFG3L1 is a pseudogene in humans (Kremmidiotis et al., 2001) and is expressed at low levels in the mouse brain (Koppen et al., 2007; Martinelli et al., 2009), it is conceivable that the regulation of OPA1 processing by the m-AAA protease is of potential pathogenic relevance. To examine whether the loss of AFG3L2 impairs OPA1 cleavage in vivo, we monitored OPA1 processing in mitochondria isolated from tissues of a recently established Afg3l2−/− mouse line (Maltecca et al., 2008). The abundance of individual OPA1 isoforms varies in different tissues, as previously described (Fig. 6 A; Akepati et al., 2008). In the absence of AFG3L2, L-OPA1 isoforms accumulated at strongly reduced levels in the brain, heart, and kidney, demonstrating that the stability of L-OPA1 depends on AFG3L2 in vivo. Notably, deletion of Afg3l2 hardly affected OPA1 isoforms in the liver (Fig. 6 A), which is consistent with a compensatory effect of AFG3L1, which is expressed at higher levels relative to AFG3L2 in the liver (Koppen et al., 2007). Figure 6. Impaired OPA1 processing in Afg3l2−/− mice. (A) Immunoblot analysis of mitochondria isolated from the brain, liver, heart, and kidney of WT (L2+/+ ) or Afg3l2−/− (L2−/− ) mice using antisera directed against OPA1 and the 70-kD subunit of complex II (CII). (B) Immunoblot analysis of OPA1 in WT or Afg3l2−/− primary MEFs grown in the presence of glucose or galactose. (C and D) Mitochondrial morphology in WT or Afg3l2−/− cells was visualized by expression of a mitochondrially targeted GFP. >100 cells were scored. Bas represent means ± SD of three independent experiments. (E and F) Stabilization of long OPA1 isoforms in Afg3l2−/− MEFs grown in glucose or galactose upon down-regulation of OMA1. Immunoblot analysis was performed using specific antisera recognizing OPA1 and complex II. Scr, scrambled RNAi. Bar, 10 µm. To analyze whether depletion of OMA1 can suppress deleterious effects of the loss of the Afg3l2 gene, we monitored OPA1 processing and mitochondrial morphology in Afg3l2−/− MEFs (Fig. 6, B–D). Deletion of Afg3l2 only slightly affected cleavage of OPA1 in MEFs grown on glucose-containing medium, most likely because of the presence of AFG3L1 in these cells (Fig. 6 B). However, L-OPA1 was absent in Afg3l2−/− MEFs grown in the presence of galactose, i.e., conditions with an increased demand for mitochondrial respiratory function (Fig. 6 B). This was accompanied by a fragmentation of the mitochondrial network in the majority of these cells (Fig. 6, C and D). Depletion of OMA1 in immortalized Afg3l2−/− MEFs, cultured either in glucose- or galactose-containing medium, stabilized L-OPA1, providing further evidence that OMA1 mediates OPA1 processing if the function of the m-AAA protease is impaired (Fig. 6 E). Mitochondrial dysfunction induces OPA1 processing by OMA1 We next examined whether OMA1 also cleaves OPA1 under other cellular conditions known to induce OPA1 processing. Current models suggest that a dysfunction of mitochondria accompanied by decreased mitochondrial ATP levels stimulates OPA1 processing and results in mitochondrial fragmentation (Duvezin-Caubet et al., 2006; Baricault et al., 2007). Therefore, we compared the accumulation of OPA1 isoforms in a human osteosarcoma cell line containing (WT) or lacking mitochondrial DNA (mtDNA; ρ0; Fig. 7 A). Although long and short OPA1 isoforms accumulated in these cells in the presence of mtDNA, short isoforms accumulated in ρ0 cells (Fig. 7 A). Strikingly, long OPA1 isoforms were detected after RNAi-mediated down-regulation of OMA1, suggesting that the loss of mtDNA induces OPA1 cleavage by OMA1. Figure 7. Mitochondrial dysfunction induces OPA1 processing by OMA1. (A) OMA1 stabilizes long OPA1 isoforms in the absence of mtDNA. Immunoblot analysis of human osteosarcoma cells (143B) using specific antisera recognizing OPA1 and the 70-kD subunit of complex II (CII). Cells lacking mtDNA (ρ0) were transfected with OMA1-specific or scrambled (Scr) siRNA and compared with parental cells containing mtDNA (WT). Cell lysates were analyzed 72 h after transfection. (B and C) Stabilization of long OPA1 isoforms against CCCP (B)- and oligomycin (OLG; C)-induced processing in OMA1-depleted MEFs. MEFs transfected with OMA1-specific or scrambled siRNA were incubated after 48 h with 20 µM CCCP or after 60 h with 2 µM oligomycin for the indicated time periods. Cell lysates were analyzed by SDS-PAGE and immunoblotted with antibodies directed against OPA1 and complex II. OPA1 is converted into short isoforms upon dissipation of Δψm with the uncoupler CCCP or upon inhibition of the F1FO-ATP synthase with oligomycin (Fig. 7, B and C; Duvezin-Caubet et al., 2006; Ishihara et al., 2006). Down-regulation of OMA1 before the addition of CCCP or oligomycin significantly stabilized long OPA1 isoforms, indicating that OPA1 processing is mediated by OMA1 under both conditions (Fig. 7, B and C). In conclusion, these experiments identify OMA1 as the protease responsible for the inducible cleavage of OPA1 at processing site S1. Discussion Mutations in m-AAA protease subunits cause neurodegeneration, but pathogenic mechanisms remain enigmatic. Our results identify the m-AAA protease as a novel regulator of mitochondrial membrane fusion and cristae morphology, suggesting that m-AAA protease–associated diseases may represent a group of neurological disorders caused by deficiencies in mitochondrial dynamics. Murine AFG3L1- and AFG3L2-containing m-AAA protease isoenzymes exert at least partially overlapping activities during membrane fusion. Therefore, the relative expression of AFG3L1 and -2 in different tissues will determine whether or not a functional impairment of one m-AAA protease subunit affects the morphology of the mitochondrial network. Notably, the m-AAA protease subunit predominantly expressed in the mouse brain is AFG3L2, whereas AFG3L1 is only present at lower levels (Koppen et al., 2007; Martinelli et al., 2009). This explains severe neurological defects (Maltecca et al., 2008) and cerebellar degeneration in mice with reduced levels of AFG3L2-containing m-AAA isoenzymes (Martinelli et al., 2009), which is reminiscent of patients with heterozygous mutations in human AFG3L2 (Cagnoli et al. 2008. Annual meeting of the American Society of Human Genetics. Abstr. 1501; DiBella et al. 2008. Annual meeting of the American Society of Human Genetics. Abstr. 216). Whereas mitochondrial fusion depends on the presence of either AFG3L1 or -2, the third m-AAA protease subunit paraplegin is not required to maintain a reticulated mitochondrial network. Deletion of Spg7-encoding paraplegin did not affect mitochondrial morphology in MEFs. Moreover, phenotypes observed upon down-regulation of AFG3L1 and -2 were not worsened in paraplegin-deficient cells. However, it should be noted that an efficient depletion of AFG3L1 and -2 inactivates paraplegin that only exists in heterooligomeric complexes with AFG3L1 and -2 (Koppen et al., 2007). Thus, it is conceivable that paraplegin-containing isoenzymes may control mitochondrial morphology in cells expressing limiting levels of AFG3L1 and -2. This may explain the cell type–specific axonal degeneration associated with mutations in paraplegin both in mouse and human (Casari et al., 1998; Ferreirinha et al., 2004). The m-AAA protease controls mitochondrial fusion via OPA1 (Fig. 8). The loss of AFG3L2 in various tissues of the mouse, down-regulation of AFG3L1 and -2 in MEFs, or the expression of a dominant-negative AFG3L2 variant in human cells decreases OPA1 stability and results in an increased processing and turnover of long OPA1 isoforms. This causes fragmentation of the tubular mitochondrial network, which is at least partially restored upon expression of an OPA1 variant lacking the cleavage site S1. Thus, our experiments link, for the first time, the function of AFG3L1 and -2 to OPA1 in vivo. Figure 8. Two peptidases regulate OPA1 cleavage at S1. Newly imported OPA1 molecules are processed by the mitochondrial processing peptidase (MPP) in the matrix space (Ishihara et al., 2006). The stability of long OPA1 isoforms depends on m-AAA protease isoenzymes containing AFG3L1 and -2, which ensure the balanced formation of long and short OPA1 isoforms upon S1 cleavage and maintain mitochondrial fusion. The m-AAA protease itself may act as a processing peptidase for OPA1 or promote constitutive cleavage by OMA1. At decreased mitochondrial ATP levels, after dissipation of Δψm, or in the absence of mtDNA, OMA1 promotes stress-induced OPA1 processing, resulting in the complete conversion of long OPA1 isoforms to short variants. Constitutive cleavage of OPA1 at site 2 is not depicted. MTS, mitochondrial targeting sequence. Strikingly, inactivation or loss of the m-AAA protease does not result in the accumulation of long OPA1 isoforms, as predicted by experiments in yeast (Duvezin-Caubet et al., 2007), but decreases their stability. The accelerated cleavage of OPA1 in the absence of the m-AAA protease is mediated by OMA1, a new member of a conserved and widespread family of membrane-bound M48 metallopeptidases. We have previously identified yeast Oma1 in the mitochondrial inner membrane as a peptidase that can substitute for the function of the m-AAA protease during degradation of a misfolded membrane protein (Käser et al., 2003). Similarly, synthetic growth defects have been observed when mutations in the bacterial AAA protease FtsH were combined with mutations in HtpX, which is a distant homologue of OMA1, indicating overlapping proteolytic activities (Shimohata et al., 2002). In agreement with these findings, we demonstrate in this study that mammalian OMA1, similar to the m-AAA protease, can cleave OPA1. The substrate specificity of OMA1 is distinct from yeast Oma1, which cleaves neither OPA1 when expressed in yeast nor the yeast OPA1 homologue Mgm1 (Duvezin-Caubet et al., 2007). Therefore, differences in substrate recognition by OMA1-like peptidases may explain why the loss of the m-AAA protease is not accompanied by increased OPA1 processing in yeast. Interestingly, mitochondrial rhomboid proteases, although highly conserved, also differ in their ability to cleave OPA1. Both yeast Pcp1 and its mammalian homologue PARL mediate processing of Mgm1, but neither of them cleave OPA1 when expressed in yeast (Duvezin-Caubet et al., 2007). The regulation of OPA1 stability and processing at site S1 by two peptidases raises the question of which peptidase mediates constitutive and stress-induced OPA1 cleavage. Our experiments reveal that OMA1 promotes OPA1 processing if mitochondrial activities are impaired (Fig. 8). The loss of mtDNA, dissipation of Δψm, or a decrease of mitochondrial ATP levels induces OPA1 processing by OMA1. The molecular mechanism of OMA1 activation under stress conditions remains to be determined. Mitochondrial dysfunction may alter the accessibility of the S1 cleavage site in OPA1, allowing stress-induced cleavage by OMA1. Alternatively, stress conditions may increase the proteolytic activity of OMA1 itself. It is also possible that stress conditions lower mitochondrial ATP levels and thereby m-AAA protease activity, resulting in the accumulation of an activator of OMA1. In contrast to the stress-induced OPA1 cleavage, constitutive cleavage of OPA1 during mitochondrial biogenesis affects only a subset of the newly imported OPA1 molecules and results in an approximately equimolar accumulation of long and short isoforms. As both forms are required for mitochondrial fusion (Song et al., 2007), their balanced formation is critical for the maintenance of the mitochondrial network. Although our experiments identify OMA1 as the peptidase responsible for stress-induced OPA1 cleavage, two possibilities must be considered for constitutive OPA1 processing. According to the first scenario, OMA1 is responsible for both constitutive and induced OPA1 cleavage. However, the m-AAA protease is required to stabilize long OPA1 isoforms and inhibit OMA1 activity under normal conditions, possibly exerting functions beyond a role as a processing enzyme. These may include the ATP-dependent membrane dislocation of OPA1 preceding its proteolytic cleavage. A similar nonproteolytic activity has been assigned to the m-AAA protease during the biogenesis of yeast cytochrome c peroxidase (Tatsuta et al., 2007). In yeast, the ATP-dependent mitochondrial import motor was proposed to regulate Mgm1 processing by the rhomboid protease in response to mitochondrial ATP levels in a process termed alternative topogenesis (Herlan et al., 2004). Constitutive OPA1 processing might be regulated in a similar manner. Notably, membrane dislocation could also be affected by an altered phospholipid composition of mitochondrial membranes, which has recently been observed in yeast mitochondria lacking the m-AAA protease (Osman et al., 2009). Several observations indeed suggest that OMA1 can cleave OPA1 in the presence of the m-AAA protease. Depletion of OMA1 in human osteosarcoma cells or in MEFs cultured in galactose-containing medium leads to the disappearance of OPA1 isoforms c and e, indicating that OMA1 can affect constitutive OPA1 processing at S1. Moreover, OMA1 is required for S1 cleavage of OPA1 overexpressed in MEFs. However, an OMA1-like peptidase appears to be absent in Caenorhabditis elegans and Drosophila melanogaster, although mitochondrial morphology depends on OPA1 in both organisms. In addition, neither down-regulation nor overexpression of OMA1 affects mitochondrial morphology in MEFs, suggesting that OMA1 is dispensable for the balanced formation of long and short OPA1 isoforms. According to the second scenario, constitutive and stress-induced OPA1 processing are mediated by two distinct peptidases, which differ in their enzymatic properties: the ATP-dependent m-AAA protease ensures the balanced accumulation of long and short isoforms under constitutive conditions, whereas OMA1 acting in an ATP-independent manner completely converts long OPA1 isoforms into short forms under stress conditions. In agreement with a role of the m-AAA protease as a processing enzyme under normal conditions, reconstitution experiments in yeast demonstrate that AFG3L1 and -2 can cleave OPA1 in a heterologous environment (Duvezin-Caubet et al., 2007). Moreover, paraplegin binds OPA1 when overexpressed and stimulates OPA1 processing (Ishihara et al., 2006). OPA1 processing by ATP-dependent and -independent peptidases under different physiological conditions would resolve the apparent caveat of previous studies suggesting that processing of OPA1 may involve the m-AAA protease but is accelerated at decreased mitochondrial ATP levels (Duvezin-Caubet et al., 2006, 2007; Ishihara et al., 2006; Baricault et al., 2007). It also offers an attractive possibility for regulation: low ATP levels may impair membrane dislocation of OPA1 possibly mediated by the m-AAA protease and thereby make OPA1 accessible for OMA1 cleavage under stress conditions. Clearly, further experiments are required to define the relative contribution of the m-AAA protease and OMA1 to constitutive OPA1 cleavage, which may also depend on relative expression levels in different tissues and cell types. Regardless, the identification of proteases involved in OPA1 processing now allows us to assess the relevance of an impaired OPA1 cleavage and mitochondrial fragmentation for the maintenance of cellular functions and for various disease states. Materials and methods Generation of immortalized MEFs lines All animal procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee. The generation of Spg7−/− mice (Ferreirinha et al., 2004) and Afg3l2Emv66/Emv66 genetic characterization (Maltecca et al., 2008) were previously described. Afg3l2Emv66/Emv66 mice were provided by G. Cox (The Jackson Laboratory, Bar Harbor, ME). Primary MEFs were established from embryonic day (E) 14.5 Spg7−/− or E13.5 Afg3l2Emv66/Emv66 and littermate WT embryos and immortalized by SV40 transformation. MEFs were cultured in DME containing 4.5 g/liter glucose and supplemented with 10% FCS. When specified, MEFs were cultured in DME with 6 mM galactose. The human osteosarcoma-derived cell line 143B and its ρ0 derivative were provided by R. Wiesner (University of Cologne, Cologne, Germany) and grown in medium supplemented with 50 µg/ml uridine. Down-regulation of m-AAA protease subunits and OMA1 RNAi-mediated knockdown of paraplegin, AFG3L1 and -2, and OMA1 was performed using specific Stealth RNAi and the nontargeting Stealth RNAi negative control (Invitrogen). The sequences are 5′-GCGCGUCAUUGCUGGUACUGCUAAA-3′ for paraplegin, 5′-CCUGCCUCCGUACGCUCUAUCAAUA-3′ for AFG3L2, 5′-GCGAAACCAUGGUGGAGAAGCCAUA-3′ for AFG3L1, and 5′-GGAUACAGUCAAAGUUGCAGAGUA-3′, 5′-AGUUCUCAGAGAGUCUUCAUGGCUA-3′, and 5′-GCUUCUUGGUCUGAGUGCAUUUGGA-3′ for murine OMA1. For human OMA1, Stealth siRNAs with the sequences 5′-TGGACTACTGCTTGCTGCAAAGGCT-3′ and 5′- TGGCAGCAAATGGAGTTCGTTGATA-3′ were used. 10–20 nM of each siRNA was transfected twice with Lipofectamine (Invitrogen). Down-regulation was monitored by immunoblot analysis of cell lysates generated after 2 d if not indicated otherwise. Expression of AFG3L2 variants and OMA1 Human Afg3l2 was cloned into pcDNA5/FRT/TO, and mutations were introduced into the WB motif (E408Q) or the proteolytic center (E575Q). Stable transformants in FlpIn T-REx 293 cells were obtained by cotransfection with the plasmid pOG44, allowing expression of Flp-recombinase (Invitrogen). Expression of fusion proteins harboring a C-terminal hexahistidine epitope tag was induced with 1 µg/ml tetracycline. After 24 h, we analyzed cell lysates by immunoblotting and assessed mitochondrial morphology. The coding region of murine Oma1 (GenBank/EMBL/DDBJ accession no. NM_025909) was cloned into the EcoRI–BamHI sites of the vector pEGFP-N2 in frame with GFP. Assessment of cell growth 5×103 FlpIn T-REx 293 cells expressing AFG3L2 variants were seeded in medium with or without 1 µg/ml tetracycline. To assess the number of living cells in culture, we used the One Solution Cell Proliferation Assay (Promega) and monitored the conversion of a tetrazolium compound to formazan, determining the absorbance at 490 nm. Fluorescence microscopy Mitochondrial morphology was examined after expression of mito-DsRed using a microscope (DeltaVision; Applied Precision; Merkwirth et al., 2008). Fluorescence images were acquired using a camera (CoolSNAP HQ/ICX285; Sony) and a Plan-Apochromat N 60× NA 1.42 oil objective (Olympus). Image stacks were deconvoluted with the SoftWoRx Imaging Suite (Applied Precision). Image files were further processed with the CorelDRAW 11 Graphics Suite software (Corel). For colocalization of OMA1-GFP and mitochondria, we stained cells with MitoTracker red CMXRos (Invitrogen) and used a microscope (Eclipse E600; Nikon) and a Plan-Apochromat A 60× NA 1.40 oil objective (Nikon). Images were acquired with a confocal microscope (Radiance 2100; Bio-Rad Laboratories). For detection of the ER, cells were stained with an antiserum directed against calnexin and examined using an imaging station (Eclipse TE2000-S [Nikon] + CARV II [BD]) and a Plan-Apochromat VC 100× NA 1.40 oil objective (Nikon), and images acquired using a camera (QuantEM 512 SC; Photometrics) and deconvoluted with MetaMorph software (MDS Analytical Technologies). Transmission electron microscopy MEFs were plated on glass coverslips, transfected with siRNA, and after 48 h, flat embedded (Merkwirth et al., 2008). Ultrathin sections (60–70 nm) were cut and mounted on pioloform-coated copper grids (Plano). Sections were stained with lead citrate and uranyl acetate and viewed with a transmission electron microscope (JEM-2100; JEOL Ltd.) operated at 80 kV. Micrographs were taken using a charge-coupled device camera (Erlangshen ES500W; Gatan Inc.). Polarographic measurements Respiration of intact cells was measured at 37°C with a Clark-type electrode oxygraph (Hansatech Inc.). 2.5 × 106 cells were assayed in 250 mM saccharose, 20 mM Hepes, pH 7.4, 10 mM KH2PO4, 4 mM MgCl2, 1 mM EDTA, 5 mM glucose, 2 mM pyruvate, and 4 mM glutamate. After recording endogenous respiration, ATP synthase was inhibited with 2 µM oligomycin followed by uncoupling of oxidative phosphorylation using CCCP with concentrations of 250–750 nM. Cellular respiration was inhibited with 2 mM KCN and was corrected to KCN-insensitive respiration. Inhibitor experiments To induce SIMH, MEFs were exposed to 1 µM CHX for 3 h 2–3 d after transfection with siRNAs and plasmids and were incubated for 5 h with the following protease inhibitors: 5 µM MG132, 0.5 mM o-phe, 50 µM pepstatin A, 0.5 mM Pefabloc SC, and 50 µM E-64d. To induce OPA1 cleavage, MEFs were treated either with 2 µM oligomycin 60 h after transfection or with 20 µM CCCP 48 h after transfection. Antibodies Polyclonal antibodies directed against murine m-AAA protease subunits were described previously (Koppen et al., 2007). An antiserum recognizing murine LON was provided by C. Suzuki (University of Medicine and Dentistry of New Jersey, Newark, NJ). The following commercially available antibodies were used: α-OPA1 (Invitrogen), α-Flag M2 (Sigma-Aldrich), α–SLP-2 (GenWay Biotech Inc.), α-calnexin (Stressgen), and α–complex II (Invitrogen). Online supplemental material Fig. S1 shows the mitochondrial morphology in paraplegin-deficient MEFs. Fig. S2 demonstrates the maintenance of the mitochondrial membrane potential in Afg3l2−/− mitochondria using TMRE fluorescence. Fig. S3 shows the stability of OPA1 in AFG3L1/AFG3L2-deficient cells lacking various mitochondrial proteases and the effect of an inhibition of cytosolic protein synthesis on OPA1 isoforms accumulating in m-AAA protease–deficient MEFs in the presence of MG132. Fig. S4 shows the quantification of OMA1 mRNA levels in MEFs after down-regulation of OMA1 by RNAi, demonstrates the assembly of AFG3L2 and -1 in m-AAA protease complexes in OMA1-deficient mitochondria by blue-native–PAGE analysis, and reveals normal processing of OMA1 in m-AAA protease–deficient mitochondria. Fig. S5 shows the accumulation of AFG3L1 and -2 in OMA1-deficient MEFs. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200906084/DC1.
Author and article information
Journal
Journal ID (nlm-ta): Sci Rep
Journal ID (iso-abbrev): Sci Rep
Title:
Scientific Reports
Publisher:
Nature Publishing Group
ISSN
(Electronic):
2045-2322
Publication date
(Electronic):
14
September
2015
Publication date Collection: 2015
Volume: 5
Electronic Location Identifier: 13989
Affiliations
[3
]School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln , Lincoln NE, 68588, USA
[4
]Eppley Institute for Research in Cancer, University of Nebraska Medical Center , Omaha, NE 68198, USA
Author notes
Article
Publisher Item ID: srep13989DOI: 10.1038/srep13989
PMC ID: 4568518
PubMed ID: 26365306
SO-VID: b4992188-2a18-41ed-936e-599305ae49bd
Copyright © Copyright © 2015, Macmillan Publishers Limited
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This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
History
Date
received
: 10
May
2015
Date
accepted
: 13
August
2015
Categories
Subject:
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ScienceOpen disciplines: Uncategorized