Oma1, a Novel Membrane-bound Metallopeptidase in Mitochondria with Activities Overlapping with the m-AAA Protease

Abstract

The integrity of the inner membrane of mitochondria is maintained by a membrane-embedded quality control system that ensures the removal of misfolded membrane proteins. Two ATP-dependent AAA proteases with catalytic sites at opposite membrane surfaces are key components of this proteolytic system. Here we describe the identification of a novel conserved metallopeptidase that exerts activities overlapping with the m-AAA protease and was therefore termed Oma1. Both peptidases are integral parts of the inner membrane and mediate the proteolytic breakdown of a misfolded derivative of the polytopic inner membrane protein Oxa1. The m-AAA protease cleaves off the matrix-exposed C-terminal domain of Oxa1 and processively degrades its transmembrane domain. In the absence of the m-AAA protease, proteolysis of Oxa1 is mediated in an ATP-independent manner by Oma1 and a yet unknown peptidase resulting in the accumulation of N- and C-terminal proteolytic fragments. Oma1 exposes its proteolytic center to the matrix side; however, mapping of Oma1 cleavage sites reveals clipping of Oxa1 in loop regions at both membrane surfaces. These results identify Oma1 as a novel component of the quality control system in the inner membrane of mitochondria. Proteins homologous to Oma1 are present in higher eukaryotic cells, eubacteria and archaebacteria, suggesting that Oma1 is the founding member of a conserved family of membrane-embedded metallopeptidases. The integrity of the inner membrane of mitochondria is maintained by a membrane-embedded quality control system that ensures the removal of misfolded membrane proteins. Two ATP-dependent AAA proteases with catalytic sites at opposite membrane surfaces are key components of this proteolytic system. Here we describe the identification of a novel conserved metallopeptidase that exerts activities overlapping with the m-AAA protease and was therefore termed Oma1. Both peptidases are integral parts of the inner membrane and mediate the proteolytic breakdown of a misfolded derivative of the polytopic inner membrane protein Oxa1. The m-AAA protease cleaves off the matrix-exposed C-terminal domain of Oxa1 and processively degrades its transmembrane domain. In the absence of the m-AAA protease, proteolysis of Oxa1 is mediated in an ATP-independent manner by Oma1 and a yet unknown peptidase resulting in the accumulation of N- and C-terminal proteolytic fragments. Oma1 exposes its proteolytic center to the matrix side; however, mapping of Oma1 cleavage sites reveals clipping of Oxa1 in loop regions at both membrane surfaces. These results identify Oma1 as a novel component of the quality control system in the inner membrane of mitochondria. Proteins homologous to Oma1 are present in higher eukaryotic cells, eubacteria and archaebacteria, suggesting that Oma1 is the founding member of a conserved family of membrane-embedded metallopeptidases. The majority of mitochondrial proteins is nuclear encoded and synthesized at cytosolic ribosomes. Import into mitochondria is mediated by various protein translocases in the outer and inner membrane that allow the passage of preproteins only in a largely unfolded conformation (1Truscott K. Brandner K. Pfanner N. Curr. Biol. 2003; 13: R326-R337Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 2Neupert W. Annu. Rev. Biochem. 1997; 66: 863-917Crossref PubMed Scopus (975) Google Scholar). Folding and assembly of mitochondrial proteins must therefore occur inside mitochondria. Little is known about the efficiency of this process, but it is clear that mitochondria, as other organelles, harbor a quality control system that ensures the recognition and removal of non-native polypeptides, preventing their potentially harmful accumulation within the organelle (3Käser M. Langer T. Semin. Cell Dev. Biol. 2000; 11: 181-190Crossref PubMed Scopus (73) Google Scholar). Notably, a functional impairment of components of this system leads to neurodegeneration in various forms of hereditary spastic paraplegia, illustrating the importance of protein quality control for mitochondrial function (4Hansen J.J. Dürr A.I. C.-R. Georgopoulos C. Ang D. Nielson M. N. Davoine C. S. Brice A. Fontaine B. Gregersen N. Bross P. Am. J. Hum. Genet. 2002; 70: 1328-1332Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 5Casari G. De-Fusco M. Ciarmatori S. Zeviani M. Mora M. Fernandez P. DeMichele G. Filla A. Cocozza S. Marconi R. Durr A. Fontaine B. Ballabio A. Cell. 1998; 93: 973-983Abstract Full Text Full Text PDF PubMed Scopus (681) Google Scholar). Molecular chaperone proteins and ATP-dependent proteases present in different subcompartments of mitochondria maintain protein quality control within the organelle (3Käser M. Langer T. Semin. Cell Dev. Biol. 2000; 11: 181-190Crossref PubMed Scopus (73) Google Scholar). In line with the endosymbiotic origin of mitochondria, many of these components exhibit significant homology to bacterial proteins. ATP-dependent proteases homologous to Lon proteases (6Wang N. Gottesman S. Willingham M.C. Gottesman S. Maurizi M.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11247-11251Crossref PubMed Scopus (177) Google Scholar, 7Suzuki C.K. Suda K. Wang N. Schatz G. Science. 1994; 264: 273-276Crossref PubMed Scopus (183) Google Scholar, 8Van Dyck L. Pearce D.A. Sherman F. J. Biol. Chem. 1994; 269: 238-242Abstract Full Text PDF PubMed Google Scholar) and, at least in some organisms, Clp proteases (9Kang S.G. Ortega J. Singh S.K. Wang N. Huang N.N. Steven A.C. Maurizi M.R. J. Biol. Chem. 2002; 277: 21095-21102Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 10Halperin T. Zheng B. Itzhaki H. Clarke A.K. Adam Z. Plant Mol. Biol. 2001; 45: 461-468Crossref PubMed Scopus (62) Google Scholar) have been identified in the mitochondrial matrix space, whereas the inner membrane harbors two AAA proteases homologous to bacterial FtsH proteases (11Langer T. Trends Biochem. Sci. 2000; 25: 207-256Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). These membrane-embedded peptidases were termed m- and i-AAA proteases to indicate their different topology in the inner membrane; the m-AAA protease is active on the matrix and the i-AAA protease on the intermembrane side of the membrane. Both proteases consist of homologous subunits conserved in all eukaryotic cells. The m-AAA protease is a hetero-oligomeric complex of Yta10 and Yta12 subunits in yeast (12Arlt H. Tauer R. Feldmann H. Neupert W. Langer T. Cell. 1996; 85: 875-885Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar) and of AFG3L2 and paraplegin subunits in human (13Atorino L. Silvestri L. Koppen M. Cassina L. Ballabio A. Marconi R. Langer T. Casari G. J. Cell Biol. 2003; (in press)PubMed Google Scholar), whereas Yme1 subunits most likely form homo-oligomeric i-AAA proteases in mitochondria of both organisms (14Weber E.R. Hanekamp T. Thorsness P.E. Mol. Biol. Cell. 1996; 7: 307-317Crossref PubMed Scopus (119) Google Scholar, 15Leonhard K. Herrmann J.M. Stuart R.A. Mannhaupt G. Neupert W. Langer T. EMBO J. 1996; 15: 4218-4229Crossref PubMed Scopus (218) Google Scholar, 16Shah Z.H. Hakkaart G.A.J. Arku B. DeJong L. Van der Speck H. Grivell L. Jacobs H.T. FEBS Lett. 2000; 478: 267-270Crossref PubMed Scopus (41) Google Scholar). Protein degradation by ATP-dependent proteases results in the formation of peptides composed of ∼5–20 amino acid residues (17Kuzela S. Goldberg A.L. Methods Enzymol. 1994; 244: 376-383Crossref PubMed Scopus (19) Google Scholar, 18Wenzel T. Eckerskorn C. Lottspeich F. Baumeister W. FEBS Lett. 1994; 349: 205-209Crossref PubMed Scopus (147) Google Scholar, 19Thompson M.W. Singh S.K. 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Biol. 2002; 323: 835-843Crossref PubMed Scopus (140) Google Scholar, 27McQuibban G.A. Saurya S. Freeman M. Nature. 2003; 423: 537-541Crossref PubMed Scopus Google Scholar). Notably, Pcp1 the processing of the mitochondrial and mitochondrial G.A. Saurya S. Freeman M. Nature. 2003; 423: 537-541Crossref PubMed Scopus Google Scholar, M. F. C. Neupert W. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Here we have the degradation of a misfolded of the polytopic membrane protein and we describe the identification of a novel metallopeptidase in the inner membrane of mitochondria. to the m-AAA protease, Oma1 is of non-native membrane proteins and therefore a novel component of the quality control system that is conserved in higher eubacteria and and in this are of The H. G. R. B. Neupert W. Langer T. EMBO J. 1998; PubMed Scopus Google K. Herrmann J.M. Stuart R.A. Mannhaupt G. Neupert W. Langer T. EMBO J. 1996; 15: 4218-4229Crossref PubMed Scopus (218) Google Scholar), and K. Neupert W. Stuart R.A. EMBO J. 2001; PubMed Scopus Google Scholar) were were on and at in of at and were by homologous the or A. A. R. P. 1994; PubMed Scopus Google Scholar). The complete of and an of were by the was by of a of Oma1 was by of the by homologous A. A. A. P. 1998; PubMed Scopus Google Scholar). The AAA and AAA AAA AAA were for of the as which was into cells. was by in of the was by the and AAA The was into the to allow in by the of were into a to the from the into the yeast the control of the was as a in the acid was by by the and the was by by the and and was by by the and were by Protein Import into were as G. Schatz G. J. Biol. Chem. 1982; Full Text PDF PubMed Google Scholar, J.M. H. Neupert W. Stuart R.A. Cell Scholar). in of the proteins were synthesized in in the presence of and imported into mitochondria for at as H. Dyck L. Langer T. Neupert W. EMBO J. 1994; 13: PubMed Scopus Google Scholar). the Oxa1 protein were for at to to the preproteins were by the of at the protease, a of was were by and of and protease mitochondria were and in an system The of imported was at were by and the proteolytic breakdown of mitochondria were in and the system at or was at the by the on were and mitochondrial proteins were by and of were by of mitochondria. were at a of in with was were for at The protease was by mitochondria were to the in and were by with with and in were by and The efficiency of was by with cytochrome intermembrane space the membrane and matrix of mitochondrial mitochondria were at a of in and for at and proteins were by for at proteins were and both were by and The was by of proteins as a of Oma1 were an for at in at a of a for at mitochondrial were a were and by and by an the homologous to yeast Oma1 were identified by Methods Enzymol. PubMed Scopus Google Scholar) in the Protein These were by in a sequences that are homologous to the the of Oma1 were in a by 1994; PubMed Scopus Google Scholar). in the identification of the conserved proteolytic domain. of transmembrane regions was by G. J. Mol. Biol. PubMed Scopus Google Scholar) and M. E. G. A. Protein 1997; PubMed Google Scholar). of in mitochondria was a C-terminal of Oxa1 J.M. Neupert W. Stuart R.A. EMBO J. 1997; PubMed Scopus Google Scholar) and an of Oxa1. of the the to amino acid residues of the was with protein to and for of in of a of Oxa1 a is a polytopic mitochondrial membrane protein with an domain the intermembrane space and a C-terminal domain to the matrix space J.M. Neupert W. Stuart R.A. EMBO J. 1997; PubMed Scopus Google Scholar, J. T. FEBS Lett. 2001; PubMed Scopus Google Scholar, M.R. Lett. 2001; PubMed Google Scholar). of has been identified M. M. K. G. Pratje E. Mol. Genet. 1994; PubMed Scopus Google Scholar), which an of in a loop in the intermembrane space, to W. M. Pratje E. Curr. Genet. 1997; PubMed Scopus Google Scholar). of mitochondria from an at of Oxa1 with mitochondria therefore the of Oxa1 and mitochondria at was within mitochondria with a of about whereas Oxa1 was these In both proteins were synthesized in in the presence of and imported into mitochondria at observed proteolysis of but of Oxa1 mitochondria were at of functional M. M. K. G. Pratje E. Mol. Genet. 1994; PubMed Scopus Google Scholar). is therefore that of by Oxa1 its degradation the protein within mitochondria to an of into the inner membrane. the of mitochondria from and the were and the of and Oxa1 was Both proteins were to of the outer membrane In with J.M. Neupert W. Stuart R.A. EMBO J. 1997; PubMed Scopus Google Scholar), C-terminal of Oxa1 with a of about were these by proteolytic removal of the domain of Oxa1 in the intermembrane In a C-terminal with a of about in of mitochondria these and The in a of the loop transmembrane regions and it for proteolytic degradation by K. Oxa1 and as as the C-terminal by of were from the membrane its membrane topology to these results the of into the inner membrane. therefore that of the protein its into the inner membrane the of of by the m-AAA but the i-AAA to identify proteases in the degradation of the of the protein was into and mitochondria the m-AAA protease or the i-AAA protease, of the proteolytic breakdown of to was to of an intermembrane space loop of Oxa1 of was however, in mitochondria the m-AAA protease Yta10 that were in mitochondria in these mitochondria the m-AAA protease is in but is for the degradation of newly imported the m- or the i-AAA protease to the of these proteases degradation of were from these at and by with N- or C-terminal peptides of Oxa1 were a C-terminal of about to the matrix-exposed domain of Oxa1 in mitochondria was by the m-AAA protease it was in mitochondria In with the studies other proteolytic of in these mitochondria. These include the and indicate the and the C-terminal and and of the i-AAA protease the degradation of only observed the accumulation of the proteolytic as in mitochondria These a to the m-AAA protease for the degradation of as as in and for the of yet protease in the proteolytic breakdown of this polytopic membrane The i-AAA protease is known to an overlapping to the m-AAA protease K. B. G. A. Neupert W. Langer T. Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). it that the i-AAA protease, for proteolytic of in the absence of the m-AAA The of this is by the of in both AAA proteases K. B. G. A. Neupert W. Langer T. Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, C. P. J. G. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). to the of the proteolytic of newly imported in mitochondria the accumulation of of mitochondria of the formation of the for for the proteolytic breakdown of newly imported in mitochondria was were of by the and the of and was these Notably, and only was to the suggesting that are in the mitochondrial matrix space of the ATP-dependent i-AAA protease which exposes its to the intermembrane space therefore these that a novel metallopeptidase which exposes its catalytic to the matrix for in to identify this proteolytic we the of for with or The to the identification of and proteins the and the Proteins with known in other mitochondria were In the the of in mitochondria was the K. M. PubMed Scopus Google Scholar). mitochondrial inner membrane proteins a mitochondrial the of the we the proteins for the of that as transmembrane to the identification of for mitochondrial both as as proteins. a in the of as it for a mitochondrial protein of that harbors a conserved domain of the metallopeptidase family A.J. Barrett A.J. of Scholar). proteins are present in higher and in eubacteria as as and but have been Notably, in higher eukaryotic have an that is in and proteins and the yeast protein encoded by was termed Oma1 overlapping with m-AAA the of we a a at the were and were by was from the mitochondrial and was in the These results were by mitochondrial protein Oma1 was synthesized in in the presence of and with mitochondria. Oma1 in a in a manner its this the of Oma1 into mitochondria as it was on the presence of a membrane the inner membrane Oma1 to of the outer membrane Oma1 a as we proteolytic processing into mitochondria of Oma1 with mitochondrial processing peptidase which form are present in the of Oma1 suggesting that Oma1 is an integral membrane In to its mitochondria were and with a the In a mitochondrial were at integral membrane proteins the in the these whereas proteins as as membrane proteins in the was from the these a membrane was to but to proteolytic degradation of the outer membrane a protein in the intermembrane space, was whereas these from these results that Oma1 is an integral of the inner membrane and exposes its the to the intermembrane Oma1 a Molecular in the of Oma1 was in were with and were to were a was in a from the to a of The formation of this complex was on is of a assembly in the mitochondrial inner membrane. the of the observed of the complex with a However, to to Oma1 forms a homo-oligomeric complex or with other yet unknown proteins. of by results identify Oma1 as a novel metallopeptidase in the mitochondrial inner membrane. a of Oma1 in the degradation of misfolded polytopic membrane was in cells, and in by homologous Oma1 exhibit on or at or The of in mitochondria Oma1 was in was imported into mitochondria from and which were at to allow proteolysis and with mitochondria, degradation of was only by Oma1 or the m-AAA protease Yta10 However, was in mitochondria both proteases These results that both Oma1 and the m-AAA protease are of this polytopic membrane protein and an overlapping Both proteases act of other as the proteolytic breakdown of in the absence of In as as in mitochondria proteolysis of is mediated by the m-AAA the C-terminal domain is whereas the transmembrane domain of is In the absence of the m-AAA protease, on the other proteolytic of in mitochondria These were by Oma1 as were in mitochondria Oma1 the of in mitochondria or both proteases by at in the absence of the m-AAA protease N- and C-terminal proteolytic of were in these mitochondria as of different on the of on the presence or absence of the m-AAA protease, whereas at in and mitochondria, of in in a significant of Oma1 is for the formation of the proteolytic and as the proteolytic which were in mitochondria both proteases Notably, is in the absence of the m-AAA protease and Oma1. The proteolytic and in mitochondria pointing to the of yet other a proteolytic of were into the of Oma1 the proteolytic The and and the catalytic acid were by Oma1 and the proteins were in cells, and the of Oxa1 was the proteolytic and at of were in the of Oma1. the integrity of the is for proteolytic Oma1 as a from these that Oma1 the misfolded polytopic membrane protein and in and for a of the m-AAA protease In to the m-AAA protease, however, Oma1 mediate the complete of but N- and C-terminal proteolytic fragments. The of yeast the m-AAA protease was of Oma1 for in the of both peptidases of at Both by of Oma1 with studies metallopeptidase indicate that the catalytic center of Oma1 is at the inner of the mitochondrial inner membrane. Oma1 cleaves misfolded at this membrane we the cleavage sites of Oma1 in the In a were that C-terminal of at amino acid residues or amino acid residues transmembrane In Oxa1 were of at amino acid residues and The proteins were synthesized in in the presence of and as and The of the proteolytic which proteolysis of newly imported was by an the In mitochondria, only the C-terminal was by the m-AAA protease, whereas the of to in a manner acid residues were as the amino of by the that the m-AAA protease within K. Herrmann J.M. Stuart R.A. Mannhaupt G. Neupert W. Langer T. EMBO J. 1996; 15: 4218-4229Crossref PubMed Scopus (218) Google Scholar). In mitochondria, the C-terminal was by cleavage of at amino acid residues that the transmembrane and that are in the matrix space the of the identified at its the amino acid residues whereas amino acid residues were as the amino of the of Oma1 cleavage sites of the proteolytic these that Oma1 is of cleaving only within loop regions in the matrix space but in a loop that harbors the and is to the intermembrane have identified a conserved as a novel component of the quality control system in the inner membrane of mitochondria. Oma1 has overlapping with the m-AAA protease and cleaves a misfolded polytopic membrane protein at Proteins homologous to Oma1 a protein family with present in higher as as in eubacteria and different in their domain all of are to integral membrane proteins and a metallopeptidase domain of the suggesting that Oma1 a novel of membrane proteins. of the inner membrane protein Oxa1 a in the loop the and transmembrane was as a to the quality control of polytopic membrane proteins. of Oxa1 in mitochondria by protease in the the loop the and transmembrane is in the that misfolded Oxa1 is in an ATP-dependent manner by the m-AAA protease in an ATP-independent by Oma1 and yet unknown peptidase resulting in the accumulation of proteolytic fragments. Notably, cleavage is a for proteolysis by the m-AAA protease but to occur of m-AAA protease of mitochondria and studies indicate that the proteolytic center of Oma1 is present on the matrix side of the inner membrane. was only in the presence of a whereas of of the intermembrane space Oma1 The is in to a transmembrane that the only C-terminal to the proteolytic the C-terminal of Oma1 was to in to The of the proteolytic by on the other to cleavage in loop regions of the protein to both the intermembrane and matrix membrane of the misfolded protein as the loop both the as as an Oma1 cleavage is in to the outer of the inner membrane. therefore that Oma1 a polytopic membrane protein at opposite membrane surfaces. is of mitochondrial AAA proteases that membrane proteins their from the membrane K. B. G. A. Neupert W. Langer T. Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Oma1 was to in a complex in the inner membrane that a within the a of membrane-embedded to however, proteolysis membrane of proteins or by Oma1 is to membrane its from the of peptidases the protease, the and Science. 2002; PubMed Scopus Google Scholar, A. B. Trends Cell Biol. 2003; 13: Full Text Full Text PDF PubMed Scopus Google Scholar). has identified the of Oma1 as the proteolytic peptidases harbor their catalytic residues within the membrane the conserved of Oma1 as as that of other family is in to a transmembrane but most likely within the membrane. peptidases to only transmembrane whereas Oma1 was to a polytopic membrane the other Oma1 from peptidases the complete of membrane the or AAA proteases T. M. C. K. Biochem. 2001; PubMed Scopus Google Scholar, B. Rev. Mol. Cell Biol. 2002; PubMed Scopus Google Scholar), as proteolysis by Oma1 on and results in the accumulation of proteolytic of a polytopic with its therefore a novel of peptidases that membrane-embedded as The of the proteolytic breakdown of the polytopic Oxa1 protein into the of membrane protein degradation by AAA other ATP-dependent AAA proteases are to into peptides L. K. T. J. Langer T. Science. 2001; PubMed Scopus (177) Google Scholar). of Oxa1 by the m-AAA protease, however, results in the accumulation of the C-terminal domain in the matrix space its that the m-AAA protease degrades the from the which is to the intermembrane space of mitochondria. therefore the that the m-AAA protease proteolysis of a polytopic membrane protein at sites which results in the of the C-terminal of Oxa1. is of the proteolytic processing of by which is to a loop formation of the within the of the M. S. Cell Biol. 2002; PubMed Scopus Google Scholar). In a the m-AAA protease mediate the processing of the newly imported form of cytochrome c peroxidase (26Esser K. Tursun B. Ingenhoven M. Michaelis G. Pratje E. J. Mol. Biol. 2002; 323: 835-843Crossref PubMed Scopus (140) Google Scholar, 27McQuibban G.A. Saurya S. Freeman M. Nature. 2003; 423: 537-541Crossref PubMed Scopus Google Scholar). studies proteins with transmembrane that their as as the of or the other AAA protease, is by the of and the of amino acid residues present at membrane K. B. G. A. Neupert W. Langer T. Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, K. A. Neupert W. Langer T. Nature. PubMed Scopus Google Scholar). The m-AAA protease misfolded an amino acid in an intermembrane space loop of this protein was matrix-exposed of the Oxa1 protein a non-native conformation which is by the m-AAA protease, the of misfolded Oxa1 degradation by the i-AAA protease is These therefore the importance of the of a of proteins with different membrane to the of membrane proteins by AAA for and for the