The Oxa1 Protein Forms a Homooligomeric Complex and Is an Essential Part of the Mitochondrial Export Translocase in Neurospora crassa

The Oxa1 protein is a ubiquitous constituent of the inner membrane of mitochondria. Oxa1 was identified in yeast as a crucial component of the protein export machinery known as the OXA translocase, which facilitates the integration of proteins from the mitochondrial matrix into the inner membrane. We have identified the Neurospora crassa Oxa1 protein which shows a sequence identity of 22% to the yeast homologue. Despite the low level of identity, the function of the homologues is conserved as the N. crassa gene fully complemented a yeast null mutant. Genetic analysis revealed that Oxa1 is essential for viability in N. crassa. Cells propagated under conditions that severely reduce Oxa1 levels grew extremely slowly and were deficient in subunits of complex I and complex IV. Isolation of the Oxa1 complex from N. crassa mitochondria revealed a 170–180-kDa complex that contained exclusively Oxa1. Since the Oxa1 monomer has a molecular weight of 43,000, our data suggest that the OXA translocase consists of a homooligomer most likely containing four Oxa1 subunits. The Oxa1 protein is a ubiquitous constituent of the inner membrane of mitochondria. Oxa1 was identified in yeast as a crucial component of the protein export machinery known as the OXA translocase, which facilitates the integration of proteins from the mitochondrial matrix into the inner membrane. We have identified the Neurospora crassa Oxa1 protein which shows a sequence identity of 22% to the yeast homologue. Despite the low level of identity, the function of the homologues is conserved as the N. crassa gene fully complemented a yeast null mutant. Genetic analysis revealed that Oxa1 is essential for viability in N. crassa. Cells propagated under conditions that severely reduce Oxa1 levels grew extremely slowly and were deficient in subunits of complex I and complex IV. Isolation of the Oxa1 complex from N. crassa mitochondria revealed a 170–180-kDa complex that contained exclusively Oxa1. Since the Oxa1 monomer has a molecular weight of 43,000, our data suggest that the OXA translocase consists of a homooligomer most likely containing four Oxa1 subunits. The biogenesis of mitochondria requires delivery of several hundred gene products to their specific locations within the organelle. A small number of mostly hydrophobic proteins is encoded on the mitochondrial genome, whereas the great majority of mitochondrial proteins is encoded in the nucleus and synthesized in the cytosol. A series of protein translocation machineries is required for the import and sorting of newly synthesized mitochondrial proteins to their individual destinations. Three of these translocases facilitate protein import from the cytosol into mitochondria: the TOM complex (translocase of the outer membrane) in the outer membrane and two inner membrane TIM complexes (translocases of the innermembrane) which have differential substrate specificity (1.Neupert W. Annu. Rev. Biochem. 1997; 66: 863-917Crossref PubMed Scopus (981) Google Scholar, 2.Herrmann J. Neupert W. Curr. Opin. Microbiol. 2000; 3: 210-214Crossref PubMed Scopus (120) Google Scholar, 3.Rehling P. Wiedemann N. Pfanner N. Truscott K.N. Crit. Rev. Biochem. Mol. Biol. 2001; 36: 291-336Crossref PubMed Scopus (64) Google Scholar). A fourth translocase mediates the insertion of its substrates from the mitochondrial matrix into the inner membrane. Since the direction of translocation is opposite that of the other three translocases, the process is referred to as mitochondrial protein export. The export machinery is referred to as the OXA translocase since the only component identified thus far is the Oxa1 protein. Oxa1 was originally shown to be required for the formation of cytochrome coxidase in Saccharomyces cerevisiae (4.Bonnefoy N. Chalvet F. Hamel P. Slonimski P.P. Dujardin G. J. Mol. Biol. 1994; 239: 201-212Crossref PubMed Scopus (182) Google Scholar, 5.Bauer M. Behrens M. Esser K. Michaelis G. Pratje E. Mol. Gen. Genet. 1994; 245: 272-278Crossref PubMed Scopus (109) Google Scholar). In yeast, the OXA translocase has now been shown to facilitate the insertion of several nuclear-encoded, matrix-targeted proteins and most, if not all, mitochondrially synthesized membrane proteins into the inner membrane (6.He S. Fox T.D. Mol. Biol. Cell. 1997; 8: 1449-1460Crossref PubMed Scopus (157) Google Scholar, 7.Hell K. Herrmann J. Pratje E. Neupert W. Stuart R.A. FEBS Lett. 1997; 418: 367-370Crossref PubMed Scopus (149) Google Scholar, 8.Hell K. Herrmann J.M. Pratje E. Neupert W. Stuart R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2250-2255Crossref PubMed Scopus (185) Google Scholar, 9.Hell K. Neupert W. Stuart R.A. EMBO J. 2001; 20: 1281-1288Crossref PubMed Scopus (218) Google Scholar). The OXA translocase has been well conserved through evolution and homologues of Oxa1 are present in bacteria, chloroplasts, and mitochondria of various organisms (4.Bonnefoy N. Chalvet F. Hamel P. Slonimski P.P. Dujardin G. J. Mol. Biol. 1994; 239: 201-212Crossref PubMed Scopus (182) Google Scholar, 10.Bonnefoy N. Kermorgant M. Groudinsky O. Minet M. Slonimski P.P. Dujardin G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11978-11982Crossref PubMed Scopus (91) Google Scholar, 11.Samuelson J.C. Chen M. Jiang F. Moller I. Wiedmann M. Kuhn A. Phillips G.J. Dalbey R.E. Nature. 2000; 406: 637-641Crossref PubMed Scopus (428) Google Scholar, 12.Moore M. Harrison M.S. Peterson E.C. Henry R. J. Biol. Chem. 2000; 275: 1529-1532Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). The best characterized family member is the Oxa1 protein of S. cerevisiae. A knockout mutant of the S. cerevisiae OXA1 gene is viable, but mutants are unable to respire and grow only on fermentable carbon sources (4.Bonnefoy N. Chalvet F. Hamel P. Slonimski P.P. Dujardin G. J. Mol. Biol. 1994; 239: 201-212Crossref PubMed Scopus (182) Google Scholar). Schizosaccharomyes pombe contains two genes encoding distinct Oxa1 proteins. Both are able to complement S. cerevisiae Oxa1 deficiency. Inactivation of both oxa1genes in S. pombe is lethal to this petite negative yeast (13.Bonnefoy N. Kermorgant M. Groudinsky O. Dujardin G. Mol. Micro. 2000; 35: 1135-1145Crossref PubMed Scopus (28) Google Scholar). The Escherichia coli homologue, YidC, is an essential component of the bacterial protein export machinery (11.Samuelson J.C. Chen M. Jiang F. Moller I. Wiedmann M. Kuhn A. Phillips G.J. Dalbey R.E. Nature. 2000; 406: 637-641Crossref PubMed Scopus (428) Google Scholar, 14.Scotti P.A. Urbanus M.L. Brunner J. de Gier J.W. von Heijne G. van der Does C. Driessen A.J. Oudega B. Luirink J. EMBO J. 2000; 19: 542-549Crossref PubMed Scopus (305) Google Scholar). To date, work on Oxa1 has concentrated on its genetics, function, and identification of substrates. The yeast Oxa1 contains five transmembrane domains with the N terminus of the protein residing in the intermembrane space and the C terminus in the matrix (15.Herrmann J.M. Neupert W. Stuart R.A. EMBO J. 1997; 16: 2217-2226Crossref PubMed Scopus (133) Google Scholar). Sequence comparisons suggest that this topology is highly conserved among Oxa1 family members, but little else is known about the structure of the OXA translocase. The E. coli YidC protein was reported to be at least partially associated with the Sec translocase (14.Scotti P.A. Urbanus M.L. Brunner J. de Gier J.W. von Heijne G. van der Does C. Driessen A.J. Oudega B. Luirink J. EMBO J. 2000; 19: 542-549Crossref PubMed Scopus (305) Google Scholar). However, there is no homologue of the Sec machinery in mitochondria of yeast (16.Glick B.S. von Heijne G. Protein Sci. 1996; 5: 2651-2652Crossref PubMed Scopus (69) Google Scholar) or other organisms with completely sequenced genomes. It is unknown whether Oxa1 is associated with other components. In this report we describe the characteristics of an N. crassa oxa1mutant and show that a homooligomeric Oxa1 complex can be purified from mitochondria following lysis with non-ionic detergents. Growth and handling of N. crassa were as described (17.Davis R.H. De Serres F.J. Methods Enzymol. 1970; 17: 79-143Crossref Scopus (940) Google Scholar). The N. crassastrains used in this study are listed in Table I. S. cerevisiae strains were isogenic to the wild type strain W303a and were cultivated at 30 °C on YP medium supplemented with 2% glucose, or 2% galactose, and 0.5% KOH-buffered lactate (18.Herrmann J.M. Fölsch H. Neupert W. Stuart R.A. Celis J.E. Cell Biology: A Laboratory Handbook. Academic Press, San Diego1994: 538-544Google Scholar). In the Δoxa1 yeast strain the complete OXA1 reading frame was replaced by the HIS3 gene (8.Hell K. Herrmann J.M. Pratje E. Neupert W. Stuart R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2250-2255Crossref PubMed Scopus (185) Google Scholar). The plasmid TPI-oxa1Nc, containing the coding sequence for N. crassa Oxa1, was transformed into this mutant resulting in strain Δoxa1 (oxa1Nc).Table IStrains used in this studyStrainGenotype1-aThe cyh-2 allele confers resistance to cycloheximide.OriginHVa cyh-2 lys-2 leu-5 mei-2FGSC number 7255MVA am132 inl inv mei-2FGSC number 7265OXB80–1As HV, but carries an ectopic copy of oxa1.Transformation of HV with plasmid Bleomycin resistant.XAB1–5OX80–7-1Sheltered heterokaryon:Cross of MV × OXB80–1 (cyh-2 lys-2 leu-5 mei-2 oxa1RIP + am132 inl inv mei-2), mating type unknown. The first nucleus may also contain an ectopic RIPed version of oxa1.OX80–15-1Sheltered heterokaryon:Cross of MV × OXB80–1 (cyh-2 lys-2 leu-5 mei-2 oxa1RIP oxa1RIP (EC) + am132 inl inv mei-2), mating type unknown.K5–15-23–1cyh-2 lys-2 leu-5 mei-2 oxa1RIPTransformation of OX80–15-1 oxa1RIP (EC), also contains an ectopic oxa1 encoding a functional version of the protein with a with plasmid The cyh-2 allele confers resistance to in a The sequence of the S. cerevisiae Oxa1 protein (4.Bonnefoy N. Chalvet F. Hamel P. Slonimski P.P. Dujardin G. J. Mol. Biol. 1994; 239: 201-212Crossref PubMed Scopus (182) Google Scholar) was used to a N. crassa homologue in a of the of N. crassa sequence data were from the sequence of the and used to a N. crassa A J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) was and several were was for was by a containing the oxa1 gene and a encoding a resistance gene from B. PubMed Scopus Google Scholar) into the and of plasmid was used to strain HV of N. crassa to a to as a substrate for is a that the of the N. crassa present in the are and by the of to in both of the sequence Annu. Rev. Genet. PubMed Scopus Google Scholar). carries an from into The plasmid was transformed into E. coli strain for the of for was to and which contain that at the to and of the coding A resistance gene from C. B. M. J. M. Genet. 36: Scholar) was into plasmid to and is a containing the sequence The was by a that contained the coding of the C terminus of oxa1 with the The to a sequence of an of the The was with with and into a containing of that been with with and with the coding of the oxa1 gene as well as the the containing the replaced the C terminus and of the oxa1 for of yeast Δoxa1 strain and contained an version of the N. crassa into the of the plasmid which of N. crassa oxa1 in yeast under of the The oxa1 gene was by the process of Annu. Rev. Genet. PubMed Scopus Google Scholar). we that null mutants of oxa1 be in N. we to strains in which nucleus contained no functional oxa1 requires of the that carries the R. J. E. E. Neurospora Scholar, R. J. E. E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, R. J. Genet. as a that the N. crassa oxa1 gene was on The and of for have been described Genet. Scholar) and are to used to the gene on H. R. Neupert W. 1994; PubMed Google Scholar). the strain was transformed with plasmid to a of oxa1 to the were on containing through of on containing and by analysis for of ectopic integration of a copy of was to as the in a to the The of oxa1 in of both of the the The from this were for and were by on the into containing two The by the of the nucleus containing the oxa1RIP gene to functional Oxa1 is or by a nucleus containing an The may also contain the ectopic copy of oxa1 on its through To for containing both wild type and RIPed oxa1 was from and used in with The products were sequenced and two and sequence with of the to of was for and sequence analysis of from this strain revealed that the nucleus contained both the and ectopic of Both contained an which at of the coding We these to be null since the proteins encoded be the first membrane were a protein and the C terminus of N. crassa Oxa1. of this protein in E. the sequence encoding to of the Oxa1 was by the and The was with and into the of the E. coli 1994; PubMed Scopus Google Scholar). The used yeast Oxa1 and were described (15.Herrmann J.M. Neupert W. Stuart R.A. EMBO J. 1997; 16: 2217-2226Crossref PubMed Scopus (133) Google Scholar). purified N. crassa complex I and a of purified complex used as a for the specificity of the were by and To facilitate of Oxa1, we strain The strain was by insertion of a oxa1 encoding a version of the into the oxa1RIP nucleus of The resulting strain is a that exclusively a version of Oxa1 at wild type from strain were in lysis and or at a of of mitochondrial protein of for 30 at the was by at × for at The was with of lysis the and for with The was into a with lysis containing and with lysis containing The protein was purified by the to in or The was with containing and with containing or was in and a in a or and in series in a were for the Oxa1 to molecular weight under both PubMed Google R.A. R. R.E. in and of yeast A. R.A. R.H. 20: PubMed Scopus Google of N. crassa M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, R.A. Mol. Cell. Biol. 5: PubMed Scopus Google and E. W. H. J. FEBS Lett. 35: PubMed Scopus Google Scholar) were to was with the from were as described for yeast (18.Herrmann J.M. Fölsch H. Neupert W. Stuart R.A. Celis J.E. Cell Biology: A Laboratory Handbook. Academic Press, San Diego1994: 538-544Google Scholar) and N. crassa N. Neupert W. EMBO J. PubMed Scopus Google Scholar). N. crassa for oxa1 was identified in a the sequence of the S. cerevisiae Oxa1 protein as the from the sequence were used to products to a of N. crassa. The oxa1 gene sequence was from a and has been in the data number A of the N. crassa sequence revealed no oxa1 The sequence of the N. crassa Oxa1 protein is shown in with its and S. cerevisiae The sequence of the N. crassa protein is 22% to both the yeast and Despite this low level of identity, the structure of the proteins is three Oxa1 proteins are of a well conserved of about that contains five transmembrane domains with highly conserved and is by and which contain little sequence However, a for structure formation is in the matrix of homologues described mutants of the S. cerevisiae oxa1 gene were to a petite (4.Bonnefoy N. Chalvet F. Hamel P. Slonimski P.P. Dujardin G. J. Mol. Biol. 1994; 239: 201-212Crossref PubMed Scopus (182) Google Scholar). Oxa1 null mutant in N. crassa was by the of to a in which nucleus contains a functional version of the other not Sequence analysis that the oxa1 present in the RIPed nucleus were null the by N. crassa for contain and five the the two be to three of nucleus nucleus and containing both To whether containing only nucleus were viable, from the were containing and to shown in Table analysis of these revealed no whereas both and were In and be following of the with plasmid encoding the version of Oxa1. these data that Oxa1 is essential for viability in with the that S. pombe strains both of their Oxa1 were also (13.Bonnefoy N. Kermorgant M. Groudinsky O. Dujardin G. Mol. Micro. 2000; 35: 1135-1145Crossref PubMed Scopus (28) Google to the oxa1RIP and in a To that Oxa1 is required for the strain was propagated in the of to the of containing the oxa1RIP allele to containing the wild type allele an complete of the whereas the strain grew only in medium revealed that in of in Oxa1 levels in the whereas no in strains under conditions The level of Oxa1 in the in medium also to be in strains nucleus no Oxa1. in Oxa1 no on the of the levels of the mitochondrial proteins and were in strains of the We an plasmid for yeast the N. crassa oxa1 gene under the of the yeast transformed into of a yeast strain an oxa1 the N. crassa gene the on carbon sources In yeast, Oxa1 is required for the insertion and of into the inner membrane. In the of Oxa1, the of not the matrix and is that no can be of Δoxa1 mitochondria (6.He S. Fox T.D. Mol. Biol. Cell. 1997; 8: 1449-1460Crossref PubMed Scopus (157) Google Scholar, 7.Hell K. Herrmann J. Pratje E. Neupert W. Stuart R.A. FEBS Lett. 1997; 418: 367-370Crossref PubMed Scopus (149) Google Scholar). from yeast N. crassa Oxa1 contain wild type of that the N. crassa protein is able to yeast Oxa1 data show that the function of Oxa1 proteins is highly conserved and that we have the functional homologue of yeast Oxa1. that the N. crassa its yeast homologue, is a component of the machinery for the insertion of proteins into the inner membrane of mitochondria. We if of Oxa1 in N. crassa the levels of subunits of complexes by shown in the levels of both and of complex were in N. crassa mitochondria. yeast, N. crassa contains a complex I of the of mitochondria with I revealed levels of the and subunits of I is the first that the function of the Oxa1 protein is required for the biogenesis of complex I. Since a number of complex I subunits are mitochondrially encoded in N. likely that the membrane insertion of at least of requires the OXA translocase. Oxa1 contains five transmembrane domains and a that is in the mitochondrial matrix (15.Herrmann J.M. Neupert W. Stuart R.A. EMBO J. 1997; 16: 2217-2226Crossref PubMed Scopus (133) Google Scholar). the of a structure in the matrix To whether this a in Oxa1 function, we a mutant version of Oxa1 that the of the protein the was transformed into the and for strains in which the the oxa1RIP nucleus was from the strains contained no Oxa1 since our was the of the protein which are in the Oxa1 is present the strains are containing only the oxa1RIP the that the are that the of the protein is present and functional in these Growth of the containing the were from strains In an Oxa1 containing only the of the not the oxa1RIP these data that the in the C terminus of the N. crassa Oxa1 is not essential for Oxa1 The yeast Oxa1 protein was reported to be of a molecular weight complex of unknown (8.Hell K. Herrmann J.M. Pratje E. Neupert W. Stuart R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2250-2255Crossref PubMed Scopus (185) Google Scholar). To study the complex we to from N. crassa mitochondria. To the we strain which exclusively a version of Oxa1. lysis of purified mitochondria with or a Oxa1 was from the protein of this was with a from mitochondria of an wild type the Oxa1 was the only that no other protein was this first of the on of N. crassa Oxa1 to and no with the protein were to of Oxa1 be of mitochondrial protein. Oxa1 at least to of mitochondrial protein To that the purified protein was Oxa1, the from a on a was and the were sequenced by The resulting sequence was to the N. crassa Oxa1 sequence at of the protein data that Oxa1 has a mitochondrial of and the for of the protein shows the for the matrix with an in A. Biochem. PubMed Scopus Google Scholar). To the of the Oxa1 complex in N. we lysis of purified mitochondria with the on a both the and version of Oxa1 were exclusively in the of Oxa1 from mitochondria in in a of about the Since the purified complex contains only Oxa1, that the protein a homooligomeric complex containing several Oxa1 subunits. of membrane proteins with is known to in complexes that contain of and associated with the proteins J. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). To the molecular weight of the Oxa1 we mitochondria in and that both the wild type and the of Oxa1 in a at about of purified Oxa1 complex from mitochondria in revealed a complex of Since the protein has a molecular weight of 43,000, these data suggest that the Oxa1 complex consists of four Oxa1 in the inner mitochondrial membrane of N. crassa. We have identified the Oxa1 protein of N. crassa. The protein shows sequence identity to other Oxa1 homologues but the number and of transmembrane is highly conserved the N. crassa protein and Oxa1 from other The function of these proteins is also since both the N. crassa and N. Kermorgant M. Groudinsky O. Minet M. Slonimski P.P. Dujardin G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11978-11982Crossref PubMed Scopus (91) Google Scholar) homologues can the of S. cerevisiae oxa1 null We have shown that the N. crassa homologue levels of yeast thus that the function of the yeast Oxa1 with to insertion of proteins into the mitochondrial inner membrane. In yeast, in Oxa1 the function of the cytochrome complex and to levels of complex and with these of Oxa1 in N. crassa in levels of cytochrome subunits. In of two subunits of complex I was to be on Oxa1. In to most S. cerevisiae not have a complex our for a of complex I subunits on Oxa1 for their these data show that the Oxa1 protein is for the biogenesis of at least four of the five complexes and likely the protein is essential in of the sequence of Oxa1 proteins revealed a in the matrix which is to the sequence in the is not are which and are in a of molecular A. Biochem. Sci. 1996; Full Text PDF PubMed Scopus Google Scholar, P. J. Cell Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). We that these domains the of in the Oxa1 complex since the complex was to be in the used the that the of the subunits is by hydrophobic the transmembrane for the of a in the Oxa1 be to a of with its substrate with mitochondrial or with that with the OXA translocase. However, since the this is able to the oxa1 mutant and to the to that of is that the function of the is not the of the among Oxa1 this was It is that protein a function that the of the in the In yeast, the protein has been shown to in function and substrate specificity with Oxa1. can also with Oxa1 since was required for the of a oxa1 at the was to a as a that substrates and facilitates their membrane insertion by the OXA complex M. K. K. Stuart R.A. Neupert W. Herrmann J.M. J. Cell Biol. 2001; PubMed Scopus Google Scholar). the of of the of Oxa1 be if a protein of function to in N. crassa. of mitochondria in revealed an Oxa1 complex of In with the in a complex with a molecular Since we not proteins with purified Oxa1 we as that the lysis conditions in protein of the the in molecular weight is likely to the of the associated can to the of membrane was shown to be for for of 30 to were J. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of in molecular weight is with our for the of the complex following in with The of the yeast Oxa1 complex mitochondria with and M. K. K. Stuart R.A. Neupert W. Herrmann J.M. J. Cell Biol. 2001; PubMed Scopus Google were to that in N. crassa in a conserved of the OXA translocase. data show that of the Oxa1 complex from in or not to of protein subunits. we the that both protein subunits from a OXA our the that Oxa1 as a most likely a However, the of a or be since we not the of to the complex the the bacterial Sec which was reported to be at least partially associated with the bacterial Oxa1 homologue, YidC (14.Scotti P.A. Urbanus M.L. Brunner J. de Gier J.W. von Heijne G. van der Does C. Driessen A.J. Oudega B. Luirink J. EMBO J. 2000; 19: 542-549Crossref PubMed Scopus (305) Google was shown to substrate van der Does C. H. A. Driessen A.J. EMBO J. 2000; 19: PubMed Scopus Google Scholar). the of YidC is was reported that YidC partially as a on der M. N. Luirink J. Driessen A.J. EMBO 2001; PubMed Scopus Google Scholar). the low sequence identity Oxa1 a homooligomeric Oxa1 complex the functional of Oxa1 proteins since with other not be for translocation data suggest a in which the OXA translocase can function as a complex exclusively of Oxa1 subunits and its in the biogenesis of mitochondria We Oxa1 to at least of mitochondrial protein. to a of of Oxa1 of mitochondrial and about for a is with that for the translocase for to for a and that of the translocase for to about for a C. M. K. E. Neupert W. Brunner M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). A of export in the inner membrane be required for of substrates or to export if the process is an of insertion be to integration of hydrophobic of into the the of of proteins their in the work the of the purified Oxa1 complex into The of the complex functional of the OXA translocation machinery highly and of the of protein export in mitochondria. We B. A. H. J. S. R. J. and J. at the Neurospora crassa the of the N. crassa oxa1 was and We and for complex I and purified complex and for reading of the

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