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• W2078397197 abstract "In most strains of Saccharomyces cerevisiae the mitochondrial gene COX1, for subunit 1 of cytochrome oxidase, contains multiple exons and introns. Processing of COX1 primary transcript requires accessory proteins factors, some of which are encoded by nuclear genes and others by reading frames residing in some of the introns of the COX1 and COB genes. Here we show that the low molecular weight protein product of open reading frame YLR204W, for which we propose the name COX24, is also involved in processing of COX1 RNA intermediates. The growth defect of cox24 mutants is partially rescued in strains harboring mitochondrial DNA lacking introns. Northern blot analyses of mitochondrial transcripts indicate cox24 null mutants to be blocked in processing of introns aI2 and aI3. The dependence of intron aI3 excision on Cox24p is also supported by the growth properties of the cox24 mutant harboring mitochondrial DNA with different intron compositions. The intermediate phenotype of the cox24 mutant in the background of intronless mitochondrial DNA, however, suggests that in addition to its role in splicing of the COX1 pre-mRNA, Cox24p still has another function. Based on the analysis of a cox14-cox24 double mutant, we propose that the other function of Cox24p is related to translation of the COX1 mRNA. In most strains of Saccharomyces cerevisiae the mitochondrial gene COX1, for subunit 1 of cytochrome oxidase, contains multiple exons and introns. Processing of COX1 primary transcript requires accessory proteins factors, some of which are encoded by nuclear genes and others by reading frames residing in some of the introns of the COX1 and COB genes. Here we show that the low molecular weight protein product of open reading frame YLR204W, for which we propose the name COX24, is also involved in processing of COX1 RNA intermediates. The growth defect of cox24 mutants is partially rescued in strains harboring mitochondrial DNA lacking introns. Northern blot analyses of mitochondrial transcripts indicate cox24 null mutants to be blocked in processing of introns aI2 and aI3. The dependence of intron aI3 excision on Cox24p is also supported by the growth properties of the cox24 mutant harboring mitochondrial DNA with different intron compositions. The intermediate phenotype of the cox24 mutant in the background of intronless mitochondrial DNA, however, suggests that in addition to its role in splicing of the COX1 pre-mRNA, Cox24p still has another function. Based on the analysis of a cox14-cox24 double mutant, we propose that the other function of Cox24p is related to translation of the COX1 mRNA. Cytochrome c oxidase (COX) 4The abbreviations used are: COX, cytochrome c oxidase; pet mutant, respiratory deficient mutant of yeast with a mutation in a nuclear gene; ρo/- mutant, respiratory deficient mutant with either large deletions in or lacking mitochondrial DNA; mtDNA, mitochondrial DNA; HA, hemagglutinin. biogenesis is a complex process that requires the expression and interaction of subunits encoded by mitochondrial and nuclear genes. In Saccharomyces cerevisiae at least 20 nuclear gene products (1Tzagoloff A. Dieckmann C.L. Microbiol. Rev. 1990; 54: 211-225Crossref PubMed Google Scholar, 2McEwen J.E. Ko C. Kloeckner-Gruissem B. Poyton R.O. J. Biol. Chem. 1986; 261: 11872-11879Abstract Full Text PDF PubMed Google Scholar) have been shown to assist COX assembly. These proteins promote steps, ranging from processing of mitochondrial COX-specific RNAs (3Seraphin B. Simon M. Faye G. EMBO J. 1988; 7: 1455-1464Crossref PubMed Scopus (50) Google Scholar, 4Manthey G.M. McEwen J.E. EMBO J. 1995; 14: 4031-4043Crossref PubMed Scopus (182) Google Scholar) and their translation (5Mulero J.J. Fox T.D. Mol. Biol. Cell. 1993; 4: 327-1335Crossref Scopus (69) Google Scholar, 6Costanzo M.C. Fox T.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2677-2681Crossref PubMed Scopus (87) Google Scholar) to recruitment, formation, and addition of the metal and heme prosthetic groups present in the catalytic subunits of the complex (7Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 20531-20535Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 8Hiser L. Di Valentin M. Hamer A.G. Hosler J.P. J. Biol. Chem. 2000; 275: 619-623Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 9Barros M.H. Nobrega F.G. Tzagoloff A. J. Biol. Chem. 2002; 277: 9997-10002Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Cox1p (subunit 1) of COX is an important constituent containing the cytochrome a and a3 centers. This subunit is encoded by the mitochondrial COX1 gene, which is transcribed as a polycistronic precursor RNA containing COX1, ATP8, ATP6, and ENS2 (10Manon S. Guerin M. Biochim. Biophys. Acta. 1989; 985: 127-132Crossref PubMed Scopus (12) Google Scholar). Most commonly used laboratory strains have a COX1 gene with variable but multiple introns (11Bonitz S.G. Coruzzi G. Thalenfeld B.E. Tzagoloff A. Macino G. J. Biol. Chem. 1980; 255: 11927-11941Abstract Full Text PDF PubMed Google Scholar). The aI3, aI4, aI5α, and aI5β introns of COX1 are group I introns, whereas aI1, aI2, and aI5γ belong to group II introns. Both types of introns have the ability to act as mobile elements with the difference that homing of group II introns depends on an RNA intermediate and homing of group I intron is a DNA-based process (12Dujon B. Gene (Amst.). 1989; 82: 91-114Crossref PubMed Scopus (397) Google Scholar, 13Lazowska J. Meunier B. Macadre C. EMBO J. 1994; 13: 4963-4972Crossref PubMed Scopus (61) Google Scholar). The mobility of group I introns is enhanced by endonucleases encoded in the introns themselves. aI3 encodes the I-SceIII endonuclease that cleaves the junction of the two flanking exons, needed for its homing but in addition appears to exert a positive effect in removal of the intron (14Guo W., W. Moran J.V. Hoffman P.W. Henke R.M. Butow R.A. Perlman P.S. J. Biol. Chem. 1995; 270: 15563-15570Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 15Szczepanek T. Macadre C. Meunier B. Lazowska J. Gene (Amst.). 1994; 139: 1-7Crossref PubMed Scopus (13) Google Scholar). Splicing of the COX1 primary transcript is assisted by protein factors referred to as maturases that are encoded by reading frames located within some of the introns of the COX1 and COB genes (16Lazowska J. Jacq C. Slonimski P.P. Cell. 1980; 2: 333-348Abstract Full Text PDF Scopus (335) Google Scholar, 17Kennell J.C. Moran J.V. Perlman P.S. Butow R.A. Lambowitz A.M. Cell. 1993; 73: 133-146Abstract Full Text PDF PubMed Scopus (119) Google Scholar, 18Wenzlau J.M. Saldanha R.J. Butow R.A. Perlman P.S. Cell. 1989; 56: 421-430Abstract Full Text PDF PubMed Scopus (165) Google Scholar, 19Seraphin B. Faye G. Hatat D. Jacq C. Gene (Amst.). 1992; 113: 1-8Crossref PubMed Scopus (25) Google Scholar, 20Lazowska J. Claisse M. Gargouri A. Kotylak Z. Spyridakis A. Slonimski P.P. J. Mol. Biol. 1989; 205: 275-289Crossref PubMed Scopus (70) Google Scholar). Excision of some intervening sequences also depends on nuclear genes such as CBP2, and SUV3 and MSS116, the latter two coding for RNA helicases (3Seraphin B. Simon M. Faye G. EMBO J. 1988; 7: 1455-1464Crossref PubMed Scopus (50) Google Scholar, 21McGraw P. Tzagoloff A. J. Biol. Chem. 1983; 258: 9459-9468Abstract Full Text PDF PubMed Google Scholar, 22Stepien P.P. Margossian S.P. Landsman D. Butow R.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6813-6817Crossref PubMed Scopus (86) Google Scholar). Cbp2p interacts with and stabilizes a splicing competent secondary structure of the cytochrome b pre-mRNA (23Gampel A. Tzagoloff A. Mol. Cell Biol. 1989; 9: 5424-5433Crossref PubMed Scopus (62) Google Scholar). Suv3p has been implicated in stabilizing the COX1 transcript by regulating turnover of group I intronic RNAs (24Golik P. Szczepanek T. Bartnik E. Stepien P.P. Lazowska J. FEMS Yeast Res. 2004; 4: 477-485Crossref PubMed Scopus (5) Google Scholar, 25Margossian S.P. Li H. Zassenhaus H.P. Butow R.A. Cell. 1996; 84: 199-209Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), whereas Mss116p has been shown to function in splicing of all mtDNA introns in S. cerevisiae and Neurospora crassa (26Huang H.R. Rowe C.E. Mohr S. Jiang Y. Lambowitz A.M. Perlman P.S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 163-168Crossref PubMed Scopus (137) Google Scholar). Some of the accessory factors appear to have several functions in mitochondrial RNA metabolism. PET309, first isolated as a COX1 translation facilitator, also affects the stability of intron-containing COX1 RNA (4Manthey G.M. McEwen J.E. EMBO J. 1995; 14: 4031-4043Crossref PubMed Scopus (182) Google Scholar) and was recently found to be associated with CBP1, a cytochrome b RNA stabilization and translation factor (27Krause K. Lopes de Souza R. Roberts D.G. Dieckmann C.L. Mol. Biol. Cell. 2004; 15: 2674-2683Crossref PubMed Scopus (48) Google Scholar). NAM2/MSL1, the mitochondrial leucyl-tRNA synthetase, is required for excision of the aI4 and bI4 introns of COB (28Herbert C.J. Labouesse M. Dujardin G. Slonimski P.P. EMBO J. 1988; 7: 473-483Crossref PubMed Scopus (126) Google Scholar, 29Labouesse M. Mol. Gen. Genet. 1990; 224: 209-221Crossref PubMed Scopus (66) Google Scholar). Excision of the aI5β intron of COX1 depends on several nuclear gene products. Already mentioned are the helicases Suv3p and Mss116p. Other factors include Pet54p, Mss18p, and Mrs1p. Pet54p was first characterized as a translational factor for COX3 mRNA (6Costanzo M.C. Fox T.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2677-2681Crossref PubMed Scopus (87) Google Scholar), but was subsequently shown to also play a role in aI5β excision (30Valencik M.L. Kloeckener-Gruissem B. Poyton R.O. McEwen J.E. EMBO J. 1989; 8: 3899-3904Crossref PubMed Scopus (35) Google Scholar). Mrs1p is required for processing of introns bI3 of COB, and aI5β and aI5γ of COX1 (31Bousquet I. Dujardin G. Poyton R.O. Slonimski P.P. Curr. Genet. 1990; 18: 117-124Crossref PubMed Scopus (40) Google Scholar). At present the only proteins known to function in processing of a single intron are Mss18p (3Seraphin B. Simon M. Faye G. EMBO J. 1988; 7: 1455-1464Crossref PubMed Scopus (50) Google Scholar) and Cpb2p (23Gampel A. Tzagoloff A. Mol. Cell Biol. 1989; 9: 5424-5433Crossref PubMed Scopus (62) Google Scholar), which target aI5β and bI5, respectively. In the course of analyzing the biochemical defects of the respiratory deficient pet mutant of S. cerevisiae, we identified a new gene, which when mutated causes the accumulation of COX1 intermediates transcripts. This gene has been designated COX24 in keeping with our previous convention for naming genes involved in expression of cytochrome oxidase. COX24 corresponds to reading frame YLR204W and was previously named QRI5 (32Simon M. Della Seta F. Sor F. Faye G. Yeast. 1992; 8: 559-567Crossref PubMed Scopus (3) Google Scholar). The phenotype of cox24 mutants and characterization of their mitochondrial RNAs lead us to propose that Cox24p plays an important role in processing of the COX1 primary transcript, but like the other aforementioned processing factors, may have another function as well. Strains and Media—The strains of yeast used in this study are listed in Table 1. The respiratory deficient mutants of complementation group G82 were derived from S. cerevisiae D273-10B/A1 by mutagenesis with nitrosoguanidine or ethyl methanesulfonate (1Tzagoloff A. Dieckmann C.L. Microbiol. Rev. 1990; 54: 211-225Crossref PubMed Google Scholar). The following media were used routinely to grow yeast: YPD (2% glucose, 1% yeast extract, 2% peptone), YPGal (2% galactose, 1% yeast extract, 2% peptone), and YEPG (2% ethanol, 3% glycerol, 1% yeast extract, 2% peptone).TABLE 1Genotypes and sources of yeast strainsStrainGenotypemtDNASourceW303-1AMATa ade2-1 his3-1,15 leu2-3, 112 trp1-1 ura 3-1ρ+—aDr. R. Rothstein, Department of Human Genetics, Columbia University, New York.W303-1BMATα ade2-1 his3-1,15 leu2-3, 112 trp1-1 ura3-1ρ+—aDr. R. Rothstein, Department of Human Genetics, Columbia University, New York.W303/ρoMATα ade2-1 his3-1,15 leu2-3, 112 trp1-1 ura3-1ρoThis studyCB11MATa ade1ρ+Ref. 33ten Berge A.M. Zoutewelle G. Needleman R.B. Mol. Gen. Genet. 1974; 131: 113-121Crossref PubMed Scopus (30) Google ScholarD273-10B/A21MATα met6ρ+ OR ER PR bI+, aI1+, aI2+, aI3+, a4+, aI5γ+Ref. 34Tzagoloff A. Akai A. Foury F. FEBS Lett. 1976; 65: 391-396Crossref PubMed Scopus (79) Google ScholaraW303/IoMATa ade2-1 his3-1,15 leu2-3, 112 trp1-1 ura3-1IoThis studyW303/IoMATα ade2-1 his3-1,15 leu2-3, 112 trp1-1 ura3-1IoThis studyJC3ρoMATa kar1-1 ade2 lys2ρoATCC 201577JC3/A21MATa kar1-1 ade2 lys2ρ+JC3ρo × D27310B/A21JC3/GF134-6DMATa kar1-1 ade2 lys2bI+, aI4+JC3ρo × GF134-6DJC11/WI04MATα kar1-1 his3bI+, aI1+, aI2+, aI3+, aI5γ+Ref. 29Labouesse M. Mol. Gen. Genet. 1990; 224: 209-221Crossref PubMed Scopus (66) Google ScholarJC11/CK5112MATα, kar1-1, his3aI2+, aI3+, aI5γ+ w+—bDr. Jaga Lazowska, Centre de Génétique Moleculaire du CNRS, Gif-sur-Yvette, FranceGF134-6DMATα his1 met6bI+, aI4+Ref. 35Seraphin B. Boulet A. Simon M. Faye G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6810-6814Crossref PubMed Scopus (102) Google ScholarW303/A21MATα ade2-1 his3-1,15 leu2-3, 112 trp1-1 ura3-1ρ+, mit+W303/ρo × JC3/A21W303/GF134-6DMATα ade2-1 his3-1,15 leu2-3,112 trp1-1 ura3-1bI+, aI4+W303/ρo × JC3/GF134-6DW303/WI04MATa ade2-1 his3-1,15 leu2-3, 112 trp1-1 ura3-1bI+, aI1+, aI2+, aI3+, aI5γ+W303/ρo × JC11/WI04W303/CK5112MATa ade2-1 his3-1,15 leu2-3, 112 trp1-1 ura3-1aI2+, aI3+, aI5γ+ w+W303/ρo × JC11/CK5112C149MAT α met6 cox24-1ρ+Ref. 1Tzagoloff A. Dieckmann C.L. Microbiol. Rev. 1990; 54: 211-225Crossref PubMed Google ScholarB149MATa ade1 cox24-1ρ+C149 × aCB11C149/UL1MAT α leu2-3,112 ura3-1 cox24-1ρ+C149 × W303-1AB149/IoMATa ade1 leu2-3,112 trp1-1 ura3-1IoB149ro × W303/IoW303ΔCOX24MATα ade2-1 his 3-1,15 leu2-3, 112 trp1-1 ura3-1 cox24::HIS3ρ+This studyaW303ΔCOX24MATa ade2-1 his3-1,15 leu2-3,112 trp1-1 ura 3-1 cox24::HIS3ρ+This studyaW303ΔCOX24/R2MATa ade2-1 his3-1,15 leu2-3,112 trp1-1 ura3-1 cox24::HIS3ρ+,R2This studyW303ΔCOX24/IoMATα ade2-1 his3-1,15 leu2-3,112 trp1-1 ura3-1 cox24::HIS3IoW303ΔCOX24 × a W303/IoaW303ΔCOX24/IoMATa ade2-1 his3-1,15 leu2-3,112 trp1-1 ura3-1 cox24::HIS3IoW303ΔCOX24 × aW303/IoaW303ΔCOX24/A21MATa ade2-1 his3-1,15 leu2-3,112 trp1-1 ura3-1 cox24::HIS3ρ+W303ΔCOX24/ρo × W303/A21aW303ΔCOX24/WI04MATa ade2-1 his3-1,15 leu2-3,112 trp1-1 ura3-1 cox24::HIS3bI+, aI1+, aI2+, aI3+, aI5γ+W303ΔCOX24/ρo × W303/WI04aW303ΔCOX24/CK5112MATa ade2-1 his3-1,15 leu2-3,112 trp1-1 ura3-1 cox24::HIS3aI2+, aI3+, aI5γ+W303ΔCOX24/ρo × W303/CK5112aW303ΔCOX24/GF134-6DMATa ade2-1 his3-1,15 leu2-3,112 trp1-1 ura3-1 cox24::HIS3bI+, aI4+W303ΔCOX24/ρo × W303/GF134-6DW303ΔCOX14MATα ade2-1 his3-1,15 leu2-3,112 trp1-1 ura3-1 cox14::TRP1ρ+Ref. 36Barrientos A. Zambrano A. Tzagoloff A. EMBO J. 2004; 23: 3472-3482Crossref PubMed Scopus (168) Google ScholarW303ΔCOX14/IoMATα ade2-1 his3-1,15 leu2-3,112 trp1-1 ura3-1 cox14::TRP1IoThis studyaW303ΔCOX14,COX24MATa ade2-1 his3-1,15 leu2-3,112 trp1-1 ura3-1 cox24::HIS3 cox14::TRP1IoaW303ΔCOX14 × W303ΔCOX24aW303ΔCOX14/B149MATa ade2-1 his3-1,15 leu2-3,112 trp1-1 ura3-1 cox24 cox14::TRP1IoW303ΔCOX14 × B149/Ioa Dr. R. Rothstein, Department of Human Genetics, Columbia University, New York.b Dr. Jaga Lazowska, Centre de Génétique Moleculaire du CNRS, Gif-sur-Yvette, France Open table in a new tab Cloning of COX24—pG82/T1, a recombinant plasmid containing COX24, was isolated by transformation of C149/UL1 with a yeast genomic library consisting of partial Sau3A fragments of yeast nuclear DNA cloned in the yeast/Escherichia coli shuttle plasmid YEp13 (37Broach J.R. Strathern J.N. Hicks J.B. Gene (Amst.). 1979; 8: 121-133Crossref PubMed Scopus (672) Google Scholar). Approximately 5 × 108 cells were transformed with 50 μg of the plasmid library by the method of Beggs (38Beggs J.D. Nature. 1978; 275: 104-109Crossref PubMed Scopus (793) Google Scholar). Disruption of COX24—A BamHI-BglII fragment of pG82/T1 containing COX24 was transferred to pUC19. The resultant plasmid (pG82/ST10) was used to replace the COX24 coding sequence with the yeast HIS3 gene. Amplification of pG82/ST10 with bi-directional primers 5-GGCAGATCTGGGCTGTAAAAACCTCTCAC and 5′-GGCAGATCTGTTTATCTCGTTCTGGTGTC resulted in a clean deletion of COX24. The linear product containing COX24 5′- and 3′-flanking regions in pUC19 was digested with BglII and ligated to HIS3 on a 1-kb BamHI fragment. The cox24::HIS3 null allele was recovered from this plasmid as a BamHI-XbaI fragment and was substituted by homologous recombination (39Rothstein R.J. Methods Enzymol. 1983; 101: 202-211Crossref PubMed Scopus (2033) Google Scholar) for the wild-type gene in respiratory competent strains W303-1B and W303-1A. Construction of a Hybrid Gene Expressing Cox24p Tagged with a Hemagglutin Epitope at the Carboxyl Terminus—pG82/T1 was used as a PCR template for amplification of the COX24-HA gene coding for Cox24p with 12 carboxyl-terminal residues consisting of three glycine residues as spacers and the hemagglutinin tag (HA). The PCR primers used for amplification were: 5′-GGCGGATC-CGTCCACCTTCGCAATATCTAC and 5′-GGCAAGCTTTCAAGCGTAGTCTGGGACGTCGTATGGGTACCCTCCTCCTCTACCCTGAGATAGCTTTCT. The PCR product was digested with a combination of BamHI and HindIII, and was cloned in the yeast/E. coli shuttle plasmids YEp352 and YIp352 (40Hill J.E. Myers A.M. Koerner T.J. Tzagoloff A. Yeast. 1986; 2: 163-167Crossref PubMed Scopus (1083) Google Scholar) yielding pG82/ST16 and pG82/ST17, respectively. W303ΔCOX24 was transformed either with uncut pG82/ST16 or with pG82/ST17 linearized at the NcoI site of the URA3 marker for integration at the homologous site in chromosomal DNA (39Rothstein R.J. Methods Enzymol. 1983; 101: 202-211Crossref PubMed Scopus (2033) Google Scholar). Miscellaneous Procedures—Standard methods were used for plasmid manipulations (41Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The COX24 gene and flanking regions were sequenced by the method of Maxam and Gilbert (42Maxam A.M. Gilbert W. Methods Enzymol. 1980; 65: 49-56Google Scholar). Yeast mitochondria were prepared by the method of Faye et al. (43Faye G. Kujawa C. Fukuhara H. J. Mol. Biol. 1974; 88: 185-203Crossref PubMed Scopus (180) Google Scholar) except that Zymolyase 20T (ICN Laboratories, Aurora, OH) instead of glusulase was used to obtain spheroplasts. Spectral analyses of mitochondrial cytochromes were performed as described previously (44Tzagoloff A. Akai A. Needleman R.B. J. Biol. Chem. 1975; 250: 8228-8235Abstract Full Text PDF PubMed Google Scholar). Protein concentrations were determined by the method of Lowry et al. (45Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Cloning of COX24—C149 is one of three independent respiratory deficient strains assigned to complementation group G82 of a pet mutant collection (1Tzagoloff A. Dieckmann C.L. Microbiol. Rev. 1990; 54: 211-225Crossref PubMed Google Scholar). Transformation of the derivative strain C149/L1 with a yeast genomic library yielded a leucine-independent and respiratory competent transformant (C149/L1/T1) that was used to isolate the recombinant plasmid pG82/T1. Complementation tests with subclones containing different regions of the nuclear DNA insert in this plasmid revealed that the gene responsible for restoring respiration in C149/L1 corresponds to reading frame YLR204W on chromosome XII (data not shown). The mutant W303ΔCOX24 harboring a null allele of the gene, henceforth referred to as COX24, was obtained by homologous recombination with a linear fragment of nuclear DNA in which the entire reading frame was replaced with HIS3. The identity of COX24 as the gene responsible for the respiratory defect of C149/L1 was confirmed by the lack of complementation of the point mutant by the null mutant W303ΔCOX24 and the sequence of the gene in C149/L1, which was found to have a single base (guanine) deletion in the codon corresponding to lysine at position 87 of the polypeptide chain. The deletion of this nucleotide creates a stop codon immediately after the mutation, thereby shortening the protein by 24 amino acids. Phenotype of the cox24 Null Mutant—C149/L1 and W303ΔCOX24 exhibit a clear growth defect on non-fermentable carbon sources (ethanol/glycerol). The spectrum of mitochondrial cytochromes in the null mutant indicated the presence of a very low level of an “a” type cytochrome with an absorption maximum of the α peak at 595 nm instead of 605 nm, the normal positions of cytochrome a and a3 bands in cytochrome oxidase absorb normally (Fig. 1, A and B). The α absorption band of cytochrome b is also reduced in the mutant but is located at the expected wavelength (560 nm). Despite the reduction of spectrally detectable cytochrome b, in vivo labeling of mitochondrial products indicated normal translation of cytochrome b (Fig. 1C). A partial deficiency of cytochrome b is a common property of COX mutants. In contrast to cytochrome b, translation of Cox1p, Cox2p, and Cox3p, the three mitochondrially encoded subunits of cytochrome oxidase, was reduced. This was most evident for Cox1p, which was present in only trace amounts in the mutant. Subunit 6 (Atp6p) of the ATPase was normally translated (Fig. 1C), ruling out a defect in transcription or processing of the large polycistronic precursor RNA containing COX1, ATP8, and ATP6 (10Manon S. Guerin M. Biochim. Biophys. Acta. 1989; 985: 127-132Crossref PubMed Scopus (12) Google Scholar). The steady-state concentrations of COX subunits in the cox24 null mutant were examined by Western blot analysis with antibodies against Cox1p, Cox2p, and Cox3p, and the nuclear encoded subunits Cox4p and Cox5p. Of these five constituents, Cox1p and Cox2p were reduced to barely detectable levels in the mutant. A very substantial decrease was also seen in Cox3p. Both Cox4p and Cox5p were reduced but to a lesser extent (Fig. 1D). This pattern is characteristic of most mutants that fail to form functional COX, independent of whether the block occurs at a pre- or post-translation step of the assembly pathway (46Barrientos A. Barros M.H. Valnot I. Rotig A. Rustin P. Tzagoloff A. Gene (Amst.). 2002; 286: 53-63Crossref PubMed Scopus (157) Google Scholar). W303ΔCOX24 gives rise to revertants with growth properties on non-fermentable substrates intermediate between wild-type and the mutant. Although the suppressor mutation(s) has been ascertained to be in mtDNA, for unknown reasons we have not been able to map the mutations by deletion analysis with cytoplasmic petites obtained from several such revertants. The partial restoration of growth of one such revertant (W303ΔCOX24/R2) correlates with an increase in the mitochondrial steady-state concentration of Cox1p (Fig. 1D). Phenotype of a cox24 Mutant with Intronless Mitochondrial DNA—The lesion in the cox24 mutant appeared most likely to be in processing and/or translation of Cox1p. The parental W303 strains into which the cox24 null allele was introduced have mitochondrial genomes with COX1 introns aI1-aI4 and aI5γ (47Muroff I. Tzagoloff A. EMBO J. 1990; 9: 2765-2773Crossref PubMed Scopus (30) Google Scholar). To probe the possible involvement of Cox24p in intron processing, the cox24 null mutation was transferred to a strain lacking mitochondrial introns by a cross of W303ΔCOX24/ρ0 (a cox24 null mutant lacking mitochondrial DNA) to the intronless strain W303/I0. Diploid cells issued from the cross yielded meiotic progeny with the cox24 null allele in an intronless mitochondrial background (aW303ΔCOX24/I0 and W303ΔCOX24/I0). These strains were able to grow on non-fermentable substrates (YPEG), albeit not as well as the wild-type strains W303-1A or W303-1A/I0 (Fig. 2A). Growth of W303ΔCOX24/I0 on glycerol/ethanol correlated with a partial restoration of Cox1p translation (Fig. 1C). Western analysis of mitochondria with a polyclonal antibody against Cox1p indicated a substantial increase in the steady-state concentration of this subunit in the mutant cells (Fig. 2B). Paradoxically, a Cox1p monoclonal antibody detected only trace amounts of Cox1p, even at very high loading of mitochondrial proteins (Fig. 2B). This suggests that the Cox1p produced in W303ΔCOX24/I0 may be different from the normal protein. COX1 Transcript Processing in cox24 Mutants—The partial rescue of the cox24 null mutation in a strain with intronless mtDNA pointed to a role of Cox24p in processing of the COX1 pre-mRNA. This is supported by the mitochondrial COX1 transcripts present in wild-type and mutant strains harboring mtDNAs with or without introns (Fig. 3A). Northern blots of total mitochondrial RNA hybridized to a probe from exon aE4 of COX1 indicated the accumulation of partially processed COX1 RNA intermediates in the cox24 null mutant with the original intron-containing mtDNA. In this background the mature COX1 mRNA was not detected. As expected, COX1 processing intermediates were absent in the wild-type or cox24 mutant with the intronless genome. In this background the only COX1 transcript detected by the probe had a size corresponding to the mature mRNA. This reinforces the conclusion that the cox24 mutation does not affect endonucleolytic cleavage of the primary polycistronic transcript. Northern blots of the same RNAs with an exon probe from the cytochrome b (COB) gene revealed the presence in the cox24 mutant of the mature size of COB mRNA. Although there was some accumulation of COB precursor transcripts in the mutant, the mRNA concentration was not appreciably less than in the wild type (Fig. 3B). The presence of the processed COB mRNA in the mutant is consistent with the in vivo labeling results showing normal translation of cytochrome b (Fig. 1C) and support a role of Cox24p confined to processing of the COX1 pre-mRNA. The function of Cox24p in splicing of the COX1 precursor was assessed by hybridization of total mitochondrial RNAs with probes from introns aI1, aI2, aI3, aI4, and aI5γ. The results of these Northern analyses indicated that the cox24 mutant is able to splice group II introns aI1 and aI5γ. Both introns are stable and accumulate in the wild-type and mutant (Fig. 4). The concentration of aI2, the third stable group II intron of COX1, is significantly lower in the mutant and is present at nearly wild-type levels in the revertant W303ΔCOX24/R2. The results with probes against group I introns aI3 and aI4 are more difficult to interpret because the excised products are unstable and normally are degraded. The aI4 probe detected high molecular weight pre-cursors that are much less abundant in wild type and are substantially reduced in the revertant. In the case of the aI3 probe, several of the partially processed intermediates seen in wild type are absent in the mutant but are partially restored in the revertant (Fig. 4). These results point to multiple roles of Cox24p in COX1 mRNA processing, including excision of both group I and group II introns. Suppression of the cox24 Null Mutant by mtDNA with Different Intron Compositions—The COX1 processing defect was also tested by examining the suppressor activities of mitochondrial genomes differing in their intron compositions. These were introduced into a ρ0 derivative of the cox24 null mutant by cytoduction (49Conde J. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 3651-3655Crossref PubMed Scopus (385) Google Scholar). As might be predicted, the absence of COB introns did not relieve the respiratory deficiency of the mutant (CK5112 in Fig. 5). This was also true of the cox24 mutant with a COX1 gene lacking the aI4 intron (WIO4). The cox24 mutant, however, was partially rescued by mtDNA lacking introns aI1, aI2, aI3, and aI5γ (GF134-6D). These results are consistent with Northern hybridizations with intron-specific probes, indicating impaired processing of aI2 and aI3 in the mutant (Fig. 4). Cox1p Expression Is Inhibited in a cox14-cox24 Double Mutant—The incomplete suppression of the cox24 mutant by a mitochondrial genome devoid of introns suggested that in addition to transcript processing Cox24p may have another function, either in translation or assembly of cytochrome oxidase. Mutations in genes required for maturation of the COX1 precursor transcript or translation of the mRNA display the absence of Cox1p among the normal complement of mitochondrial translation products. Translation of Cox1p is also compromised by mutations that prevent assembly of the subunits into the functional complex (36Barrientos A. Zambrano A. Tzagoloff A. EMBO J. 2004; 23: 3472-3482Crossref PubMed Scopus (168) Google Scholar). Synthesis of Cox1p, in all such assembly defective mutants can be rescued when the original mutation is combined with a cox14 null mutation (36Barrientos A. Zambrano A. Tzagoloff A. EMBO J. 2004; 23: 3472-3482Crossref PubMed Scopus (168) Google Scholar). This circumstance permits mutations that prevent formation or translation of the Cox1p mRNA to be discriminated from mutations affecting a post-translational assembly step. A regulatory model of Cox1p translation has been invoked to explain the epistatic effect of the cox14 mutation over other mutations affecting post-translational events in COX assembly (36Barrientos A. Zambrano A. Tzagoloff A. EMBO J. 2004; 23: 3472-3482Crossref PubMed Scopus (168) Google Scholar). This experimental paradigm was used to examine expression of Cox1p in a cox24-cox14 double mutant with intronless mtDNA. The results of in vivo translation assays indicated that not only did inclusion of the cox14 mutation fail to enhance Cox1p synthesis over and above that seen in the single" @default.
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• W2078397197 title "COX24 Codes for a Mitochondrial Protein Required for Processing of the COX1 Transcript" @default.
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