The Pentatricopeptide Repeats Present in Pet309 Are Necessary for Translation but Not for Stability of the Mitochondrial COX1 mRNA in Yeast
2007; Elsevier BV; Volume: 283; Issue: 3 Linguagem: Inglês
10.1074/jbc.m708437200
ISSN1083-351X
AutoresFaviola Tavares-Carreón, Yolanda Camacho‐Villasana, Angélica Zamudio-Ochoa, Miguel Shingú-Vázquez, Alfredo Torres‐Larios, Xóchitl Pérez-Martínez,
Tópico(s)Metalloenzymes and iron-sulfur proteins
ResumoPet309 is a protein essential for respiratory growth. It is involved in translation of the yeast mitochondrial COX1 gene, which encodes subunit I of the cytochrome c oxidase. Pet309 is also involved in stabilization of the COX1 mRNA. Mutations in a similar human protein, Lrp130, are associated with Leigh syndrome, where cytochrome c oxidase activity is affected. The sequence of Pet309 reveals the presence of at least seven pentatricopeptide repeats (PPRs) located in tandem in the central portion of the protein. Proteins containing PPR motifs are present in mitochondria and chloroplasts and are in general involved in RNA metabolism. Despite the increasing number of proteins from this family found to play essential roles in mitochondria and chloroplasts, little is understood about the mechanism of action of the PPR domains present in these proteins. In a series of in vivo analyses we constructed a pet309 mutant lacking the PPR motifs. Although the stability of the COX1 mRNA was not affected, synthesis of Cox1 was abolished. The deletion of one PPR motif at a time showed that all the PPR motifs are required for COX1 mRNA translation and respiratory growth. Mutations of basic residues in PPR3 caused reduced respiratory growth. According to a molecular model, these residues are facing a central cavity that could be involved in mRNA-binding activity, forming a possible path for this molecule on Pet309. Our results show that the RNA metabolism function of Pet309 is found in at least two separate domains of the protein. Pet309 is a protein essential for respiratory growth. It is involved in translation of the yeast mitochondrial COX1 gene, which encodes subunit I of the cytochrome c oxidase. Pet309 is also involved in stabilization of the COX1 mRNA. Mutations in a similar human protein, Lrp130, are associated with Leigh syndrome, where cytochrome c oxidase activity is affected. The sequence of Pet309 reveals the presence of at least seven pentatricopeptide repeats (PPRs) located in tandem in the central portion of the protein. Proteins containing PPR motifs are present in mitochondria and chloroplasts and are in general involved in RNA metabolism. Despite the increasing number of proteins from this family found to play essential roles in mitochondria and chloroplasts, little is understood about the mechanism of action of the PPR domains present in these proteins. In a series of in vivo analyses we constructed a pet309 mutant lacking the PPR motifs. Although the stability of the COX1 mRNA was not affected, synthesis of Cox1 was abolished. The deletion of one PPR motif at a time showed that all the PPR motifs are required for COX1 mRNA translation and respiratory growth. Mutations of basic residues in PPR3 caused reduced respiratory growth. According to a molecular model, these residues are facing a central cavity that could be involved in mRNA-binding activity, forming a possible path for this molecule on Pet309. Our results show that the RNA metabolism function of Pet309 is found in at least two separate domains of the protein. Biogenesis of the mitochondrial cytochrome c oxidase (COX) 2The abbreviations used are: COXcytochrome c oxidaseUTRuntranslated regionPPRpentatricopeptide repeatTPRtetratricopeptide repeatHAhemagglutinin. complex depends on a large set of proteins. In the yeast Saccharomyces cerevisiae more than 20 nuclear genes have been found to be necessary for assembly and maintenance of the functional COX (1Barrientos A. Korr D. Tzagoloff A. EMBO J. 2002; 21: 43-52Crossref PubMed Scopus (141) Google Scholar, 2Tzagoloff A. Dieckmann C.L. Microbiol. Rev. 1990; 54: 211-225Crossref PubMed Google Scholar, 3Herrmann J.M. Funes S. Gene (Amst.). 2005; 354: 43-52Crossref PubMed Scopus (104) Google Scholar). The enzyme in mammals and yeast is composed of 13 and 12 subunits, respectively. The core of the enzyme is formed by subunits Cox1, Cox2, and Cox3, which are encoded in the mitochondrial DNA. Expression of the mitochondrial-encoded subunits is highly regulated by proteins involved in transcription, transcript stability and processing, translation, and assembly into the mitochondrial inner membrane (3Herrmann J.M. Funes S. Gene (Amst.). 2005; 354: 43-52Crossref PubMed Scopus (104) Google Scholar, 4Costanzo M.C. Fox T.D. Annu. Rev. Genet. 1990; 24: 91-113Crossref PubMed Google Scholar, 5Dieckmann C.L. Staples R.R. Int. Rev. Cytol. 1994; 152: 145-181Crossref PubMed Scopus (77) Google Scholar). cytochrome c oxidase untranslated region pentatricopeptide repeat tetratricopeptide repeat hemagglutinin. In humans, deficiency in COX assembly is associated with mitochondrial disorders. The majority of these are caused by autosomal recessive mutations that affect COX assembly factors (6Barrientos A. Barros M.H. Valnot I. Rotig A. Rustin P. Tzagoloff A. Gene. 2002; 286: 53-63Crossref PubMed Scopus (157) Google Scholar, 7Shoubridge E.A. Hum. Mol. Genet. 2001; 10: 2277-2284Crossref PubMed Scopus (154) Google Scholar). An example of such a factor is the mRNA-binding protein Lrp130 (8Mili S. Pinol-Roma S. Mol. Cell Biol. 2003; 23: 4972-4982Crossref PubMed Scopus (123) Google Scholar, 9Xu F. Morin C. Mitchell G. Ackerley C. Robinson B.H. Biochem. J. 2004; 382: 331-336Crossref PubMed Scopus (139) Google Scholar). Mutations in the LRP130 gene have been associated with the neurodegenerative disorder Leigh syndrome of the French Canadian type (10Mootha V.K. Lepage P. Miller K. Bunkenborg J. Reich M. Hjerrild M. Delmonte T. Villeneuve A. Sladek R. Xu F. Mitchell G.A. Morin C. Mann M. Hudson T.J. Robinson B. Rioux J.D. Lander E.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 605-610Crossref PubMed Scopus (475) Google Scholar). These patients lack fully functional COX activity, associated with defects in the COX1 and COX3 transcripts (9Xu F. Morin C. Mitchell G. Ackerley C. Robinson B.H. Biochem. J. 2004; 382: 331-336Crossref PubMed Scopus (139) Google Scholar). It has been proposed that PET309 is the yeast homologue of LRP130 (10Mootha V.K. Lepage P. Miller K. Bunkenborg J. Reich M. Hjerrild M. Delmonte T. Villeneuve A. Sladek R. Xu F. Mitchell G.A. Morin C. Mann M. Hudson T.J. Robinson B. Rioux J.D. Lander E.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 605-610Crossref PubMed Scopus (475) Google Scholar), with 37% of similarity over 300 amino acids. Both genes seem to participate in mRNA processing and may have similar functions in mitochondria. Pet309 is a translational activator necessary for Cox1 synthesis. It specifically acts on the 5′-UTR of the COX1 mRNA to activate translation. In addition, it is required to stabilize the pre-COX1 transcript (11Manthey G.M. McEwen J.E. EMBO J. 1995; 14: 4031-4043Crossref PubMed Scopus (179) Google Scholar). It has been observed that translational activators specific for the COX1, COX2, and COX3 mRNAs interact with each other and with the mitochondrial inner membrane (12Brown N.G. Costanzo M.C. Fox T.D. Mol. Cell Biol. 1994; 14: 1045-1053Crossref PubMed Scopus (68) Google Scholar, 13Manthey G.M. Przybyla-Zawislak B.D. McEwen J.E. Eur. J. Biochem. 1998; 255: 156-161Crossref PubMed Scopus (62) Google Scholar, 14Green-Willms N.S. Butler C.A. Dunstan H.M. Fox T.D. J. Biol. Chem. 2001; 276: 6392-6397Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 15Naithani S. Saracco S.A. Butler C.A. Fox T.D. Mol. Biol. Cell. 2003; 14: 324-333Crossref PubMed Scopus (128) Google Scholar), suggesting that the activators promote that translation initiation takes place close to the insertion and assembly sites of the three COX subunits in the mitochondrial inner membrane (15Naithani S. Saracco S.A. Butler C.A. Fox T.D. Mol. Biol. Cell. 2003; 14: 324-333Crossref PubMed Scopus (128) Google Scholar, 16Sanchirico M.E. Fox T.D. Mason T.L. EMBO J. 1998; 17: 5796-5804Crossref PubMed Scopus (101) Google Scholar). Both Lrp130 and Pet309 contain several pentatricopeptide repeats (PPRs). These repeats belong to a protein family that is very large in plants, with at least 442 members in Arabidopsis thaliana. However, there are fewer examples of these proteins in fungi, animals, and protists (17Lurin C. Andres C. Aubourg S. Bellaoui M. Bitton F. Bruyere C. Caboche M. Debast C. Gualberto J. Hoffmann B. Lecharny A. Le Ret M. Martin-Magniette M.L. Mireau H. Peeters N. Renou J.P. Szurek B. Taconnat L. Small I. Plant Cell. 2004; 16: 2089-2103Crossref PubMed Scopus (1003) Google Scholar, 18Pusnik M. Small I. Read L.K. Fabbro T. Schneider A. Mol. Cell Biol. 2007; 27: 6876-6888Crossref PubMed Scopus (87) Google Scholar). Pet309 is the only yeast translational activator that has been found to contain PPR motifs. In general, PPR proteins are usually found to localize in mitochondria and chloroplasts. It is known from the small set of PPR proteins studied to date that they participate mostly in different steps of sequence-specific RNA metabolism. They are implicated in precursor transcript stability and processing (which includes splicing and editing) (19Nakamura T. Meierhoff K. Westhoff P. Schuster G. Eur. J. Biochem. 2003; 270: 4070-4081Crossref PubMed Scopus (102) Google Scholar, 20Yamazaki H. Tasaka M. Shikanai T. Plant J. 2004; 38: 152-163Crossref PubMed Scopus (111) Google Scholar, 21Schmitz-Linneweber C. Williams-Carrier R.E. Williams-Voelker P.M. Kroeger T.S. Vichas A. Barkan A. 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However, in a few examples specific RNA-binding activity or their natural RNA targets has been demonstrated (21Schmitz-Linneweber C. Williams-Carrier R.E. Williams-Voelker P.M. Kroeger T.S. Vichas A. Barkan A. Plant Cell. 2006; 18: 2650-2663Crossref PubMed Scopus (233) Google Scholar, 26Gillman J.D. Bentolila S. Hanson M.R. Plant J. 2007; 49: 217-227Crossref PubMed Scopus (70) Google Scholar). These proteins play essential roles in plant embryogenesis, cytoplasmic male sterility restoration, and chloroplast to nucleus retrograde signaling (for examples see Refs. 17Lurin C. Andres C. Aubourg S. Bellaoui M. Bitton F. Bruyere C. Caboche M. Debast C. Gualberto J. Hoffmann B. Lecharny A. Le Ret M. Martin-Magniette M.L. Mireau H. Peeters N. Renou J.P. Szurek B. Taconnat L. Small I. Plant Cell. 2004; 16: 2089-2103Crossref PubMed Scopus (1003) Google Scholar, 27Bentolila S. Alfonso A.A. Hanson M.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10887-10892Crossref PubMed Scopus (394) Google Scholar, 28Koussevitzky S. Nott A. Mockler T.C. Hong F. Sachetto-Martins G. Surpin M. Lim J. Mittler R. Chory J. Science. 2007; 316: 715-719Crossref PubMed Google Scholar). PPRs are degenerated 35-amino acid motifs proposed to consist of two antiparallel α helices. There is no structural information about PPR proteins, but models based on the closely related TPR (tetratricopeptide repeat) proteins suggest that the tandem repeats of these domains form a solenoid-like structure with a hydrophilic cavity where the phosphate skeletons of RNA might interact (29Small I.D. Peeters N. Trends Biochem. Sci. 2000; 25: 46-47Abstract Full Text Full Text PDF PubMed Google Scholar). Despite the growing number of PPR proteins discovered and characterized to date, very little is understood about the specific role of the PPR motifs present in these proteins. Yeast Pet309 provides a useful model of a PPR protein to elucidate the mechanism of action of the PPR motifs. Pet309 is predicted to contain at least seven PPR motifs located in the central portion of the protein. To test the function of the repeats present in Pet309, a set of deletions of the PPR motifs was constructed and analyzed. A model of the Pet309 PPR region was generated, and site-directed mutagenesis was carried out on residues that are predicted to be necessary for mRNA binding. It was shown that all the seven PPR repeats present in Pet309 are necessary for COX1 mRNA translation, and that mutation of basic residues that could be facing the inner cavity of the PPR structure decrease Pet309 activity. Surprisingly, the COX1 mRNA levels were not affected by the PPR deletions, showing that the mRNA stability function of Pet309 is independent of the PPR domains. Strains, Media, and Genetic Methods—The S. cerevisiae strains used in this study are listed in Table 1. All strains are derived from strain D273-10B. Genetic manipulation and standard media recipes were as previously described (30Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1988Google Scholar). Yeast were cultured in complete fermentable media (1% yeast extract, 2% Bacto-peptone) or synthetic complete media (0.67% yeast nitrogen base, supplemented with the appropriate amino acids), containing 2% glucose, 2% raffinose or 3% ethanol-3% glycerol. The pet309Δ::LEU2 deletion construct was obtained by PCR. Strains XPM201 and XPM10b were transformed with the PCR product (31Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar), and correct integration of the pet309Δ::LEU2 construct was confirmed by PCR.TABLE 1S. cerevisiae strains used in this studyStrain nameNuclear (mitochondrial) genotypeReferenceXPM232Matα ura3-52 leu2-3, 112 lys2 arg8::hisG pet309Δ::LEU2 (ρ+, ΔΣai)This studyXPM231Matα ura3-52 leu2-3, 112 lys2 arg8::hisG pet309Δ::LEU2 (ρ+, cox1Δ::ARG8m)This studyXPM201Matα ura3-52 leu2-3, 112 lys2 arg8::hisG (ρ+, ΔΣai)This studyXPM10bMatα ura3-52 leu2-3, 112 lys2 arg8::hisG (ρ+, cox1Δ::ARG8m)52Perez-Martinez X. Broadley S.A. Fox T.D. EMBO J. 2003; 22: 5951-5961Crossref PubMed Scopus (153) Google ScholarSB5Matα ura3Δ ade2 PET309::3xHA (ρ+)S. A. Broadley Open table in a new tab Plasmid Constructs—Total DNA from the strain SB5 was used to amplify the PET309::HA sequence, including 310 and 205 bp of the PET309 5′- and 3′-UTR, respectively. This product was ligated into XbaI-XhoI sites of pBluescript to generate plasmid pXP96. In addition, the product was subcloned into the XbaI-XhoI sites of yeast expression vectors pRS416 (32Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) and YEp352 (33Hill J.E. Myers A.M. Koerner T.J. Tzagoloff A. Yeast. 1986; 2: 163-167Crossref PubMed Scopus (1082) Google Scholar) to generate pXP97 and pXP104, respectively. All pet309 mutant sequences were generated by fusion PCR (34Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6833) Google Scholar), using Accuzyme DNA polymerase (Bioline) and pXP96 as the DNA template. The PCR products obtained from the PPR region of pet309 were ligated into PstI-EcoRI pXP96. After sequencing the constructs the complete pet309 genes were subcloned into XbaI-XhoI pXP97 or pXP104 to generate yeast expression plasmids. Analysis of Mitochondrial Proteins—Mitochondria were isolated from late logarithmic phase cells grown on synthetic complete media without uracil, containing 2% raffinose. Crude mitochondria were isolated and purified by centrifugation on 5–25% Nycodenz gradients (35Glick B.S. Pon L.A. Methods Enzymol. 1995; 260: 213-223Crossref PubMed Scopus (287) Google Scholar). Mitochondria separation into membrane and soluble fractions, and alkaline carbonate extractions of membranes were as described (36Glick B.S. Methods Enzymol. 1995; 260: 224-231Crossref PubMed Scopus (56) Google Scholar, 37Fujiki Y. Hubbard A.L. Fowler S. Lazarow P.B. J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1383) Google Scholar, 38Diekert K. de Kroon A.I. Kispal G. Lill R. Methods Cell Biol. 2001; 65: 37-51Crossref PubMed Google Scholar). Mitoplasting and proteinase K treatment were carried out as previously described (38Diekert K. de Kroon A.I. Kispal G. Lill R. Methods Cell Biol. 2001; 65: 37-51Crossref PubMed Google Scholar). Total cellular extracts were isolated from cells grown to mid-log phase on synthetic complete media without uracil, containing 2% raffinose (39Yaffe M.P. Methods Enzymol. 1991; 194: 627-643Crossref PubMed Scopus (150) Google Scholar). Proteins were separated by SDS-PAGE on a 12.5% gel (40Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207218) Google Scholar). Western blots were probed with anti-HA-horseradish peroxidase (Roche Biochemicals), anti-Cox1 (Molecular probes), anti-citrate synthase, anti-Arg8p, anti-Yme1p (the three provided by T. D. Fox), anti-cytochrome c1 (provided by D. González-Halphen) or anti-glucose-6-phosphate dehydrogenase (Sigma) antibodies. Secondary goat anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase (Sigma or Bio-Rad) was detected with the ECL or ECL+ kits (GE Healthcare). Synthesis of Mitochondrial Proteins—Translation in isolated mitochondria in the presence of [35S]methionine was performed as described (41Westermann B. Herrmann J.M. Neupert W. Methods Cell Biol. 2001; 65: 429-438Crossref PubMed Google Scholar). After translation, mitochondria were washed with 0.6 m sorbitol, 20 mm HEPES, pH 7.4, and the radiolabeled proteins were separated on a SDS-PAGE gel, blotted into protran nitrocellulose membrane, and analyzed with a Typhoon 8600 PhosphorImager (Amersham Biosciences). Northern Blot Analyses—Total RNA was prepared using the TRIzol reagent (Invitrogen) from yeast cultures grown to late log phase on raffinose-synthetic complete media lacking uracil. RNA was blotted to Hybond XL membrane (GE Healthcare). Blots were probed sequentially with the radioactively labeled COX1 exon 4, COX2 and with the 15S rRNA gene (42Shen Z.H. Fox T.D. Nucleic Acids Res. 1989; 17: 4535-4539Crossref PubMed Scopus (53) Google Scholar) to standardize the loading. Blots were analyzed with a Typhoon 8600 PhosphorImager and quantitated with ImageQuaNT 5.1 software. Modeling for the PPR Region in Pet309—A search using the TPRpred server (43Karpenahalli M.R. Lupas A.N. Soding J. BMC Bioinformatics. 2007; 8: 2Crossref PubMed Scopus (152) Google Scholar) against the whole Pet309 sequence revealed the presence of 11 putative PPR motifs located between residues 312 and 759. The six motifs with the lowest p value (∼1e-07-1e-06, which indicates 1 × 10-7 to 1 × 10-6) correspond to one segment of the protein comprising residues 347–560. Using this fragment of the sequence, the HHpred server (44Soding J. Biegert A. Lupas A.N. Nucleic Acids Res. 2005; 33 (web server issue): W244-W248Crossref PubMed Scopus (2654) Google Scholar) revealed an alignment with the six tandem TPRs of the crystal structure of PilF (45Kim K. Oh J. Han D. Kim E.E. Lee B. Kim Y. Biochem. Biophys. Res. Commun. 2006; 340: 1028-1038Crossref PubMed Scopus (47) Google Scholar) (PDB code 2ho1) from Pseudomonas aeruginosa, with 10% identity extending over 213 residues. Only five residue insertions of one residue each occur in the alignment, all at the level of the junctions between the repeats. The three-dimensional model of the PPR repeats was constructed using SWISSMODEL (46Schwede T. Kopp J. Guex N. Peitsch M.C. Nucleic Acids Res. 2003; 31: 3381-3385Crossref PubMed Scopus (4506) Google Scholar) alignment interface mode. Fig. 6 was created with PyMOL, and the electrostatic potentials were calculated with the program APBS (47Baker N.A. Sept D. Joseph S. Holst M.J. McCammon J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10037-10041Crossref PubMed Scopus (5877) Google Scholar). The PPR Region of Pet309 Is Necessary for Respiratory Growth—A comparison of the Pet309 sequence against the TPRpred server reveals the presence of 11 PPR motifs in the central portion of the protein. To understand the role of the PPR domain we created a deletion from residues 347 to 632 (pet309Δppr), which corresponds to the 7 most strongly predicted PPR motifs (having the lowest p values) (Fig. 1A). To facilitate detection of Pet309, the protein was tagged at its C terminus with three tandem copies of an HA epitope. The presence of the triple-epitope in wild-type Pet309 did not interfere with the respiratory growth of cells as judged by the ability of PET309-HA to fully complement the pet- phenotype of a pet309Δ::LEU2 mutant. Both the wild-type PET309-HA and the pet309Δppr-HA genes were cloned in the vectors pRS416 and YEp352 to allow expression in yeast in low copy or multiple copy plasmids, respectively. The plasmids were transformed into a yeast pet309Δ::LEU2 mutant, and the respiratory growth of the resulting strains was examined (Fig. 1B). The wild-type PET309-HA supported normal growth on non-fermentable carbon sources, whereas the pet309Δppr-HA strain could not grow on a non-fermentable carbon source. A similar phenotype was observed in cells expressing the single copy or multiple copy expression plasmids, suggesting that overexpression of the mutant protein did not compensate for the absence of the PPR domain of Pet309. To investigate the basis of the non-respiratory phenotype of the pet309Δppr-HA strain we first looked to see if the mutant protein was localized in mitochondria. Mitochondrial and post-mitochondrial supernatant fractions were obtained from strains transformed with the low copy and high copy plasmids bearing the wild-type PET309-HA or the mutant pet309Δppr-HA genes (Fig. 2A). The protein Pet309 was specifically recognized by the anti-HA antibody as a 118-kDa band for the wild-type PET309-HA or 85-kDa band for the pet309Δppr-HA strain. These polypeptides were not detectable in mitochondria bearing the untagged PET309 gene or the empty plasmids (data not shown). Greater accumulation of the Pet309 polypeptides was observed under multiple copy expression. These polypeptides were not detectable in the post-mitochondrial supernatant fractions, indicating that the Pet309Δppr-HA protein co-purified with mitochondria. Next, we investigated whether the Pet309Δppr-HA protein was membrane-bound or soluble. Mitochondria from strains bearing the PET309-HA or the pet309Δppr-HA, low copy plasmids were sonicated and centrifuged. Both the wild-type Pet309-HA and the mutant Pet309Δppr-HA proteins were present in the membrane pellet (Fig. 2B) and absent from the soluble supernatant. Alkaline Na2CO3 extraction of the mitochondrial membranes solubilized the wild-type Pet309-HA and the Pet309Δppr-HA proteins (Fig. 2C), indicating that both behave as peripheral membrane proteins. To examine the submitochondrial location of the Pet309 proteins, purified mitochondria were converted to mitoplasts by osmotic shock treatment and were subjected to protease digestion. Both Pet309-HA and Pet309Δppr-HA proteins were protected from proteinase K treatment in mitochondria and in mitoplasts (Fig. 2D). This result indicates that both proteins are facing the matrix side of the inner membrane. These results are different from what was observed by Manthey (13Manthey G.M. Przybyla-Zawislak B.D. McEwen J.E. Eur. J. Biochem. 1998; 255: 156-161Crossref PubMed Scopus (62) Google Scholar), who reported that Pet309-c-Myc was an integral inner membrane protein. However, in that work, a high copy plasmid was used to overexpress the Pet309-c-Myc protein. Overexpression of translational activators has been associated with problems in mitochondrial gene expression (48Tzschoppe K. Kohlwein S.D. Rodel G. Biol. Chem. 2000; 381: 1175-1183Crossref PubMed Scopus (8) Google Scholar, 49Fiori A. Perez-Martinez X. Fox T.D. Mol. Microbiol. 2005; 56: 1689-1704Crossref PubMed Scopus (24) Google Scholar) and could affect their interaction with the mitochondrial inner membrane. For this reason we analyzed the Pet309-HA proteins expressed from low copy plasmids. Taken together, these results indicate that the respiratory defect of pet309Δppr-HA mutants is not due to a mitochondrial mislocalization of the protein. The association of the mutant protein to the mitochondrial inner membrane and its submitochondrial localization were not altered by the absence of the PPR domains. These observations, together with the capacity of the mutated protein to stabilize the COX1 mRNA (see below) strongly suggest that the Pet309Δppr-HA protein is not misfolded. The PPR Motifs in Pet309 Are Required for Translation of the COX1 mRNA—Pet309 had been previously demonstrated to be necessary for the translation and stability of the COX1 mRNA (11Manthey G.M. McEwen J.E. EMBO J. 1995; 14: 4031-4043Crossref PubMed Scopus (179) Google Scholar). We investigated the effect of the PPR domain deletion on expression of the COX1 gene. Western blot analysis of mitochondrial protein extracts showed no accumulation of the Cox1 protein in the pet309Δppr-HA mutant (Fig. 3A). The mutant did not accumulate Cox1 even in pet309Δppr-HA high copy expression. Interestingly, overexpression of the wild-type Pet309-HA led to a substantial decrease in the Cox1 accumulation (3.5-fold). This observation is in agreement with the idea that overexpression of translational activators can lead to defects on the biogenesis of their target genes (49Fiori A. Perez-Martinez X. Fox T.D. Mol. Microbiol. 2005; 56: 1689-1704Crossref PubMed Scopus (24) Google Scholar). Overexpression of Pet309 could lead to formation of inactive Pet309 aggregates that could affect accumulation of Cox1 (48Tzschoppe K. Kohlwein S.D. Rodel G. Biol. Chem. 2000; 381: 1175-1183Crossref PubMed Scopus (8) Google Scholar). To investigate the effect of the pet309Δppr-HA mutation on COX1 translation, we first analyzed [35S]methionine-labeled proteins from mitochondria carrying the pet309Δppr-HA mutation in low copy or high copy expression plasmids (Fig. 3B). Labeling of Cox1 was reduced to undetectable levels by the pet309Δppr-HA mutation even in overexpression conditions. As expected, labeling of Cox1 in strains with the wild-type PET309-HA was normal, whereas a null mutation (pet309Δ) completely prevented Cox1 labeling. These results suggest that the PPR domain of Pet309 is necessary for the COX1 mRNA translation. To corroborate this, we created a pet309Δ::LEU2 strain in which the mitochondrial reporter gene ARG8m replaced the COX1 coding sequence (cox1Δ::ARG8m). The ARG8m product is a matrix-soluble protein involved in arginine biosynthesis (50Steele D.F. Butler C.A. Fox T.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5253-5257Crossref PubMed Scopus (151) Google Scholar). This reporter has been widely used to analyze translation of mitochondrial genes (49Fiori A. Perez-Martinez X. Fox T.D. Mol. Microbiol. 2005; 56: 1689-1704Crossref PubMed Scopus (24) Google Scholar, 51Bonnefoy N. Bsat N. Fox T.D. Mol. Cell Biol. 2001; 21: 2359-2372Crossref PubMed Scopus (53) Google Scholar, 52Perez-Martinez X. Broadley S.A. Fox T.D. EMBO J. 2003; 22: 5951-5961Crossref PubMed Scopus (153) Google Scholar, 53Williams E.H. Fox T.D. RNA. 2003; 9: 419-431Crossref PubMed Scopus (18) Google Scholar). Translation of cox1Δ::ARG8m has been demonstrated to be dependent upon Pet309 (52Perez-Martinez X. Broadley S.A. Fox T.D. EMBO J. 2003; 22: 5951-5961Crossref PubMed Scopus (153) Google Scholar). In cells carrying the wild-type PET309-HA, the cox1Δ::ARG8m gene supported growth in Argmedium (Fig. 3C). In contrast, cells bearing the high copy or low copy (data not shown) pet309Δppr-HA gene required arginine to grow, confirming that the PPR domain present in Pet309 is necessary for the COX1 mRNA translation. The PPR Motifs in Pet309 Are Not Required for Stabilization of the COX1 mRNA—Pet309 is also involved in the COX1 mRNA stability, as null mutants show a reduced accumulation of the mature COX1 mRNA (11Manthey G.M. McEwen J.E. EMBO J. 1995; 14: 4031-4043Crossref PubMed Scopus (179) Google Scholar). This effect is particularly strong when the COX1 gene has introns, but it is also observed with the intronless COX1 gene (11Manthey G.M. McEwen J.E. EMBO J. 1995; 14: 4031-4043Crossref PubMed Scopus (179) Google Scholar). We analyzed whether deletion of the PPR repeats present in Pet309 could affect the COX1 mRNA accumulation. Levels of the COX1 mRNA in cells bearing the intronless COX1 gene were analyzed by Northern blot and normalized to the mitochondrial 15S rRNA (Fig. 4). In wild-type cells expressing the high copy PET309-HA gene, the COX1 mRNA signal was increased 2-fold as compared with the low copy PET309-HA cells. A similar pattern was obtained for the pet309Δppr-HA cells. This effect was specific for COX1, because the COX2 mRNA levels were not affected in any sample. It has been suggested that high levels of translational activators could stabilize their target mRNAs (49Fiori A. Perez-Martinez X. Fox T.D. Mol. Microbiol. 2005; 56: 1689-1704Crossref PubMed Scopus (24) Google Scholar). This result indicates that Pet309 lacking the PPR repeats still has the capacity to stabilize the COX1 mRNA. In contrast, the null pet309 mutant showed a reduced accumulation of the COX1 mRNA as compared with the PET309-HA or the pet309Δppr-HA cells. We conclude that the PPR domains present in Pet309 are necessary for translation of the COX1 mRNA. However, the absence of these repeats does not affect the COX1 mRNA stability. Moreover, high expression of the pet309Δppr-HA protein caused accumulation of the COX1 mRNA, as observed for the wild-type Pet309-HA protein. Each One of the Seven PPR Repeats
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