A “Petite Obligate” Mutant of Saccharomyces cerevisiae
2006; Elsevier BV; Volume: 281; Issue: 24 Linguagem: Inglês
10.1074/jbc.m513805200
ISSN1083-351X
AutoresStéphane Duvezin‐Caubet, Malgorzata Rak, Linnka Lefebvre‐Legendre, Emmanuel Tétaud, Nathalie Bonnefoy, Jean‐Paul di Rago,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoWithin the mitochondrial F1F0-ATP synthase, the nucleus-encoded δ-F1 subunit plays a critical role in coupling the enzyme proton translocating and ATP synthesis activities. In Saccharomyces cerevisiae, deletion of the δ subunit gene (Δδ) was shown to result in a massive destabilization of the mitochondrial genome (mitochondrial DNA; mtDNA) in the form of 100% ρ–/ρ° petites (i.e. cells missing a large portion (>50%) of the mtDNA (ρ–) or totally devoid of mtDNA (ρ°)). Previous work has suggested that the absence of complete mtDNA (ρ+) in Δδ yeast is a consequence of an uncoupling of the ATP synthase in the form of a passive proton transport through the enzyme (i.e. not coupled to ATP synthesis). However, it was unclear why or how this ATP synthase defect destabilized the mtDNA. We investigated this question using a nonrespiratory gene (ARG8m) inserted into the mtDNA. We first show that retention of functional mtDNA is lethal to Δδ yeast. We further show that combined with a nuclear mutation (Δatp4) preventing the ATP synthase proton channel assembly, a lack of δ subunit fails to destabilize the mtDNA, and ρ+ Δδ cells become viable. We conclude that Δδ yeast cannot survive when it has the ability to synthesize the ATP synthase proton channel. Accordingly, the ρ–/ρ° mutation can be viewed as a rescuing event, because this mutation prevents the synthesis of the two mtDNA-encoded subunits (Atp6p and Atp9p) forming the core of this channel. This is the first report of what we have called a "petite obligate" mutant of S. cerevisiae. Within the mitochondrial F1F0-ATP synthase, the nucleus-encoded δ-F1 subunit plays a critical role in coupling the enzyme proton translocating and ATP synthesis activities. In Saccharomyces cerevisiae, deletion of the δ subunit gene (Δδ) was shown to result in a massive destabilization of the mitochondrial genome (mitochondrial DNA; mtDNA) in the form of 100% ρ–/ρ° petites (i.e. cells missing a large portion (>50%) of the mtDNA (ρ–) or totally devoid of mtDNA (ρ°)). Previous work has suggested that the absence of complete mtDNA (ρ+) in Δδ yeast is a consequence of an uncoupling of the ATP synthase in the form of a passive proton transport through the enzyme (i.e. not coupled to ATP synthesis). However, it was unclear why or how this ATP synthase defect destabilized the mtDNA. We investigated this question using a nonrespiratory gene (ARG8m) inserted into the mtDNA. We first show that retention of functional mtDNA is lethal to Δδ yeast. We further show that combined with a nuclear mutation (Δatp4) preventing the ATP synthase proton channel assembly, a lack of δ subunit fails to destabilize the mtDNA, and ρ+ Δδ cells become viable. We conclude that Δδ yeast cannot survive when it has the ability to synthesize the ATP synthase proton channel. Accordingly, the ρ–/ρ° mutation can be viewed as a rescuing event, because this mutation prevents the synthesis of the two mtDNA-encoded subunits (Atp6p and Atp9p) forming the core of this channel. This is the first report of what we have called a "petite obligate" mutant of S. cerevisiae. The mitochondrial inner membrane contains the ATP synthase, which utilizes a transmembrane proton gradient to catalyze ATP synthesis from inorganic phosphate and ADP. The ATP synthase has two major structural domains, an F0 component, which forms a proton-permeable pore across the membrane, and a peripheral, matrix-localized, F1 component, where the ATP is synthesized (1Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1595) Google Scholar). The F1 domain comprises five different subunits, all nucleus-encoded, with α3β3γ1δ1ϵ1 stoichiometry. The three α subunits and the three β subunits alternate in position within a hexamer that contains the adenine nucleotide processing sites (2Abrahams J.P. Leslie A.G. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2753) Google Scholar, 3Bianchet M.A. Hullihen J. Pedersen P.L. Amzel L.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11065-11070Crossref PubMed Scopus (224) Google Scholar, 4Stock D. Leslie A.G. Walker J.E. Science. 1999; 286: 1700-1705Crossref PubMed Scopus (1087) Google Scholar). The γ, δ, and ϵ subunits form a subcomplex of F1, named the central stalk, linking the α3β3 subcomplex to the ATP synthase proton channel (5Gibbons C. Montgomery M.G. Leslie A.G.W. Walker J.E. Nat. Struct. Biol. 2000; 7: 1055-1061Crossref PubMed Scopus (436) Google Scholar). During catalysis, the central stalk rotates together with a transmembrane ring of 10–12 c subunits in the F0 (6Fillingame R.H. Nat. Struct. 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In the course of this rotation, the γ subunit sequentially interacts with the three αβ pairs in a way that favors ATP synthesis in the catalytic sites, as required by the binding change mechanism (1Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1595) Google Scholar). In the yeast Saccharomyces cerevisiae, null mutations of the δ or γ subunit massively destabilize the mitochondrial genome in the form of 100% cytoplasmic petites, which are cells with a large (>50%) deletion in mitochondrial DNA (mtDNA) 6The abbreviations used are: mtDNA, mitochondrial DNA; Δδ, deletion of the S. cerevisiae ATP16 gene, encoding ATP synthase subunit δ; DASPMI, 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide; CCCP, carbonyl cyanide m-chlorophenylhydrazone. (ρ–) or completely lacking mtDNA (ρ°) (13Giraud M.-F. Velours J. Eur. J. Biochem. 1994; 245: 813-818Crossref Scopus (69) Google Scholar, 14Lai-Zhang J. Xiao Y. Mueller D.M. EMBO J. 1999; 18: 58-64Crossref PubMed Scopus (56) Google Scholar). This is an intriguing observation, since many other mutations impairing mitochondrial oxidative phosphorylation, including a null mutation of the catalytic β subunit of the ATP synthase, have no major effect on the stability of the mitochondrial genome (14Lai-Zhang J. Xiao Y. Mueller D.M. EMBO J. 1999; 18: 58-64Crossref PubMed Scopus (56) Google Scholar). Defects in the synthesis of the δ or γ subunit were shown to result in isolated mitochondria in a major uncoupling of mitochondrial respiration mediated by partial F1F0 assemblies in which proton translocation is not coupled to ATP synthesis in the F1 (15Xiao Y. Metzl M. Mueller D.M. J. Biol. Chem. 2000; 275: 6963-6968Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 16Duvezin-Caubet S. Caron M. Giraud M.-F Velours J. di Rago J.-P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13235-13240Crossref PubMed Scopus (44) Google Scholar). In media containing oligomycin, a specific inhibitor of the ATP synthase assumed to block the enzyme proton channel, mtDNA maintenance could be observed in growing Δδ or Δγ yeast, providing in vivo evidence that an ATP synthase uncoupling may be the cause of the loss of mtDNA in cells unable to properly express the δ or γ subunit (16Duvezin-Caubet S. Caron M. Giraud M.-F Velours J. di Rago J.-P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13235-13240Crossref PubMed Scopus (44) Google Scholar, 17Mueller D.M. J. Bioenerg. Biomembr. 2000; 32: 391-400Crossref PubMed Scopus (33) Google Scholar). However, how the ATP synthase uncoupling caused by the absence of the δ or γ subunit destabilizes the mtDNA is still unknown. It has been hypothesized that the loss of the mtDNA allows Δδ or Δγ yeast to survive by eliminating the ATP synthase F0 component, which is in part encoded by the mtDNA (16Duvezin-Caubet S. Caron M. Giraud M.-F Velours J. di Rago J.-P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13235-13240Crossref PubMed Scopus (44) Google Scholar, 17Mueller D.M. J. Bioenerg. Biomembr. 2000; 32: 391-400Crossref PubMed Scopus (33) Google Scholar). The idea is that the F0-mediated mitochondrial uncoupling in Δδ or Δγ yeast would be lethal by preventing essential mitochondrial reactions from occurring. As a consequence, Δδ or Δγ yeast would need to inactivate the ATP synthase proton channel in order to survive. However, in apparent contradiction with this idea, although a lack in the third central stalk subunit (ϵ) also results in a major F0-mediated mitochondrial uncoupling, only partial effects were observed on the mtDNA in Δϵ mutant (18Guelin E. Chevallier J. Rigoulet M. Guerin B. Velours J. J. Biol. Chem. 1993; 268: 161-167Abstract Full Text PDF PubMed Google Scholar). Indeed, cultures of this mutant usually contain 30% ρ+ cells, showing that retention of functional mtDNA in Δϵ yeast is not lethal. Furthermore, it has been reported that a lack in the δ subunit is viable in Kluyveromyces lactis, a "petite negative" yeast that cannot survive the ρ–/ρ° mutation (19Hansbro P.M. Clark-Walker Chen X. J.G.D. Curr. Genet. 1998; 33: 46-51Crossref PubMed Scopus (6) Google Scholar). These observations raise the possibility that destabilization of the mtDNA in Δδ and Δγ S. cerevisiae strains results from an increase in the frequency of the ρ–/ρ° mutation rather than from the impossibility of these mutants surviving when they contain functional mtDNA. Fox and co-workers (20Steele D.F. Butler C.A. Fox T.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5253-5257Crossref PubMed Scopus (151) Google Scholar) have developed elegant approaches for the study of mitochondria based on the insertion into the mtDNA of nonrespiratory genes like ARG8m. This gene is a mitochondrial version of the nuclear ARG8 gene encoding a mitochondrial protein involved in arginine biosynthesis. In this study, we have used ARG8m to better understand why and how a lack in δ subunit destabilizes the mtDNA. To this end, we have inserted ARG8m into a noncoding region of the mitochondrial genome (i.e. keeping intact all of the genes that normally reside in yeast mitochondria). Thus, in a Δarg8 nucleus and in the absence of external arginine, this mtDNA is required not only for respiration but also for arginine biosynthesis. With this system, we clearly show that the ATP synthase proton translocating activity is lethal to yeast cells missing the δ subunit and demonstrate that the ρ–/ρ° mutation is a suppressor allowing survival of Δδ yeast. This is the first report of what we have called a "petite obligate" mutant. We discuss the results in relation with potential mechanisms regulating the assembly of the ATP synthase. Strains, Media, and Genetic Techniques—The S. cerevisiae strains used are listed in Table 1. Escherichia coli XL1-Blue strain (Stratagene) was used for the cloning and propagation of plasmids. Complete glucose (YPGA), galactose (YPGALA), or glycerol (N3) and minimal media for growing yeast were prepared as described in Ref. 22Lefebvre-Legendre L. Balguerie A. Duvezin-Caubet S. Giraud M.-F. Slonimski P.P. di Rago J.-P. Mol. Microbiol. 2003; 47: 1329-1339Crossref PubMed Scopus (38) Google Scholar. The yeast sporulation medium and the procedure for converting yeast into ρ° strains by ethidium bromide treatment have been described (27di Rago J.P. Netter P. Slonimski P.P. J. Biol. Chem. 1990; 265: 15750-15757Abstract Full Text PDF PubMed Google Scholar). Yeast transformation (28Gietz D. St. Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425-1426Crossref PubMed Scopus (2894) Google Scholar), crosses, and tetrad dissection (29Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics: A Laboratory Course Manual. 1990; (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)Google Scholar) were performed as described previously.TABLE 1Yeast strains used in this studyStrainNuclear genotype, plasmidMtDNAOriginKL14-4A/60Mata his1 trp2ρ°SlonimskiDFS160Matα leu2Δ ura3-52 ade2-101 arg8ΔURA3 kar1-1ρ°Ref. 20Steele D.F. Butler C.A. Fox T.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5253-5257Crossref PubMed Scopus (151) Google ScholarNB40-3CMata lys2 leu2-3,112 ura3-52 his3ΔHinDIII arg8::hisGρ+cox2-62Ref. 20Steele D.F. Butler C.A. Fox T.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5253-5257Crossref PubMed Scopus (151) Google ScholarNB151-1BMata pet111::LEU2 lys2 ura3-52 his3ΔHinDIII arg8::hisG leu2ρ°Ref. 20Steele D.F. Butler C.A. Fox T.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5253-5257Crossref PubMed Scopus (151) Google ScholarPVY10/60Matα atp4ΔURA3 his3 met6 ura3ρ°J. VeloursSDC12Matα/ Mat a lys2/+ leu2Δ/ leu2-3,112 ura3-52/ura3-52 ade2-101/+ his3ΔHinDIII/+ arg8ΔURA3/arg8::hisG kar1-1/+ρ+ARG8mThis studySDC13Matα/ Mata atp16ΔKANR/ + lys2/ + leu2Δ/ leu2-3,112 ura3-52/ura3-52 ade2-101/+ his3ΔHinDIII/+ arg8ΔURA3/arg8::hisG kar1-1/+ρ+ARG8mThis studySDC15Matα/ Mata lys2/+ leu2Δ/ leu2-3,112 ura3-52/ura3-52 ade2-101/+ his3ΔHinDIII/+ arg8ΔURA3/arg8::hisG kar1-1/+ρ+This studySDC22MATα ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 can1-100 arg8::HIS3ρ+ARG8mRef. 16Duvezin-Caubet S. Caron M. Giraud M.-F Velours J. di Rago J.-P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13235-13240Crossref PubMed Scopus (44) Google ScholarSDC3Matα/ Mata pet111::LEU2/+ lys2/lys2 leu2/? ura3-52/ura3-52 ade2-101/+ his3ΔHinDIII/ his3ΔHinDIII arg8ΔURA3/arg8::hisGρ+ARG8mThis studySDC12-4BMatα lys2 leu2 ura3-52 his3ΔHinDIII ade2-101 arg8ΔURA3ρ+ARGmThis studySDC12-21AMata lys2 leu2 ura3-52 his3ΔHinDIII arg8::hisGρ+ARGmThis studySDC13-14B/60Mata atp16::KANR lys2 leu2 ura3-52 his3ΔHinDIII arg8::hisGρ°This studySDC16-7CMatα atp4ΔURA3 ura3 his3 met6 arg8::hisGρ+ARG8mThis studySDC17-31BMatα atp4ΔURA3 atp16ΔKANR lys2 ura3 his3 met6 arg8::hisGρ+ARG8mThis studySDC29-10CMATa arg8::HIS3 atp16::KanMX4 atp4::TRP1 ade2-1 ura3-1 his3-11,15 leu2-3,112 trp1 can1-100, pSDC13ρ+ARG8mThis studySDC33arg8::HIS3 atp16::KanMX4 atp4::TRP1, pSDC13, pSDC20ρ+ARG8mThis studyYSE15a/60MATa ade2-1 his3-11,15 Δtrp1 leu2-3,112 ura3-1 atp4::TRP1 can1-100ρ°This studySDC6MATα ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 arg8::HIS3 atp16::KANR can1-100, pSDC13ρ+ARG8mRef. 16Duvezin-Caubet S. Caron M. Giraud M.-F Velours J. di Rago J.-P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13235-13240Crossref PubMed Scopus (44) Google Scholar Open table in a new tab Construction of a COX2-ARG8m Transcriptional Unit and Its Insertion Upstream of COX2 in the mtDNA—To create an ARG8m construct that could direct integration into intergenic regions of the mtDNA, we took advantage of the EcoRI site engineered on plasmid pPT24, 285 bases upstream of the COX2 start codon (i.e. upstream of the promoter) (23Thorsness P.E Fox T.D. Genetics. 1993; 134: 21-28Crossref PubMed Google Scholar). Using standard cloning procedures and two-step PCR strategies (24Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 61-68Crossref PubMed Scopus (2640) Google Scholar) with pPT24 (containing COX2) and pDS24 (containing ARG8m) (20Steele D.F. Butler C.A. Fox T.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5253-5257Crossref PubMed Scopus (151) Google Scholar) as templates, we constructed an EcoRI cassette containing the ARG8m open reading frame precisely flanked on the 5′ side by 143 bp of the COX2 promoter region and on the 3′ side by 119 bp of the COX2 terminator. This ARG8m cassette was inserted into the EcoRI site of pPT24 in the forward orientation to yield plasmid pSDC10. The initial COX2 locus of pPT24 (from 485 bases upstream to 2015 bases downstream of the start codon) is therefore separated in two parts in pSDC10, the region –485 to –285 before the first EcoRI site and the region –285 to +2015 after the ARG8m cassette and the second EcoRI site (see Fig. 1A). The plasmid pSDC10 was introduced by co-transformation with the nuclear selectable LEU2 plasmid pFL46 into the ρ° strain DFS160 by microprojectile bombardment using a biolistic PDS-1000/He particle delivery system (Bio-Rad) as described (25Bonnefoy N. Fox T.D. Methods Cell Biol. 2001; 65: 381-396Crossref PubMed Google Scholar). Mitochondrial transformants were identified among the Leu+ nuclear transformants by their ability to produce both respiring and arginine-prototrophic diploid clones when mated to the nonrespiring and arginine auxothrophic NB40-3C strain, bearing deletions in both the mitochondrial COX2 gene (cox2-62) and in the nuclear ARG8 gene. The DNA recombination events leading to the simultaneous integration of ARG8m and COX2 in NB40-3C mtDNA are illustrated in Fig. 1A. One respiratory growth-competent and arginine-prototrophic clone (called SDC12) was retained for further analyses, and the ARG8m integration was verified molecularly by Southern analysis and sequencing. A control diploid strain, SDC15, isogenic to SDC12 but carrying the wild-type mtDNA was constructed by crossing DFS160 with NB80, the COX2 equivalent of NB40-3C (26Bonnefoy N. Fox T.D. Mol. Gen. Genet. 2000; 262: 1036-1046Crossref PubMed Scopus (61) Google Scholar). Construction of ARG8m Strains Carrying atp16 (δ) and/or atp4 Disruptions—We first created a diploid strain (SDC13) heterozygous for a null mutation of the ATP synthase δ subunit gene (Δδ), homozygous for a null mutation of ARG8 (Δarg8) and containing the ARG8m mtDNA (Δδ/+Δarg8/Δarg8 [ρ+ ARG8m]), by deleting one of the two δ subunit gene copies in SDC12 with the atp16::KanMX4 cassette described previously (16Duvezin-Caubet S. Caron M. Giraud M.-F Velours J. di Rago J.-P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13235-13240Crossref PubMed Scopus (44) Google Scholar). A Δδ Δarg8 [ρ+ ARG8m] spore derived from SDC13 was converted to ρ° by ethidium bromide treatment (SDC13-14B/60) and crossed with strain SDC16-7C (Δatp4 Δarg8 [ρ+ ARG8m]), a spore derived from the cross of PVY10/60 (Δatp4 ARG8 ρ°) with a spore of SDC12 (SDC12–21A: Δarg8 [ρ+ ARG8m]). The resulting diploid was sporulated to generate the haploid strain SDC17-31B (Δδ Δatp4 Δarg8 [ρ+ ARG8m]). Construction of a Strain Expressing Atp4p and the δ Subunit from Regulatable Promoters—The ATP4 gene from strain BMA64-1B was precisely replaced by the TRP1 marker, using the PCR strategy already described (30Baudin-Baillieu A. Guillemet E. Cullin C. Lacroute F. Yeast. 1997; 13: 353-356Crossref PubMed Scopus (55) Google Scholar). One Δatp4 transformant was converted to ρ°, and its mating type was changed using a plasmid-borne HO gene to give YSE15/60. This Δatp4[ρ°] strain was crossed with SDC6, a W303-based Δδ Δarg8 [ρ+ ARG8m] strain containing a plasmid (pSDC13) expressing subunit δ under the control of a doxycycline-repressible promoter (16Duvezin-Caubet S. Caron M. Giraud M.-F Velours J. di Rago J.-P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13235-13240Crossref PubMed Scopus (44) Google Scholar). The resulting diploid was sporulated, and a Δarg8 Δatp4 Δδ spore containing pSDC13 and the ARG8m mtDNA was isolated (SDC29-10c). This strain was finally transformed with plasmid pSDC20, a pAS24-borne plasmid containing ATP4 cloned as a BamHI-PstI fragment under the control of the GAL10 promoter, to give SDC33. Biochemistry and Cell Biology Techniques—Mitochondrial protein synthesis analysis was as described in Ref. 48Lefebvre-Legendre L. Vaillier J. Benabdelhak H. Velours J.Slonimski di Rago P. P.J.P. J. Biol. Chem. 2001; 276: 6789-6796Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar. Oxygen consumption rates were measured with a Clark electrode in the growth medium as described previously (31Rigoulet M. Guerin B. FEBS Lett. 1979; 102: 18-22Crossref PubMed Scopus (65) Google Scholar). SDS-PAGE was done according to Laemmli (32Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207200) Google Scholar). Western blot analyses were performed as described previously (33Arselin G. Vaillier J. Graves P.V. Velours J. J. Biol. Chem. 1996; 271: 20284-20290Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Polyclonal antibodies raised against yeast Atp4p, subunit δ, Atp9p, and Atp6p were used after 1:10,000 dilutions, and those against Arg8p were used after a 1:2000 dilution. Nitrocellulose membranes were incubated with peroxidase-labeled antibodies at a 1:10,000 dilution and revealed with the ECL reagent of Amersham Biosciences. Epifluorescence microscopy of DASPMI-stained cells was carried out with a Leica DMRXA microscope fitted with a ×100 immersion objective and a standard fluorescein isothiocyanate filter. Acetylornithine aminotransferase (Arg8p) is a nuclear encoded mitochondrial protein involved in ornithine and arginine biosynthesis (34Jauniaux J.C. Urrestarazu L.A. Wiame J.M. J. Bacteriol. 1978; 133: 1096-1107Crossref PubMed Google Scholar, 35Heimberg H. Boyen A. Crabeel M. Glansdorff N. Gene (Amst.). 1990; 90: 69-78Crossref PubMed Scopus (49) Google Scholar). Although it is normally synthesized in the cytosol and then imported into mitochondria, Fox and co-workers (20Steele D.F. Butler C.A. Fox T.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5253-5257Crossref PubMed Scopus (151) Google Scholar, 36He S. Fox T.D. Mol. Biol. Cell. 1997; 8: 1449-1460Crossref PubMed Scopus (157) Google Scholar, 37Bonnefoy N. Bsat N. Fox T.D. Mol. Cell. Biol. 2001; 21: 2359-2372Crossref PubMed Scopus (53) Google Scholar, 38Perez-Martinez X. Broadley S.A. Fox T.D. EMBO J. 2003; 22: 5951-5961Crossref PubMed Scopus (153) Google Scholar) have shown that Arg8p can be synthesized as well directly inside the mitochondrion from a recoded gene, ARG8m, inserted into the mtDNA. In the present study, we have inserted ARG8m in the intergenic region upstream of the COX2 gene, using a protocol described by Mireau et al. (39Mireau H. Arnal N. Fox T.D. Mol. Genet. Genomics. 2003; 270: 1-8Crossref PubMed Scopus (15) Google Scholar). The ARG8m open reading frame was flanked with the 5′- and 3′-untranslated regions of COX2 and then integrated into the mtDNA by homologous recombination (see "Materials and Methods" and Fig. 1A). We have isolated in this way a diploid strain (SDC12) with the expected integration of ARG8m in mtDNA and homozygous for a null mutation in ARG8 (Δarg8/Δarg8 [ρ+ ARG8m]). SDC12 accumulated normal levels of Arg8p (Fig. 1B), grew well on media devoid of arginine, and was respiratory competent (Fig. 1C). As expected, arginine was required for SDC12 to grow in the presence of ethidium bromide, an intercalating agent inducing the loss of mtDNA (Fig. 1C). The mtDNA in SDC12 showed a good stability. Indeed, after about 15 generations in complete 10% glucose (i.e. conditions where arginine biosynthesis and oxidative phosphorylation are not required for growth), over 98% of the cells were still respiratory competent and arginine-prototrophic. A good mtDNA stability was also observed in SDC12 meiotic segregants. In SDC12, the ARG8m gene is under control of the COX2 5′-untranslated region. Therefore, ARG8m expression should depend on Pet111p, a nucleus-encoded Cox2p translational activator (40Dunstan H.M. Green-Willms N.S. Fox T.D. Genetics. 1997; 147: 87-100Crossref PubMed Google Scholar, 41Green-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). To make sure that this was the case, an SDC12 meiotic segregant (SDC12-4B) was crossed with a ρ° Δpet111 strain (NB151-3C Δarg8 pet111::LEU2), to give SDC3 (Δarg8/Δarg8 Δpet111/+ [ρ+ ARG8m]). All the spores from SDC3 are necessarily Δarg8 and should therefore not grow in media lacking arginine if they cannot express ARG8m. As expected, the ascii usually contained two spores, both respiratory growth-deficient and leucine-prototrophic, as a result of the pet111 deletion (not shown). These spores were also arginine-auxotrophic. Following their transformation with a plasmid-borne wild type PET111 gene, respiratory competence and arginine prototrophy were recovered, demonstrating the requirement for Pet111p to express ARG8m. Elimination of the δ subunit gene in S. cerevisiae always results in 100% ρ–/ρ° populations, and evidence suggests that this conversion into petites is caused by an uncoupling of the ATP synthase (13Giraud M.-F. Velours J. Eur. J. Biochem. 1994; 245: 813-818Crossref Scopus (69) Google Scholar, 14Lai-Zhang J. Xiao Y. Mueller D.M. EMBO J. 1999; 18: 58-64Crossref PubMed Scopus (56) Google Scholar, 15Xiao Y. Metzl M. Mueller D.M. J. Biol. Chem. 2000; 275: 6963-6968Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 16Duvezin-Caubet S. Caron M. Giraud M.-F Velours J. di Rago J.-P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13235-13240Crossref PubMed Scopus (44) Google Scholar, 17Mueller D.M. J. Bioenerg. Biomembr. 2000; 32: 391-400Crossref PubMed Scopus (33) Google Scholar). Below, we describe experiments we performed with the ARG8m system to determine how this ATP synthase defect impairs mtDNA maintenance. Δδ/+ Yeast Is Genetically Unstable—We first aimed to analyze the segregation of the δ subunit deletion (atp16::KanMX4) and ARG8m markers upon sporulation of SDC13, a diploid heterozygous for the δ deletion, homozygous for Δarg8, and containing the ARG8m mtDNA. It has been reported that diploid strains with a heterozygous mutation in the δ subunit gene (i) have an increased propensity to produce ρ–/ρ° petites (30–40%), (ii) grow slowly on respiratory substrates, and (iii) exhibit partially uncoupled mitochondria, indicating that the reduced δ subunit gene dosage from 2 to 1 has semidominant negative effects (15Xiao Y. Metzl M. Mueller D.M. J. Biol. Chem. 2000; 275: 6963-6968Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). To see whether such defects occurred also in our Δδ/+ SDC13 strain, SDC13 cells were plated for single colonies and incubated for 6 days on glucose plates (Fig. 2). Very small size colonies, probably corresponding to ρ–/ρ° cells were observed, but in a rather limited proportion (less than 10%). Two other distinct types of colonies were observed. One (named SDC13-G), representing about 20% of the population, consisted of grande colonies like those formed by the corresponding wild type (SDC12). The other type (SDC13-I), which was the most frequent (70%), consisted of colonies with an intermediate size, suggesting a reduced but not complete loss of respiratory capacity. In addition, these colonies had a scalloped shape. This trait is typical of strains with an increased propensity to produce ρ–/ρ° petites (47Ephrussi B. Hottinguer H. Chimenes Y. Ann. Inst. Pasteur. 1949; 76: 351-367Google Scholar), but it could also reflect a reduced cell viability. When SDC13-I subclones were plated again for single colonies, a similar colonial heterogeneity was observed, whereas SDC13-G subclones gave essentially grande colonies. Similar observations were made with other, genetically independent, SDC13 isolates (all checked by Southern blot). Sporulation and Tetrad Analysis of Δδ/+ ARG8m Yeast—One SDC13-I subclone and one SDC13-G subclone were sporulated, and tetrads were dissected on rich glucose plates. In the case of SDC13-I, a rather large proportion of the tetrads (42%, n = 40; see Table 2) were incomplete with only two or three viable spores. A lower proportion of incomplete tetrads (20%, n = 20, Table 2) was obtained from SDC13-G. A number of complete tetrads showing a correct segregation of the mating types was analyzed, 11 for SDC13-G and nine for SDC13-I. Geneticin resistance segregated 2:2 in all SDC13-G tetrads. However, only two tetrads showed the expected co-segregation of Geneticin resistance with respiratory growth deficiency. In the nine other SDC13-G tetrads, all spores were respiratory competent. In the case of SDC13-I, four tetrads showed the expected 2:2 co-segregation of Geneticin resistance and respiratory deficiency. In three other tetrads, all spores grew on glycerol and were Geneticin-sensitive. In the remaining two tetrads, Geneticin resistance segregated 2:2, but all spores were respiratory competent.TABLE 2Tetrad analyses of heterozygous Δδ/+ yeastStrainPhenotypes in complete tetradsNumber of analyzed tetrads4 Gly+ G418s2 Gly- G418r 2 Gly+ G418s2 Gly+ G418r 2 Gly+ G418sGly+G418r%%SDC121010010SDC13-I34277339SDC13-G029915011 Open table in a new tab All spores from SDC13 are necessarily Δarg8 and depend therefore on their capacity to remain ρ+ and to express ARG8m in order to proliferate in media lacking arginine. With no exception, all of the spores that were at the same time respiratory deficient and Geneticin-resistant, thus presumably deleted for the δ subunit, were arginine-auxotrophic. Test crosses with a ρ° tester revealed that the early progenies of these spores were entirely composed of ρ–/ρ° cells. Not surprisingly, all of the spores that were respiratory competent were able to grow in media lacking arginine. Tetrads from SDC13-I were also dissected on a glucose medium lacking arginine. Many were incomplete, but this was seen as well, although to a lesser extent, with ascii from the wild type SDC12 strain. It might be that germination on minimal medium is less efficient. However, significantly, none of the SDC13-I spores that germinated in the absence of external arginine was Δδ. Altogether, the results of this first set of experiments indicated that Δδ cells were strictly unable to propagate/express the mtDNA or that they were not viable when th
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