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• W2135457913 abstract "Perturbation of cardiolipin (CL) synthesis in yeast cells leads to defective respiratory chain function and mitochondrial DNA loss, both of which have been implicated in aging in mammals. The availability of yeast CL mutants enabled us to directly investigate the role of CL synthesis in aging. In this report, we show that the replicative life span of pgs1Δ, which lacks both CL and the precursor phosphatidylglycerol (PG), was significantly decreased at 30 °C. The life span of crd1Δ, which has PG but not CL, was unaffected at 30 °C but reduced at 37 °C. Life span extension induced by calorie restriction was not affected by the loss of CL. However, mild heat and osmotic stress, which extend life span in wild type cells, did not increase longevity in CL mutants, suggesting that the stress response is perturbed in these mutants. Consistent with this, longevity defects in pgs1Δ were alleviated by down-regulation of the high osmolarity glycerol stress response pathway, as well as by promoting cell integrity with the osmotic stabilizer sorbitol or via genetic suppression with the kre5W1166X mutant. These findings show for the first time that perturbation of CL synthesis leads to decreased longevity in yeast, which is restored by altering signaling through stress response pathways. Perturbation of cardiolipin (CL) synthesis in yeast cells leads to defective respiratory chain function and mitochondrial DNA loss, both of which have been implicated in aging in mammals. The availability of yeast CL mutants enabled us to directly investigate the role of CL synthesis in aging. In this report, we show that the replicative life span of pgs1Δ, which lacks both CL and the precursor phosphatidylglycerol (PG), was significantly decreased at 30 °C. The life span of crd1Δ, which has PG but not CL, was unaffected at 30 °C but reduced at 37 °C. Life span extension induced by calorie restriction was not affected by the loss of CL. However, mild heat and osmotic stress, which extend life span in wild type cells, did not increase longevity in CL mutants, suggesting that the stress response is perturbed in these mutants. Consistent with this, longevity defects in pgs1Δ were alleviated by down-regulation of the high osmolarity glycerol stress response pathway, as well as by promoting cell integrity with the osmotic stabilizer sorbitol or via genetic suppression with the kre5W1166X mutant. These findings show for the first time that perturbation of CL synthesis leads to decreased longevity in yeast, which is restored by altering signaling through stress response pathways. The anionic mitochondrial phospholipid cardiolipin (CL) 5The abbreviations used are: CLcardiolipinPGphosphatidylglycerolmtDNAmitochondrial DNAPKCprotein kinase CMAPKmitogen-activated protein kinaseHOGhigh osmolarity glycerolCRcalorie restrictionSCsynthetic complete mediumWTwild typeDAPI4′,6-diamidino-2-phenylindole. is ubiquitous in eukaryotes. The importance of CL is underscored by the association of aberrant CL metabolism with several disorders, including Barth syndrome (1.Vreken P. Valianpour F. Nijtmans L.G. Grivell L.A. Plecko B. Wanders R.J. Barth P.G. Biochem. Biophys. Res. Commun. 2000; 279: 378-382Crossref PubMed Scopus (310) Google Scholar), thyroid dysfunction (2.Schlame M. Shanske S. Doty S. König T. Sculco T. DiMauro S. Blanck T.J. J. Lipid Res. 1999; 40: 1585-1592Abstract Full Text Full Text PDF PubMed Google Scholar, 3.Schlame M. Rua D. Greenberg M.L. Prog. Lipid Res. 2000; 39: 257-288Crossref PubMed Scopus (665) Google Scholar), diabetes (4.Ferreira F.M. Seiça R. Oliveira P.J. Coxito P.M. Moreno A.J. Palmeira C.M. Santos M.S. Biochim. Biophys. Acta. 2003; 1639: 113-120Crossref PubMed Scopus (48) Google Scholar), myocardial ischemia (5.Lesnefsky E.J. Slabe T.J. Stoll M.S. Minkler P.E. Hoppel C.L. Am. J. Physiol. Heart Circ. Physiol. 2001; 280: H2770-2778Crossref PubMed Google Scholar), and aging (6.Paradies G. Ruggiero F.M. Biochim. Biophys. Acta. 1990; 1016: 207-212Crossref PubMed Scopus (97) Google Scholar, 7.Maftah A. Ratinaud M.H. Dumas M. Bonté F. Meybeck A. Julien R. Mech. Ageing Dev. 1994; 77: 83-96Crossref PubMed Scopus (43) Google Scholar). The unique cellular localization of CL in the mitochondrial inner membrane suggests that it is closely associated with mitochondrial function, biogenesis, and genome stability (3.Schlame M. Rua D. Greenberg M.L. Prog. Lipid Res. 2000; 39: 257-288Crossref PubMed Scopus (665) Google Scholar, 8.Jiang F. Ryan M.T. Schlame M. Zhao M. Gu Z. Klingenberg M. Pfanner N. Greenberg M.L. J. Biol. Chem. 2000; 275: 22387-22394Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 9.Janitor M. Subík J. Curr. Genet. 1993; 24: 307-312Crossref PubMed Scopus (48) Google Scholar, 10.Daum G. Biochim. Biophys. Acta. 1985; 822: 1-42Crossref PubMed Scopus (710) Google Scholar). cardiolipin phosphatidylglycerol mitochondrial DNA protein kinase C mitogen-activated protein kinase high osmolarity glycerol calorie restriction synthetic complete medium wild type 4′,6-diamidino-2-phenylindole. The identification of yeast genes encoding CL biosynthetic enzymes and subsequent construction of deletion mutants deficient in CL biosynthesis facilitated in vivo studies to elucidate the role of CL in mitochondrial function and cell viability. Disruption of PGS1 encoding the first enzyme of the CL pathway, phosphatidylglycerolphosphate synthase, results in the complete loss of both phosphatidylglycerol (PG) and CL (11.Chang S.C. Heacock P.N. Clancey C.J. Dowhan W. J. Biol. Chem. 1998; 273: 9829-9836Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Mutants lacking the CRD1 gene encoding CL synthase contain no detectable CL but accumulate the upstream precursor PG (11.Chang S.C. Heacock P.N. Clancey C.J. Dowhan W. J. Biol. Chem. 1998; 273: 9829-9836Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 12.Jiang F. Rizavi H.S. Greenberg M.L. Mol. Microbiol. 1997; 26: 481-491Crossref PubMed Scopus (155) Google Scholar, 13.Tuller G. Hrastnik C. Achleitner G. Schiefthaler U. Klein F. Daum G. FEBS Lett. 1998; 421: 15-18Crossref PubMed Scopus (124) Google Scholar, 14.Pfeiffer K. Gohil V. Stuart R.A. Hunte C. Brandt U. Greenberg M.L. Schägger H. J. Biol. Chem. 2003; 278: 52873-52880Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 15.Zhong Q. Gohil V.M. Ma L. Greenberg M.L. J. Biol. Chem. 2004; 279: 32294-32300Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Perturbation of CL synthesis in crd1Δ mutant cells leads to mitochondrial dysfunction via uncoupling, instability of electron transport chain supercomplexes, and decreased function of individual complexes (14.Pfeiffer K. Gohil V. Stuart R.A. Hunte C. Brandt U. Greenberg M.L. Schägger H. J. Biol. Chem. 2003; 278: 52873-52880Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 16.Robinson N.C. J. Bioenerg. Biomembr. 1993; 25: 153-163Crossref PubMed Scopus (228) Google Scholar, 17.Ostrander D.B. Zhang M. Mileykovskaya E. Rho M. Dowhan W. J. Biol. Chem. 2001; 276: 25262-25272Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 18.McMillin J.B. Dowhan W. Biochim. Biophys. Acta. 2002; 1585: 97-107Crossref PubMed Scopus (240) Google Scholar). Mitochondria from the crd1Δ mutant exhibit decreased coupling during osmotic stress, heat stress, or increased respiration rate and are coupled only under optimal conditions (19.Koshkin V. Greenberg M.L. Biochem. J. 2002; 364: 317-322Crossref PubMed Scopus (113) Google Scholar, 20.Koshkin V. Greenberg M.L. Biochem. J. 2000; 347: 687-691Crossref PubMed Scopus (110) Google Scholar). In addition, crd1Δ mutant cells tend to lose mitochondrial DNA (mtDNA) and become petite (ρ0) after prolonged growth at elevated temperatures (15.Zhong Q. Gohil V.M. Ma L. Greenberg M.L. J. Biol. Chem. 2004; 279: 32294-32300Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The pgs1Δ mutant, which lacks both PG and CL, loses mtDNA even when grown at 30 °C (21.Zhong Q. Gvozdenovic-Jeremic J. Webster P. Zhou J. Greenberg M.L. Mol. Biol. Cell. 2005; 16: 665-675Crossref PubMed Scopus (47) Google Scholar). Because respiratory function and mtDNA stability have been implicated in aging (22.Bonawitz N.D. Shadel G.S. Cell Cycle. 2007; 6: 1574-1578Crossref PubMed Scopus (45) Google Scholar, 23.Laun P. Bruschi C.V. Dickinson J.R. Rinnerthaler M. Heeren G. Schwimbersky R. Rid R. Breitenbach M. Nucleic Acids Res. 2007; 35: 7514-7526Crossref PubMed Scopus (27) Google Scholar), yeast CL mutants provide excellent models to elucidate the role of CL in these processes. In addition to its central role in respiratory function, CL is also required for essential cellular processes that are not directly associated with respiration, several of which affect aging. The role of CL in essential functions was first suggested by the temperature sensitivity phenotype of CL mutants during growth on glucose, a carbon source that can be utilized by fermentation in the absence of respiratory function (15.Zhong Q. Gohil V.M. Ma L. Greenberg M.L. J. Biol. Chem. 2004; 279: 32294-32300Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 21.Zhong Q. Gvozdenovic-Jeremic J. Webster P. Zhou J. Greenberg M.L. Mol. Biol. Cell. 2005; 16: 665-675Crossref PubMed Scopus (47) Google Scholar, 24.Jiang F. Gu Z. Granger J.M. Greenberg M.L. Mol. Microbiol. 1999; 31: 373-379Crossref PubMed Scopus (51) Google Scholar). Perturbation of the CL pathway in crd1Δ and pgs1Δ mutants leads to decreased growth at elevated temperatures (15.Zhong Q. Gohil V.M. Ma L. Greenberg M.L. J. Biol. Chem. 2004; 279: 32294-32300Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). At 37 °C, the crd1Δ mutant can grow but cannot form colonies from single cells, whereas pgs1Δ cannot grow at all (15.Zhong Q. Gohil V.M. Ma L. Greenberg M.L. J. Biol. Chem. 2004; 279: 32294-32300Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). One essential process requiring the CL pathway is cell wall biogenesis, which plays a role in aging in yeast (25.Kaeberlein M. Guarente L. Genetics. 2002; 160: 83-95Crossref PubMed Google Scholar). A link between the CL biosynthetic pathway and the cell wall was first indicated by a report that disruption of the promoter of PGS1, the gene that mediates the committed and rate-limiting step of CL biosynthesis (11.Chang S.C. Heacock P.N. Clancey C.J. Dowhan W. J. Biol. Chem. 1998; 273: 9829-9836Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), resulted in cell wall defects (26.Lussier M. White A.M. Sheraton J. di Paolo T. Treadwell J. Southard S.B. Horenstein C.I. Chen-Weiner J. Ram A.F. Kapteyn J.C. Roemer T.W. Vo D.H. Bondoc D.C. Hall J. Zhong W.W. Sdicu A.M. Davies J. Klis F.M. Robbins P.W. Bussey H. Genetics. 1997; 147: 435-450Crossref PubMed Google Scholar). Consistent with this, pgs1Δ temperature sensitivity was alleviated by supplementation with sorbitol, which provides osmotic support for the cell wall, and by genetic suppression with kre5W1166X, a mutant that up-regulates cell wall-associated pathways (21.Zhong Q. Gvozdenovic-Jeremic J. Webster P. Zhou J. Greenberg M.L. Mol. Biol. Cell. 2005; 16: 665-675Crossref PubMed Scopus (47) Google Scholar). Further studies indicated that pgs1Δ exhibits cell wall defects due to defective activation of the protein kinase C (PKC)-activated Slt2 mitogen-activated protein kinase (MAPK) pathway (27.Zhong Q. Li G. Gvozdenovic-Jeremic J. Greenberg M.L. J. Biol. Chem. 2007; 282: 15946-15953Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). This pathway regulates cell wall biosynthesis and progression through the cell cycle in response to cell surface stress (28.Heinisch J.J. Biochim. Biophys. Acta. 2005; 1754: 171-182Crossref PubMed Scopus (47) Google Scholar, 29.Heinisch J.J. Lorberg A. Schmitz H.P. Jacoby J.J. Mol. Microbiol. 1999; 32: 671-680Crossref PubMed Scopus (292) Google Scholar). In response to stress, cells are under the coordinate control of both the PKC-Slt2 and high osmolarity glycerol (HOG) signaling pathways. The PKC-Slt2 pathway functions to withstand increased turgor pressure resulting from heat or hypotonic stress (i.e. shifting cells from high to low osmolarity) by increasing the expression of genes for cell wall remodeling (30.Hohmann S. Microbiol. Mol. Biol. Rev. 2002; 66: 300-372Crossref PubMed Scopus (1296) Google Scholar, 31.Davenport K.R. Sohaskey M. Kamada Y. Levin D.E. Gustin M.C. J. Biol. Chem. 1995; 270: 30157-30161Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 32.García-Rodríguez L.J. Valle R. Durán A. Roncero C. FEBS Lett. 2005; 579: 6186-6190Crossref PubMed Scopus (50) Google Scholar). In contrast, the HOG pathway is activated when cells are shifted from low to high osmolarity (hypertonic conditions). This pathway is mediated by Hog1p and two independent membrane sensors, Sho1p and Sln1p (33.Rep M. Krantz M. Thevelein J.M. Hohmann S. J. Biol. Chem. 2000; 275: 8290-8300Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). Hog1p is activated by phosphorylation, whereupon it moves into the nucleus and phosphorylates the transcriptional regulators Msn2p/Msn4p, Sko1p, etc. (30.Hohmann S. Microbiol. Mol. Biol. Rev. 2002; 66: 300-372Crossref PubMed Scopus (1296) Google Scholar). Glycerol and trehalose synthesis is subsequently induced, causing increased intracellular turgor pressure, which counterbalances the high external osmolarity (30.Hohmann S. Microbiol. Mol. Biol. Rev. 2002; 66: 300-372Crossref PubMed Scopus (1296) Google Scholar). In this light, cell wall defects resulting from a defective PKC-Slt2 pathway, as observed in pgs1Δ, could be lethal if exacerbated by HOG signaling. The MAPK and stress pathways discussed above are highly conserved from yeast to mammals and have been shown to affect life span in yeast in several ways (34.Swieciło A. Krawiec Z. Wawryn J. Bartosz G. Biliński T. Acta Biochim. Pol. 2000; 47: 355-364Crossref PubMed Scopus (28) Google Scholar, 35.Gustin M.C. Albertyn J. Alexander M. Davenport K. Microbiol. Mol. Biol. Rev. 1998; 62: 1264-1300Crossref PubMed Google Scholar, 36.Shama S. Lai C.Y. Antoniazzi J.M. Jiang J.C. Jazwinski S.M. Exp. Cell Res. 1998; 245: 379-388Crossref PubMed Scopus (112) Google Scholar, 37.Kaeberlein M. Andalis A.A. Fink G.R. Guarente L. Mol. Cell Biol. 2002; 22: 8056-8066Crossref PubMed Scopus (120) Google Scholar). First, regulation of MAPK pathways in response to heat and osmotic stress exerts complex effects on chronological and replicative life span (38.Fabrizio P. Pletcher S.D. Minois N. Vaupel J.W. Longo V.D. FEBS Lett. 2004; 557: 136-142Crossref PubMed Scopus (161) Google Scholar). Activation of the transcriptional activators Msn2p/Msn4p by Hog1p induces the general stress response and leads to the induction of many downstream targets, including superoxide dismutase (33.Rep M. Krantz M. Thevelein J.M. Hohmann S. J. Biol. Chem. 2000; 275: 8290-8300Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). Although this pathway is beneficial to cells under stress, increased expression of the transcription factor Msn2p/Msn4p and/or superoxide dismutase SOD2 decreases the replicative life span of yeast (38.Fabrizio P. Pletcher S.D. Minois N. Vaupel J.W. Longo V.D. FEBS Lett. 2004; 557: 136-142Crossref PubMed Scopus (161) Google Scholar, 39.Harris N. Costa V. MacLean M. Mollapour M. Moradas-Ferreira P. Piper P.W. Free Radic. Biol. Med. 2003; 34: 1599-1606Crossref PubMed Scopus (80) Google Scholar). Second, increased cell integrity extends yeast replicative life span, possibly by preventing cell lysis in old cells (40.Kaeberlein M. Andalis A.A. Liszt G.B. Fink G.R. Guarente L. Genetics. 2004; 166: 1661-1672Crossref PubMed Scopus (44) Google Scholar). In addition to these factors, the replicative life span of yeast cells is regulated by calorie restriction (CR) and stress (34.Swieciło A. Krawiec Z. Wawryn J. Bartosz G. Biliński T. Acta Biochim. Pol. 2000; 47: 355-364Crossref PubMed Scopus (28) Google Scholar, 41.Anderson R.M. Bitterman K.J. Wood J.G. Medvedik O. Sinclair D.A. Nature. 2003; 423: 181-185Crossref PubMed Scopus (612) Google Scholar), both of which involve activation of Sir2p. Sir2p mediates chromatin silencing and promotes longevity by suppressing the formation of extrachromosomal ribosomal DNA circles (37.Kaeberlein M. Andalis A.A. Fink G.R. Guarente L. Mol. Cell Biol. 2002; 22: 8056-8066Crossref PubMed Scopus (120) Google Scholar, 42.Gallo C.M. Smith Jr., D.L. Smith J.S. Mol. Cell Biol. 2004; 24: 1301-1312Crossref PubMed Scopus (167) Google Scholar, 43.Sinclair D.A. Guarente L. Cell. 1997; 91: 1033-1042Abstract Full Text Full Text PDF PubMed Scopus (1187) Google Scholar, 44.Lin S.J. Defossez P.A. Guarente L. Science. 2000; 289: 2126-2128Crossref PubMed Scopus (1488) Google Scholar). In this report, we show for the first time that loss of the mitochondrial anionic phospholipids CL and PG leads to decreased replicative life span. The longevity defects in CL mutants can be alleviated by genetic or osmotic remediation of the cell wall and, strikingly, by down-regulation of the HOG pathway. Our findings suggest that mitochondrial anionic phospholipids play a novel role in the cellular stress response. All chemicals used were reagent grade or better. PCR was performed using the Taq polymerase enzyme kit from Promega (Madison, WI). The Wizard Plus Miniprep DNA purification system was from Promega (Madison, WI). All other buffers and enzymes were purchased from Sigma. Glucose, yeast extract, and peptone were purchased from Difco. The Saccharomyces cerevisiae strains used in this work are listed in Table 1. Synthetic complete medium (SC) contained the amino acids and the nucleotides adenine (20.25 mg/liter), arginine (20 mg/liter), histidine (20 mg/liter), leucine (60 mg/liter), lysine (200 mg/liter), methionine (20 mg/liter), threonine (300 mg/liter), tryptophan (20 mg/liter), and uracil (20 mg/liter), vitamins, salts (essentially components of Difco Vitamin Free Yeast Base without amino acids), and glucose (2%). Synthetic drop-out medium contained all ingredients except uracil (Ura−), histidine (His−), or lysine and methionine (Lys−Met−). Sporulation medium contained potassium acetate (1%), glucose (0.05%), and essential amino acids. Complex medium (YPD) contained yeast extract (1%), peptone (2%), and glucose (2%). Complex YPDS medium was YPD supplemented with 1 m sorbitol. Solid medium contained agar (2%). Limited glucose media for CR contained 0.5% rather than 2% glucose.TABLE 1Yeast strains used in this studyPlasmid or strainCharacteristics or genotypeSource or referenceFGY3MATα, ura 3-52, lys2-801, ade2-101, trp1Δ1, his3Δ200, leu2Δ1Ref. 12.Jiang F. Rizavi H.S. Greenberg M.L. Mol. Microbiol. 1997; 26: 481-491Crossref PubMed Scopus (155) Google ScholarFGY2Isogenic with FGY3 except crd1Δ ::URA3Ref. 12.Jiang F. Rizavi H.S. Greenberg M.L. Mol. Microbiol. 1997; 26: 481-491Crossref PubMed Scopus (155) Google ScholarZQY1Isogenic with FGY3 except pgs1Δ ::TRP1Ref. 71.Zhong Q. Greenberg M.L. Biochem. Soc. Trans. 2005; 33: 1158-1161Crossref PubMed Google ScholarFGY3_A (ρ0)Isogenic with FGY3 except MATa, ADE2, ρ0This studyQZY24B (ρ0)Isogenic with FGY3_A except pgs1Δ ::TRP1Ref. 71.Zhong Q. Greenberg M.L. Biochem. Soc. Trans. 2005; 33: 1158-1161Crossref PubMed Google ScholarQZY11A (ρ0)Isogenic with QZY24B except kre5W1166XRef. 71.Zhong Q. Greenberg M.L. Biochem. Soc. Trans. 2005; 33: 1158-1161Crossref PubMed Google ScholarBY4742MATα his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0EuroscarfaEuroscarf, European Saccharomyces cerevisiae Archive for Functional analysis.sho1Δρ0Isogenic with BY4742 except sho1 ::KanMXEuroscarfcan1Δρ0 (WT ρ0)Isogenic with BY4742 except can1Δ ρ0This study9F (pgs1Δρ0)Isogenic with can1Δ ρ0 except pgs1 ::URA3This studypgs1Δhsp12Δ (ρ0)Isogenic with 9F except hsp12 ::KanMXThis studypgs1Δsho1Δ (ρ0)Isogenic with 9F except sho1 ::KanMXThis studypPS1739CEN, URA3, HOG1-GFPRef. 72.Ferrigno P. Posas F. Koepp D. Saito H. Silver P.A. EMBO J. 1998; 17: 5606-5614Crossref PubMed Scopus (352) Google Scholara Euroscarf, European Saccharomyces cerevisiae Archive for Functional analysis. Open table in a new tab Single cells from each strain were obtained and aligned on a YPD or YPDS plate, ∼60 cells/plate. At 2–3-h intervals, cells were visualized under the dissection microscope to identify mature buds. Buds were removed and counted to determine the number of daughter cells produced by each mother cell, indicative of the replicative life span. Life spans were quantified at optimal or elevated temperatures by microscopic determination of the number of times a mother cell produced a daughter cell, and the average replicative life spans (in generations) were determined as described (36.Shama S. Lai C.Y. Antoniazzi J.M. Jiang J.C. Jazwinski S.M. Exp. Cell Res. 1998; 245: 379-388Crossref PubMed Scopus (112) Google Scholar, 37.Kaeberlein M. Andalis A.A. Fink G.R. Guarente L. Mol. Cell Biol. 2002; 22: 8056-8066Crossref PubMed Scopus (120) Google Scholar). The statistical significance of differences in life spans was determined by analysis of variance (p < 0.05) and shown for all experiments in Table 2.TABLE 2Life spans of WT and CL mutant strainsGenetic backgroundStrain, medium, temperatureMeanMaxS.E.np < 0.05StandardFGY3WT, YPD, 30 °C16.7400.78117AWT, YPD, 37 °C17390.83145BWT, YPD, 37 °CaExperiments in which virgin cells were obtained at 37 °C.15.4310.7597CWT, YPDS, 30 °C25.4520.79163ADWT, YPD, heat shock25.2470.81119AEWT, osmotic shock25.9661.06115AFWT, CR, 30 °C26.5490.63190Acrd1Δ, YPD, 30 °C15.9320.71113Gcrd1Δ, YPD, 37 °C8.7280.51127B, GHcrd1Δ, YPD, 37 °CaExperiments in which virgin cells were obtained at 37 °C.6.4200.38175C, G, Hcrd1Δ, YPDS, 30 °C24.1500.81112Gcrd1Δ, YPD, heat shock16.7300.58120E, Gbp value larger than 0.05 versus its corresponding standard(s).crd1Δ, osmotic shock12.6520.75146F, Gbp value larger than 0.05 versus its corresponding standard(s).crd1Δ, CR, 30 °C23.2520.5208Gpgs1Δ, YPD, 30 °C5.7170.31106AIpgs1Δ, YPDS, 30 °C15.1580.31106D, Ipgs1Δ, CR, 30 °C3.190.13242IFGY3_A ρ0WT, YPD, 30 °C19340.79101AJpgs1Δ, YPD, 30 °C7.1130.26109I, JKpgs1Δkre5W1166X, YPD, 30 °C15.3360.77107J, KBY4742 can1Δ ρ0WT, YPD, 30 °C25.3671.39108Lpgs1Δ, YPD, 30 °C17.1310.54113LMpgs1Δhsp12Δ, YPD, 30 °C19440.98114Mbp value larger than 0.05 versus its corresponding standard(s).pgs1Δsho1Δ, YPD, 30 °C24.1510.88118Ma Experiments in which virgin cells were obtained at 37 °C.b p value larger than 0.05 versus its corresponding standard(s). Open table in a new tab A pgs1Δ strain was generated in the BY4742 (MATα) background by disrupting PGS1 and linking the pgs1Δ mutation to the dominant selectable marker URA3 and the reporter construct MFA1pr-HIS3, which is only expressed in the MATa background. Seventy-seven mutants, each with a deletion in a stress response gene, were obtained from the deletion collection generated in the BY4741 (MATa) background (Research Genetics). The gene mutated in each strain was replaced by a kanamycin (Geneticin) resistance marker (KanMX). After pgs1Δ was mated with each of the 77 deletion strains, diploids were selected on Met−Lys− double drop-out medium, and sporulation was induced. MATa spores were selected on His− drop-out medium, and double mutant meiotic progeny were selected on Ura− drop-out medium with 200 mg/liter Geneticin, as described (45.Tong A.H. Evangelista M. Parsons A.B. Xu H. Bader G.D. Pagé N. Robinson M. Raghibizadeh S. Hogue C.W. Bussey H. Andrews B. Tyers M. Boone C. Science. 2001; 294: 2364-2368Crossref PubMed Scopus (1651) Google Scholar). Growth of double mutants was examined at 39 °C to identify suppressors of pgs1Δ temperature sensitivity. Yeast cells were grown in liquid medium at 30 °C, harvested in early stationary phase, fixed in 70% ethanol at room temperature for 30 min, and stained with 1 μg/ml DAPI for 5 min, as described (21.Zhong Q. Gvozdenovic-Jeremic J. Webster P. Zhou J. Greenberg M.L. Mol. Biol. Cell. 2005; 16: 665-675Crossref PubMed Scopus (47) Google Scholar). Cells were viewed with an Olympus BX41 epifluorescence microscope using a WU filter and a ×100 oil immersion objective, and images were captured with a Q-color3 camera and represent at least 200 observed cells. Dually phosphorylated Hog1p was determined as described previously with minor modifications (46.Tamás M.J. Rep M. Thevelein J.M. Hohmann S. FEBS Lett. 2000; 472: 159-165Crossref PubMed Scopus (76) Google Scholar, 47.Krantz M. Nordlander B. Valadi H. Johansson M. Gustafsson L. Hohmann S. Eukaryot. Cell. 2004; 3: 1381-1390Crossref PubMed Scopus (51) Google Scholar). Yeast cells were grown in YPD medium to A550 of ∼1.0 and then treated with NaCl (0.8 m). Cell extracts were prepared, and proteins were separated by SDS-PAGE and visualized as described previously (46.Tamás M.J. Rep M. Thevelein J.M. Hohmann S. FEBS Lett. 2000; 472: 159-165Crossref PubMed Scopus (76) Google Scholar, 47.Krantz M. Nordlander B. Valadi H. Johansson M. Gustafsson L. Hohmann S. Eukaryot. Cell. 2004; 3: 1381-1390Crossref PubMed Scopus (51) Google Scholar). Dually phosphorylated Hog1p was detected by anti-phospho-p38 MAPK (Thr180/Tyr182) (9211S; 1:1000; New England Biolabs). Anti-Hog1p was used to detect total Hog1p (Hog1 yC-20; 1:5000; Santa Cruz Biotechnology). Plasmid pPS1739 expressing HOG1-GFP (kindly provided by Dr. Pamela Silver) was transformed into wild type and pgs1Δ cells in the FGY3 background. Transformants were grown to A550 of ∼1.0 in SC Ura− supplemented with 1 m sorbitol liquid medium (sorbitol is required for growth of pgs1Δ). Cells were treated with NaCl (0.8 m) for 2 min, stained with DAPI, and visualized as described above. Dually phosphorylated Slt2p was determined as discussed previously with minor modifications (27.Zhong Q. Li G. Gvozdenovic-Jeremic J. Greenberg M.L. J. Biol. Chem. 2007; 282: 15946-15953Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Midlog phase cells were diluted to A550 of ∼0.3 and grown at 30 or 37 °C for 2 h. Cell extracts were prepared, and proteins were separated by SDS-PAGE and visualized as described previously (27.Zhong Q. Li G. Gvozdenovic-Jeremic J. Greenberg M.L. J. Biol. Chem. 2007; 282: 15946-15953Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Dually phosphorylated Slt2p (Thr202/Tyr204) was detected by anti-phospho-p44/42 MAPK (Thr202/Tyr204) (9101S; 1:1000; Cell Signaling Technology), and anti-Mpk1p was used to detect total Slt2p (Mpk1 yN-19; 1:100; Santa Cruz Biotechnology). To address the hypothesis that a functional CL pathway is required for normal life span, virgin cells of isogenic wild type, crd1Δ, and pgs1Δ strains in the FGY3 genetic background were obtained at 30 °C, and life span was determined as described under “Experimental Procedures.” Because pgs1Δ mutant cells lose mtDNA when grown at 30 °C, life spans of isogenic wild type ρ0 cells were analyzed for comparison. In some strain backgrounds, ρ0 cells were reported to have increased replicative life spans, possibly due to up-regulation of retrograde signaling (48.Kirchman P.A. Kim S. Lai C.Y. Jazwinski S.M. Genetics. 1999; 152: 179-190Crossref PubMed Google Scholar). Consistent with this, loss of mtDNA from wild type cells in the FGY3 genetic background causes a small extension in replicative life span (Fig. 1A). At 30 °C, pgs1Δ was found to have a shortened life span of 5.7 generations, whereas crd1Δ cells displayed a normal life span of 15.9 generations (similar to the wild type life span of 16.7 generations) (Fig. 1A). When virgin cells were obtained at 30 °C and life span was determined at 37 °C, the life span of crd1Δ cells was significantly reduced to 8.7 (Fig. 1B). Life span was further reduced slightly when virgin cells were obtained at 37 °C (Fig. 1C). The pgs1Δ mutant failed to survive under these conditions. Thus, disruption of the first step of the CL pathway (pgs1Δ), resulting in loss of both PG and CL, leads to a shortened life span at 30 °C and the inability to replicate at elevated temperatures, whereas disruption of the last step of the pathway (crd1Δ), which results in the ability to synthesize PG but not CL, affects life span at elevated temperatures but not at 30 °C. As discussed above, CR resulting from low glucose in the media can induce life span extension in yeast (44.Lin S.J. Defossez P.A. Guarente L. Science. 2000; 289: 2126-2128Crossref PubMed Scopus (1488) Google Scholar). To determine if perturbation of the CL pathway affects life span extension in response to CR, we examined the life span of crd1Δ on low glucose (CR) medium at 30 °C. CR increased life span from 16.7 to 26.5 generations in wild type cells and from 15.9 to 23.2 in the crd1Δ mutant (compare Figs. 1A and 2), indicating that CL is not required for life span extension induced by CR. However, the life span of pgs1Δ was reduced rather than extended by CR (Fig. 2), suggesting that more severe mitochondrial defects in pgs1Δ prevent life span extension triggered by CR. Mild stress conditions, including heat and osmotic shock, are known to extend yeast life span (34.Swieciło A. Krawiec Z. Wawryn J. Bartosz G. Biliński T. Acta Biochim. Pol. 2000; 47: 355-364Crossref PubMed Scopus (28) Google Scholar, 36.Shama S. Lai C.Y. Antoniazzi J.M. Jiang J.C. Jazwinski S.M. Exp. Cell Res. 1998; 245: 379-388Crossref PubMed Scopus (112) Google Scholar, 37.Kaeberlein M. Andalis A.A. Fink G.R. Guarente L. Mol. Cell Biol. 2002; 22: 8056-8066Crossref PubMed Scopus (120) Google Scholar). To determine if mild stress extended the life span of CL mutants, virgin cells were obtained at 30 °C, heat-shocked by immediate exposure to elevated temperature (37 °C) for 2 generations (4 h), and then s" @default.
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• W2135457913 date "2009-07-01" @default.
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• W2135457913 title "Loss of Cardiolipin Leads to Longevity Defects That Are Alleviated by Alterations in Stress Response Signaling" @default.
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• W2135457913 doi "https://doi.org/10.1074/jbc.m109.003236" @default.
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