Absence of Cardiolipin Results in Temperature Sensitivity, Respiratory Defects, and Mitochondrial DNA Instability Independent of pet56

Abstract

Cardiolipin (CL) is a dimeric phospholipid localized primarily in the mitochondrial membrane. Previous studies have shown that yeast cells containing a disruption of CRD1, the structural gene encoding CL synthase, exhibit temperature-sensitive colony formation and multiple mitochondrial defects. A recent report (Zhang, M., Su, X., Mileykovskaya, E., Amoscato, A. A., and Dowhan, W. (2003) J. Biol. Chem. 278, 35204–35210) suggested that defects associated with CL deficiency may result from the reduced expression of PET56 in crd1Δ mutant backgrounds and should be reevaluated. In the current study, we present evidence that CL deficiency leads to mitochondrial DNA instability, loss of viability, and defects in oxidative phosphorylation at elevated temperatures. The observed mutant phenotypes are characteristic of crd1Δ mutant cells of both PET56 and pet56 backgrounds and are complemented by an episomal copy of CRD1 but not by expression of the PET56 gene. Phosphatidylglycerol is elevated in crd1Δ mutant cells when grown in the presence of fermentable and non-fermentable carbon sources, although the extent of the increase is higher in nonfermentable medium. An increase in the ratio of phosphatidylethanolamine to phosphatidylcholine was also apparent in the mutant. These findings demonstrate that CRD1, independent of PET56, is required for optimal mitochondrial function and for an essential cellular function at elevated temperatures. Cardiolipin (CL) is a dimeric phospholipid localized primarily in the mitochondrial membrane. Previous studies have shown that yeast cells containing a disruption of CRD1, the structural gene encoding CL synthase, exhibit temperature-sensitive colony formation and multiple mitochondrial defects. A recent report (Zhang, M., Su, X., Mileykovskaya, E., Amoscato, A. A., and Dowhan, W. (2003) J. Biol. Chem. 278, 35204–35210) suggested that defects associated with CL deficiency may result from the reduced expression of PET56 in crd1Δ mutant backgrounds and should be reevaluated. In the current study, we present evidence that CL deficiency leads to mitochondrial DNA instability, loss of viability, and defects in oxidative phosphorylation at elevated temperatures. The observed mutant phenotypes are characteristic of crd1Δ mutant cells of both PET56 and pet56 backgrounds and are complemented by an episomal copy of CRD1 but not by expression of the PET56 gene. Phosphatidylglycerol is elevated in crd1Δ mutant cells when grown in the presence of fermentable and non-fermentable carbon sources, although the extent of the increase is higher in nonfermentable medium. An increase in the ratio of phosphatidylethanolamine to phosphatidylcholine was also apparent in the mutant. These findings demonstrate that CRD1, independent of PET56, is required for optimal mitochondrial function and for an essential cellular function at elevated temperatures. Cardiolipin (CL), 1The abbreviations used are: CL, cardiolipin; PG, phosphatidylglycerol; SCD, synthetic complete dextrose; PE, phosphatidylethanolamine. 1The abbreviations used are: CL, cardiolipin; PG, phosphatidylglycerol; SCD, synthetic complete dextrose; PE, phosphatidylethanolamine. a unique phospholipid with dimeric structure, is ubiquitous in eukaryotes and is primarily found in the mitochondrial inner membrane (1Schlame M. Rua D. Greenberg M.L. Prog. Lipid Res. 2000; 39: 257-288Google Scholar). It plays a key role in mitochondrial bioenergetics by optimizing the activities of enzymes in the oxidative phosphorylation pathway (1Schlame M. Rua D. Greenberg M.L. Prog. Lipid Res. 2000; 39: 257-288Google Scholar), stabilizing the supercomplexes of Saccharomyces cerevisiae respiratory chain complexes III and IV (2Pfeiffer K. Gohil V. Stuart R.A. Hunte C. Brandt U. Greenberg M.L. Schagger H. J. Biol. Chem. 2003; 278: 52873-52880Google Scholar), preventing rate-dependent uncoupling, and providing osmotic stability in yeast mitochondria (3Koshkin V. Greenberg M.L. Biochem. J. 2002; 364: 317-322Google Scholar). CL is also involved in mitochondrial biogenesis, possibly via assisting protein import into mitochondria (4Jiang F. Ryan M.T. Schlame M. Zhao M. Gu Z. Klingenberg M. Pfanner N. Greenberg M.L. J. Biol. Chem. 2000; 275: 22387-22394Google Scholar) and maintaining optimal mitochondrial internal structure (5Kawasaki K. Kuge O. Chang S.C. Heacock P.N. Rho M. Suzuki K. Nishijima M. Dowhan W. J. Biol. Chem. 1999; 274: 1828-1834Google Scholar). Defective remodeling of CL is associated with Barth syndrome, a severe genetic disorder characterized by cardiomyopathy, neutropenia, skeletal myopathy, and respiratory chain defects (6Vreken P. Valianpour F. Nijtmans L.G. Grivell L.A. Plecko B. Wanders R.J. Barth P.G. Biochem. Biophys. Res. Commun. 2000; 279: 378-382Google Scholar). The phenotype of Barth syndrome is dependent upon multiple factors that are not well understood (7Barth P.G. Scholte H.R. Berden J.A. Van der Klei-Van Moorsel J.M. Luyt-Houwen I.E. Van't Veer-Korthof E.T. Van der Harten J.J. Sobotka-Plojhar M.A. J. Neurol. Sci. 1983; 62: 327-355Google Scholar, 8Barth P.G. Van den Bogert C. Bolhuis P.A. Scholte H.R. van Gennip A.H. Schutgens R.B. Ketel A.G. J. Inherit. Metab. Dis. 1996; 19: 157-160Google Scholar). Elucidation of the functions of CL will help to clarify the abnormalities associated with this disorder. We have observed that the yeast crd1Δ mutant, which lacks CL synthase and has no detectable CL, loses cell viability during growth at elevated temperatures (4Jiang F. Ryan M.T. Schlame M. Zhao M. Gu Z. Klingenberg M. Pfanner N. Greenberg M.L. J. Biol. Chem. 2000; 275: 22387-22394Google Scholar, 9Jiang F. Gu Z. Granger J.M. Greenberg M.L. Mol. Microbiol. 1999; 31: 373-379Google Scholar). Crd1Δ mutant cells cannot form colonies at 37 °C from single cells seeded on YPD (1% yeast extract, 2% peptone, and 2% dextrose) plates (9Jiang F. Gu Z. Granger J.M. Greenberg M.L. Mol. Microbiol. 1999; 31: 373-379Google Scholar). In addition, crd1Δ mutant cells grown in fermentable or non-fermentable carbon sources also segregate large numbers of petites (respiratory incompetent cells) after prolonged culture at elevated temperatures (4Jiang F. Ryan M.T. Schlame M. Zhao M. Gu Z. Klingenberg M. Pfanner N. Greenberg M.L. J. Biol. Chem. 2000; 275: 22387-22394Google Scholar). CL is, thus, essential for maintaining mitochondrial DNA stability and cell viability in both fermentable and non-fermentable media. These phenotypes of crd1Δ cells are similar to those of the yeast phosphatidylserine decarboxylase mutant psd1Δ, which contain reduced mitochondrial phosphatidylethanolamine. Psd1Δ cells exhibit a high propensity for loss of mitochondrial DNA, markedly reduced growth on non-fermentable media, and temperature sensitivity at 37 °C, similarly to crd1Δ (10Trotter P.J. Pedretti J. Voelker D.R. J. Biol. Chem. 1993; 268: 21416-21424Google Scholar, 11Birner R. Burgermeister M. Schneiter R. Daum G. Mol. Biol. Cell. 2001; 12: 997-1007Google Scholar, 12Storey M.K. Wu W.I. Voelker D.R. Biochim. Biophys. Acta. 2001; 1532: 234-247Google Scholar). A recent study reported that the defects associated with the crd1Δ mutation were attributable to the presence of a pet56 mutation in the crd1Δ strain background (13Zhang M. Su X. Mileykovskaya E. Amoscato A.A. Dowhan W. J. Biol. Chem. 2003; 278: 35204-35210Google Scholar). Pet56p catalyzes the formation of 2′-O-methylguanosine at a specific nucleotide in the peptidyl transferase center of the RNA of the large mitochondrial ribosomal subunit (14Sirum-Connolly K. Mason T.L. Science. 1993; 262: 1886-1889Google Scholar). Reduced expression of PET56 was found to slow growth, decrease mitochondrial DNA stability, and decrease cell viability at elevated temperatures (14Sirum-Connolly K. Mason T.L. Science. 1993; 262: 1886-1889Google Scholar, 15Rieger K.J. Kaniak A. Coppee J.Y. Aljinovic G. Baudin-Baillieu A. Orlowska G. Gromadka R. Groudinsky O. Di Rago J.P. Slonimski P.P. Yeast. 1997; 13: 1547-1562Google Scholar). It was suggested that the growth defects of crd1Δ, which resemble the phenotypes of pet56, were due to reduced PET56 expression rather than lack of CL (13Zhang M. Su X. Mileykovskaya E. Amoscato A.A. Dowhan W. J. Biol. Chem. 2003; 278: 35204-35210Google Scholar). However, as mitochondrial defects were observed in crd1Δ as compared with isogenic wild type cells, the presence of a pet56 mutation in both crd1Δ and wild type could not explain the mitochondrial defects. In the current study, we examined the effect of PET56 expression on temperature sensitivity, mitochondrial DNA loss, and respiratory function in the crd1Δ mutant. We determined that the mitochondrial defects observed in the crd1Δ mutant are independent of the pet56 mutation and that the expression of CRD1 but not PET56 complements all the defects. Materials—All chemicals used were reagent grade or better. [α-32P]UTP and [32P]orthophosphate were purchased from PerkinElmer Life Sciences. The polymerase chain reaction was performed using the MasterTag kit from Eppendorf. The Wizard Plus Miniprep DNA purification system, the PGEM-T Easy Vector system, and riboprobe system kits were from Promega. All other buffers and enzymes were purchased from Sigma. Glucose, yeast extract, and peptone were purchased from Difco. Yeast Strains and Growth Media—The S. cerevisiae strains used in this work are listed in Table I. Synthetic complete dextrose (SCD) medium contained the amino acids 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) as well as vitamins, salts (essentially components of Difco vitamin-free yeast base without amino acids), and glucose (2%). Synthetic dropout medium without leucine (Leu–medium) contained the same ingredients as SCD except leucine. Complex media contained yeast extract (1%), peptone (2%), and dextrose (2%) (YPD) or glycerol (3%) and ethanol (1%) (YPGE). Solid medium contained agar (2%) in addition to the above.Table IPlasmids and yeast strains used in this studyPlasmid or strainsCharacteristics or genotypeSource or ref.pGEM-T EASYCloning vector, ampRPromegapGEM-1631Derivative of pGEM-T EASY with genomic sequence of PET56This studypGEM-PET56Derivative of pGEM-T EASY with coding sequence of PET56This studypRS415Centromere plasmid, LEU2(30Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Google Scholar)pRS415-CRD1Derivative of pRS415, expresses CRD1 from its own promoterGuiling LipRS415-PET56Derivative of pRS415, expresses PET56 from its own promoterThis studyFGY3MAT α, ura 3-52, lys2-801, ade2-101, trp1Δ1, his3Δ200, leu2Δ1(31Jiang F. Rizavi H.S. Greenberg M.L. Mol. Microbiol. 1997; 26: 481-491Google Scholar)FGY2MAT α, ura 3-52, lys2-801, ade2-101, trp1Δ1, his3Δ200, leu2Δ1, crd1Δ::URA3(31Jiang F. Rizavi H.S. Greenberg M.L. Mol. Microbiol. 1997; 26: 481-491Google Scholar)BY4741MAT a, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0InvitrogenBY4741 crd1ΔMAT a, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, crd1Δ::KanMXInvitrogen Open table in a new tab Plasmid Construction—A 1631-bp PET56 sequence was amplified from yeast chromosomal DNA using PET56 5′ (5′-TCTGTCATCTTTGCCTTCGT-3′) and PET56 3′ (5′-TGCGAATTACGTGTTCAACG-3′). The PCR reaction products were cloned into the pGEM-T EASY vector. The resulting recombinant plasmid, pGEM-1631, was digested with SpeI and EcoRI. The DNA fragment containing PET56 was cloned into the pRS415 vector. The resulting recombinant plasmid pRS415-PET56 contains the complete PET56 coding sequence with 245-bp upstream and 147-bp downstream sequences. To make the PET56 riboprobe, PGEM-1631 was digested with SphI and NdeI, and the coding sequence of PET56 between this two restriction sites was cloned into the pGEM-T EASY vector. The resulting recombinant plasmid pGEM-PET56 was linearized with NdeI for synthesis of the PET56 riboprobe. Northern Analysis—Cells were grown to the mid-logarithmic phase in YPD, and RNA was isolated by hot phenol extraction (16Collart M.A. Oliviero S. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley Interscience, John Wiley and Sons, Inc., New York1994: 13.12.1-13.12.2Google Scholar), fractionated on an agarose gel, and transferred to a nylon membrane. The blots were hybridized with 32P-labeled PET56 riboprobes, followed by a riboprobe for the constitutively expressed actin filament protein gene ACT1 to normalize for loading variation. RNA probes for Northern analysis were synthesized using the Promega riboprobe system from plasmids linearized with restriction enzymes as follows (listed as plasmid, restriction enzyme, and RNA polymerase): (i) pGEM-PET56, NdeI, and T7 (PET56); and (ii) pPLg, BamHI, and SP6 (ACT1) (17Anderson M.S. Lopes J.M. J. Biol. Chem. 1996; 271: 26596-26601Google Scholar). The results were quantitated by phosphorimaging. Measurement of Cell Viability and Petite Formation—Yeast cells from a 5-ml overnight pre-culture were inoculated into a 10-ml prewarmed YPD medium. Cultures were incubated with continuous agitation for 5–10 days. Aliquots were serially diluted, spread on YPD plates, and incubated at 30 °C. After colonies formed, cells were replicated to YPGE to assess mitochondrial function. Petite formation was calculated based on the number of colonies that grew on YPD (total viable cells) and YPGE (respiratory competent). Isolation of Mitochondria and Measurement of Oxidative Phosphorylation—Isolation of mitochondria was performed as described V. Greenberg M.L. Biochem. J. 2000; Scholar). yeast cells were grown in medium containing non-fermentable glycerol or carbon The medium used for was and The mitochondrial was in the medium at The reaction medium for oxidative phosphorylation used was and was used as a respiratory were with the was from using analysis of yeast strains was as described (2Pfeiffer K. Gohil V. Stuart R.A. Hunte C. Brandt U. Greenberg M.L. Schagger H. J. Biol. Chem. 2003; 278: 52873-52880Google Scholar). Yeast cells were grown at 30 °C in YPD or YPGE media from a of after were with and to for at or to as described K. S. J. Biol. Chem. Scholar). were at the growth phase and digested by culture in containing glycerol and at temperature for to extraction was in of in and by C. X. S. J.P. J. Scholar) on plates in were by with in was by and by The of into an phospholipid is expressed as the of into of crd1Δ of pet56 examined colony formation in crd1Δ cells in both PET56 and pet56 strain strains have in the which a with However, in the is his3Δ1, a of the internal coding sequence of the S. Sci. U. S. A. Scholar). In contains the in which the of PET56 and is in PET56 is expressed in wild type and isogenic crd1Δ cells in the background but not in the the of and isogenic crd1Δ cells with PET56 genomic DNA containing the complete and coding sequence on a single copy PET56 expression to higher than those observed in cells of wild type and isogenic crd1Δ strains were on YPD or SCD medium and incubated at elevated temperatures. shown in crd1Δ cells could not form colonies at elevated temperatures. YPD, the mutant could not form colonies at °C, a temperature for the wild °C the mutant all of which were not synthetic colony formation was at °C. In in a PET56 background the crd1Δ mutant colony formation at elevated temperatures. To the temperature-sensitive phenotype of crd1Δ was due to the lack of CL, the crd1Δ were with a single copy plasmid the wild type expressed from its In both PET56 and pet56 genetic the wild type CRD1 gene the growth of the crd1Δ mutant at elevated temperatures on YPD and SCD of the wild type PET56 gene no effect on temperature The crd1Δ mutant viability after prolonged growth in culture and in pet56 and PET56 Viability was to wild type in mutant cells a containing the CRD1 gene not in both PET56 and pet56 strain of the wild type PET56 gene not of CRD1 but not PET56 crd1Δ from loss of viability prolonged culture at elevated temperatures. The crd1Δ and isogenic wild or with pRS415 or pRS415-PET56 were grown overnight in were inoculated into YPD at and incubated at the temperatures. cells were determined as described wild Petite in crd1Δ at of pet56 formation was in crd1Δ cells in PET56 and pet56 in the of cells in the crd1Δ mutant in the pet56 background was than wild type After prolonged growth of cells at 37 °C, the of crd1Δ mutant cells mitochondrial DNA, the of petites the wild type cells was In the PET56 background as crd1Δ mutant cells a higher of formation than the wild type when grown at °C. In both strain expression of CRD1 to a in the number of cells in the crd1Δ mutant to wild type the expression of PET56 no Oxidative in crd1Δ of PET56 function was in crd1Δ mutant cells in the PET56 genetic in wild type and mutant mitochondria at and °C and °C in crd1Δ mutant mitochondria were well as by a of from to However, at °C in in which wild type mitochondria were well oxidative phosphorylation in mutant mitochondria was mitochondria also in at °C. in both pet56 (3Koshkin V. Greenberg M.L. Biochem. J. 2002; 364: 317-322Google Scholar, 15Rieger K.J. Kaniak A. Coppee J.Y. Aljinovic G. Baudin-Baillieu A. Orlowska G. Gromadka R. Groudinsky O. Di Rago J.P. Slonimski P.P. Yeast. 1997; 13: 1547-1562Google Scholar) and PET56 backgrounds the crd1Δ mutation leads to In cells CRD1, was at °C and in crd1Δ was elevated in crd1Δ cells in both PET56 and pet56 backgrounds and with (2Pfeiffer K. Gohil V. Stuart R.A. Hunte C. Brandt U. Greenberg M.L. Schagger H. J. Biol. Chem. 2003; 278: 52873-52880Google Scholar, G. C. G. U. F. Daum G. Scholar), we observed in phosphatidylethanolamine which at the of phosphatidylcholine in crd1Δ increase in the ratio was in YPGE than in YPD media and in crd1Δ cells in the pet56 background than in PET56, in which was in the mid-logarithmic phase in YPGE medium. The phospholipid of crd1Δ and isogenic wild type cells that in crd1Δ are similar to CL in wild However, the increase in was higher in YPGE than in YPD of crd1Δ and isogenic wild type strains grown in YPD medium to the growth Open table in a new tab Table of crd1Δ and isogenic wild type strains grown in YPGE medium to the growth Open table in a new tab CL plays a role in mitochondrial function. Defective CL due to a mutation in the gene leads to the severe genetic disorder as Barth syndrome S. P. E. Bolhuis P.A. D. 1996; 12: Scholar). The of cardiomyopathy, skeletal myopathy, and neutropenia, is not with the of mutation in the that the phenotype is dependent on multiple factors that to not well understood R. F. X. S. K. S. J. J.A. Mol. Metab. 2002; Scholar). An of the function of CL may help to explain the of observed in Barth studies have shown that the yeast crd1Δ mutant, which has no CL, a of mitochondrial and cellular temperature growth, loss of mitochondrial DNA, respiratory function and membrane and import of into the mitochondria (1Schlame M. Rua D. Greenberg M.L. Prog. Lipid Res. 2000; 39: 257-288Google Scholar, K. Gohil V. Stuart R.A. Hunte C. Brandt U. Greenberg M.L. Schagger H. J. Biol. Chem. 2003; 278: 52873-52880Google Scholar, V. Greenberg M.L. Biochem. J. 2002; 364: 317-322Google Scholar, F. Ryan M.T. Schlame M. Zhao M. Gu Z. Klingenberg M. Pfanner N. Greenberg M.L. J. Biol. Chem. 2000; 275: 22387-22394Google Scholar, V. Greenberg M.L. Biochem. J. 2000; Scholar). (13Zhang M. Su X. Mileykovskaya E. Amoscato A.A. Dowhan W. J. Biol. Chem. 2003; 278: 35204-35210Google Scholar) reported that phenotypes in the mutant were by reduced expression of PET56 and that phenotypes associated with the crd1Δ mutant should be reevaluated. In this we examined the role of pet56 in crd1Δ mutant phenotypes and the (i) viability and loss of mitochondrial DNA at elevated temperature are independent of expression of CRD1 but not PET56 complements the growth defects and mitochondrial DNA (ii) and oxidative phosphorylation are in crd1Δ mutant mitochondria in PET56 and pet56 genetic Oxidative phosphorylation is in the mutant at °C or in the reaction medium. The crd1Δ mutant and in nonfermentable medium. phospholipid is apparent in both PET56 and pet56 although is in the In all crd1Δ mutant phenotypes are independent of Yeast strains with to of the strains used for the current study, wild type strain higher temperatures than the temperature sensitivity of crd1Δ in the genetic background is not apparent °C, a temperature that is for growth of the wild may explain studies not temperature sensitivity in the crd1Δ mutant. PET56, as well as other may well to in which the expression of this and other may phenotypes associated with The phenotype the of with isogenic wild type The phenotypes reported for the crd1Δ mutant (1Schlame M. Rua D. Greenberg M.L. Prog. Lipid Res. 2000; 39: 257-288Google Scholar, K. Gohil V. Stuart R.A. Hunte C. Brandt U. Greenberg M.L. Schagger H. J. Biol. Chem. 2003; 278: 52873-52880Google Scholar, V. Greenberg M.L. Biochem. J. 2002; 364: 317-322Google Scholar, F. Ryan M.T. Schlame M. Zhao M. Gu Z. Klingenberg M. Pfanner N. Greenberg M.L. J. Biol. Chem. 2000; 275: 22387-22394Google Scholar, V. Greenberg M.L. Biochem. J. 2000; Scholar) were all by with an isogenic wild type that the reduced expression of pet56 in strains no on The current study this We that was elevated in crd1Δ cells in YPD medium (4Jiang F. Ryan M.T. Schlame M. Zhao M. Gu Z. Klingenberg M. Pfanner N. Greenberg M.L. J. Biol. Chem. 2000; 275: 22387-22394Google Scholar). findings were reported by G. C. G. U. F. Daum G. Scholar), not in crd1Δ cells grown in YPD medium. In Chang S.C. Heacock P.N. Mileykovskaya E. Voelker D.R. Dowhan W. J. Biol. Chem. Scholar) and (13Zhang M. Su X. Mileykovskaya E. Amoscato A.A. Dowhan W. J. Biol. Chem. 2003; 278: 35204-35210Google Scholar) that was elevated in the mutant in YPD medium. In all the were at a single in the growth However, growth phase is to phospholipid S. M. H. H. J. Cell Biol. Scholar). effect is for mitochondrial and as mitochondrial is to resulting in reduced G. C. G. U. F. Daum G. Scholar, S. M. H. H. J. Cell Biol. Scholar, S. R.A. Greenberg M.L. J. Scholar). To this phospholipid were at growth in two crd1Δ strains by using a CL and by from the to the phase in YPD medium in YPGE medium the high which is with G. C. G. U. F. Daum G. Scholar, S. M. H. H. J. Cell Biol. Scholar). in crd1Δ cells were with wild type of CL However, the wild type CL were two higher in YPGE as compared with the YPD medium in the the of in crd1Δ cells was apparent in the nonfermentable medium and with the report G. C. G. U. F. Daum G. Scholar), the crd1Δ mutation in a increase in the in the background and a increase in the PET56 background result that cells may a of CL and PE, and the increase in when CL is may a to this In the increase in CL that membrane may this as well A.G. J.A. Dowhan W. B. J. Biol. Chem. 1993; 268: Scholar). that a may to a of in Molecular of the of in CL may help to this in of CL with to temperature The mutant, which lacks and CL, the severe growth defects at elevated temperatures. The crd1Δ mutant, which make but not CL, temperature-sensitive colony formation but at elevated temperatures when cells are YPD In the mutant, which CL, CL are than those in wild type and are in acids of wild type mutant has the severe temperature on glucose or glycerol ethanol but not on ethanol at elevated temperatures Z. Valianpour F. S. F.M. Wanders R.J. Greenberg M.L. Mol. Microbiol. Scholar). sensitivity of the functions may explain temperature sensitivity in non-fermentable the for the loss of viability on glucose is not of of this phenotype or synthetic may on the essential functions of These studies are in We John Lopes for providing its isogenic crd1Δ mutant, and the plasmid We also for providing the plasmid and for the