FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2031189145> ?p ?o ?g. }

• W2031189145 endingPage "32300" @default.
• W2031189145 startingPage "32294" @default.
• W2031189145 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 previously (18Koshkin V. Greenberg M.L. Biochem. J. 2000; 347: 687-691Google Scholar). Briefly, yeast cells were grown in complex medium containing non-fermentable (3% glycerol or 3% glycerol/1% ethanol) carbon sources. The medium used for isolation was 0.6 m mannitol, 20 mm Hepes/KOH, pH 7.1, 1 mm EGTA, and 0.2% (w/v) bovine serum albumin. The final mitochondrial pellet was suspended in the isolation medium at ∼10 mg/ml protein. The reaction medium for oxidative phosphorylation used was 0.6 m mannitol, 20 mm Hepes/KOH, pH 7.1, 10 mm potassium phosphate, pH 7.1, 2 mm MgCl2, 1 mm EGTA, and 0.1% (w/v) bovine serum albumin. NADH (2 mm) was used as a respiratory substrate. Respiration rates were measured with the Clark-type electrode. Phosphorylation was estimated from ADP-stimulated respiration using total oxygen consumption. Steady State Phospholipid Determination—Phospholipid analysis of yeast strains was carried out essentially 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 starting A550 of 0.025. Immediately after inoculation, cultures were supplemented with 10 μCi 32P ml–1 and allowed to grow for at least five or six generations to achieve steady state labeling as described (19Atkinson K. Fogel S. Henry S.A. J. Biol. Chem. 1980; 255: 6653-6661Google Scholar). Cells were harvested at the indicated growth phase and digested by zymolyase (1.5–2.5 mg/ml culture in 50 mm Tris-SO4 buffer, pH 7.5, containing 1.2 m glycerol and 100 mm sodium thioglycolate) at room temperature for 15 min to yield spheroplasts. Phospholipid extraction was carried out in 20 volumes of 2:1 chloroform/methanol partitioned in sterile water and separated by one-dimensional thin layer chromatography (20Leray C. Pelletier X. Hemmendinger S. Cazenave J.P. J. Chromatogr. 1987; 420: 411-416Google Scholar) on LK5 silica gel 150-Å plates in chloroform/ethanol/water/triethylamine (30: 35:7:35; v/v). Phospholipids were identified by co-migration with known standards. 32Pi in individual phospholipids was visualized by phosphorimaging and quantified by ImageQuant software (Molecular Dynamics). The incorporation of radiolabel (32Pi) into an individual phospholipid is expressed as the percentage of radiolabel incorporated into total phospholipids. Temperature Sensitivity of crd1Δ Is Independent of pet56 —We examined colony formation in crd1Δ cells in both PET56 (BY4741) and pet56 (FGY3) strain backgrounds. Both strains have mutations in the HIS3 gene, which shares a common promoter with PET56. However, in BY4741 the HIS3 allele is his3Δ1, a 187-bp deletion of only the internal coding sequence of HIS3, leaving the promoter intact (21Scherer S. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4951-4955Google Scholar). In contrast, FGY3 contains the his3Δ200 allele in which the common promoter region of PET56 and HIS3 is deleted. As seen in Fig. 1A, PET56 is fully expressed in wild type and isogenic crd1Δ cells in the BY4741 background but not in the FGY3 background. As expected, the transformation of FGY3 and isogenic crd1Δ cells with PET56 genomic DNA containing the complete promoter and coding sequence on a single copy vector restored PET56 expression to levels slightly higher than those observed in BY4741. Single cells of wild type (BY4741) and isogenic crd1Δ strains were plated on YPD or SCD medium and incubated at elevated temperatures. As shown in Fig. 1B, crd1Δ cells could not form colonies at elevated temperatures. On YPD, the mutant could not form colonies at 41 °C, a permissive temperature for the wild type. At 39 °C the mutant formed small colonies, all of which were petite (data not shown). On synthetic medium, colony formation was defective even at 39 °C. In summary, in a PET56 background the crd1Δ mutant exhibited defective colony formation at elevated temperatures. To determine whether the temperature-sensitive phenotype of crd1Δ was due to the lack of CL, the crd1Δ mutants were transformed with pRS415-CRD1, a single copy plasmid bearing the wild type CRD1gene expressed from its original promoter. In both PET56 and pet56 genetic backgrounds, the wild type CRD1 gene restored the growth of the crd1Δ mutant at elevated temperatures on YPD and SCD (Fig. 2). Expression of the wild type PET56 gene had no effect on temperature sensitivity. The crd1Δ mutant lost viability after prolonged growth in liquid culture (24 and 72 h in pet56 and PET56 backgrounds, respectively (Fig. 3). Viability was restored to wild type levels in mutant cells expressing a vector containing the CRD1 gene (but not empty vector) in both PET56 and pet56 strain backgrounds. Expression of the wild type PET56 gene did not affect viability.Fig. 3Expression of CRD1 but not PET56 protects crd1Δ from loss of viability following prolonged culture at elevated temperatures. The crd1Δ mutants and isogenic wild types FGY3 (pet56) or BY4741 (PET56) transformed with vector pRS415 pRS415-CRD1, or pRS415-PET56 were grown overnight in Leu–medium. Cells were inoculated into YPD at 106 cells/ml and incubated at the indicated temperatures. Viable cells were determined as described under “Experimental Procedures.” WT, wild type.View Large Image Figure ViewerDownload (PPT) Increased Petite Formation in crd1Δ Mutants at Elevated Temperatures Is Independent of pet56 —Petite formation was measured in crd1Δ cells in PET56 and pet56 backgrounds. As seen in Fig. 4, the percentage of petite cells in the crd1Δ mutant in the pet56 background was greater than among wild type cells. After prolonged growth (194 h) of cells at 37 °C, the majority of crd1Δ mutant cells had lost mitochondrial DNA, whereas the percentage of petites among the wild type cells was <10%. In the PET56 background as well, crd1Δ mutant cells exhibited a higher frequency of petite formation than did the wild type when grown at 39 °C. In both strain backgrounds, expression of CRD1 led to a reduction in the number of petite cells in the crd1Δ mutant to wild type levels, whereas the expression of PET56 had no effect. Uncoupled Oxidative Phosphorylation in crd1Δ Is Independent of PET56 —Mitochondrial function was assayed in crd1Δ mutant cells in the PET56 genetic background. Fig. 5 demonstrates oxygen consumption in wild type and mutant mitochondria at 25 and 40 °C under isotonic and hypotonic conditions. At 25 °C in isotonic conditions, crd1Δ mutant mitochondria were well coupled as indicated by a clear transition of respiration from state 3 to state 4. However, at 40 °C in isotonic medium, conditions in which wild type mitochondria were well coupled, oxidative phosphorylation in mutant mitochondria was completely uncoupled. Mutant mitochondria also exhibited defective coupling in hypotonic conditions, even at 25 °C. Therefore, 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 (Fig. 5), the crd1Δ mutation leads to defective coupling. In cells expressing CRD1, coupling was restored at 40 °C and under hypotonic conditions. Increased PG Levels in crd1Δ Cells—Phosphatidylglycerol (PG) was elevated in crd1Δ cells in both PET56 and pet56 backgrounds (Tables II and III). Consistent with previous reports (2Pfeiffer K. Gohil V. Stuart R.A. Hunte C. Brandt U. Greenberg M.L. Schagger H. J. Biol. Chem. 2003; 278: 52873-52880Google Scholar, 22Tuller G. Hrastnik C. Achleitner G. Schiefthaler U. Klein F. Daum G. FEBS Lett. 1998; 421: 15-18Google Scholar), we observed significant changes in phosphatidylethanolamine (PE) levels, which increased at the expense of phosphatidylcholine (PC) in crd1Δ cells. This increase in the PE/PC ratio was more pronounced in YPGE (Table III) than in YPD (Table II) media and in crd1Δ cells in the pet56 background than in PET56, in which it was evident only in the mid-logarithmic phase in YPGE medium. The comparative phospholipid profile of crd1Δ and isogenic wild type cells showed that PG levels in crd1Δ are similar to CL in wild type. However, the increase in PG was higher in YPGE than in YPD media.Table IIPhospholipid composition of crd1Δ and isogenic wild type strains grown in YPD medium to the indicated growth phaseGrowth phaseStrainsPDMECLPAPEPGPSPIPCPE/PCEarly logarithmicFGY3 (WT)2.93 ± 0.302.39 ± 0.284.26 ± 0.1118.67 ± 1.000.25 ± 0.175.36 ± 0.3924.16 ± 0.6440.03 ± 0.890.47FGY2 (crd1Δ)2.24 ± 0.25ND3.55 ± 0.2419.79 ± 0.682.42 ± 0.394.98 ± 0.3324.40 ± 0.5341.39 ± 0.720.48BY4741 (WT)2.62 ± 0.202.30 ± 0.343.74 ± 0.2018.71 ± 0.330.11 ± 0.006.26 ± 0.3323.29 ± 0.4941.79 ± 0.510.45ydl142c (crd1Δ)2.09 ± 0.19ND3.60 ± 0.1820.09 ± 0.871.83 ± 0.315.99 ± 0.2724.03 ± 0.6041.44 ± 0.780.48Mid-logarithmicFGY3 (WT)2.54 ± 0.362.86 ± 0.434.30 ± 0.6118.20 ± 1.210.25 ± 0.145.15 ± 0.9324.68 ± 0.5238.74 ± 1.190.47FGY2 (crd1Δ)1.99 ± 0.10ND3.52 ± 0.7619.47 ± 0.462.27 ± 0.114.98 ± 1.1524.47 ± 0.8741.09 ± 0.540.47BY4741 (WT)1.66 ± 0.143.31 ± 0.174.67 ± 0.1618.70 ± 1.300.17 ± 0.035.45 ± 0.9324.35 ± 0.7939.41 ± 0.530.47ydl142c (crd1Δ)1.62 ± 0.18ND3.93 ± 0.3117.77 ± 0.322.09 ± 0.194.96 ± 0.8926.17 ± 0.8541.56 ± 0.550.43Early stationaryFGY3 (WT)0.69 ± 0.093.84 ± 0.324.52 ± 0.1814.81 ± 0.630.32 ± 0.184.97 ± 0.4622.74 ± 1.4142.26 ± 1.220.35FGY2 (crd1Δ)0.54 ± 0.11ND4.72 ± 0.2419.70 ± 0.593.28 ± 0.235.34 ± 0.4721.94 ± 0.8942.39 ± 0.350.46BY4741 (WT)0.65 ± 0.085.06 ± 0.745.21 ± 0.1117.50 ± 0.360.36 ± 0.054.81 ± 0.5522.33 ± 1.0041.61 ± 0.850.42ydl142c (crd1Δ)0.83 ± 0.04ND5.27 ± 0.4718.30 ± 0.783.35 ± 0.394.99 ± 0.6522.47 ± 1.6840.75 ± 0.930.45Late stationaryFGY3 (WT)ND7.24 ± 0.584.75 ± 0.9417.24 ± 1.250.42 ± 0.032.87 ± 1.1021.95 ± 0.6742.44 ± 1.500.41FGY2 (crd1Δ)0.39 ± 0.13ND5.23 ± 0.6422.81 ± 1.236.56 ± 0.752.66 ± 0.1819.83 ± 0.5239.43 ± 0.900.58BY4741 (WT)ND7.46 ± 0.545.98 ± 0.7515.24 ± 1.150.44 ± 0.091.78 ± 0.3522.76 ± 1.1043.80 ± 2.150.35ydl142c (crd1Δ)0.63 ± 0.05ND6.66 ± 0.7818.75 ± 0.555.25 ± 0.262.49 ± 0.1121.66 ± 0.6440.95 ± 1.230.46 Open table in a new tab Table IIIPhospholipid composition of crd1Δ and isogenic wild type strains grown in YPGE medium to the indicated growth phaseGrowth phaseStrainsPDMECLPAPEPGPSPIPCPE/PCEarly logarithmicFGY3 (WT)ND7.07 ± 0.823.68 ± 0.3522.27 ± 2.110.48 ± 0.274.55 ± 1.6222.59 ± 0.5937.18 ± 1.310.60FGY2 (crd1Δ)0.38 ± 0.11ND4.67 ± 0.8828.00 ± 0.787.74 ± 0.381.87 ± 1.0621.20 ± 1.6533.91 ± 1.820.83BY4741 (WT)ND7.80 ± 0.903.92 ± 0.1520.99 ± 0.790.64 ± 0.152.55 ± 0.8023.69 ± 2.1438.12 ± 2.240.55ydl142c (crd1Δ)0.15 ± 0.01ND3.94 ± 0.1022.55 ± 1.687.23 ± 0.202.57 ± 0.4422.90 ± 1.9739.06 ± 1.210.58Mid-logarithmicFGY3 (WT)ND6.91 ± 0.633.46 ± 0.3817.29 ± 0.990.47 ± 0.294.17 ± 0.7021.84 ± 0.9043.93 ± 1.320.39FGY2 (crd1Δ)0.22 ± 0.05ND3.98 ± 0.2725.27 ± 2.337.65 ± 0.563.47 ± 0.3120.80 ± 1.4836.83 ± 1.630.69BY4741 (WT)ND7.72 ± 0.423.92 ± 1.1016.23 ± 3.540.63 ± 0.222.24 ± 0.8322.46 ± 0.9244.59 ± 1.310.36ydl142c (crd1Δ)0.15 ± 0.01ND3.94 ± 0.1019.74 ± 0.778.08 ± 0.232.94 ± 1.5921.68 ± 0.6141.58 ± 0.620.47Early stationaryFGY3 (WT)ND6.33 ± 1.005.01 ± 0.5112.28 ± 2.220.47 ± 0.123.30 ± 1.4223.00 ± 2.2446.79 ± 4.270.26FGY2 (crd1Δ)0.15 ± 0.04ND4.88 ± 0.1519.51 ± 1.597.75 ± 0.243.02 ± 0.7619.58 ± 1.8441.89 ± 2.780.47BY4741 (WT)ND7.08 ± 0.415.44 ± 0.5412.90 ± 0.350.57 ± 0.181.12 ± 0.2524.21 ± 2.045.68 ± 1.140.28ydl142c (crd1Δ)NDND5.40 ± 0.1314.94 ± 2.727.88 ± 0.172.19 ± 0.1923.88 ± 0.2042.86 ± 2.560.35Late stationaryFGY3 (WT)ND5.39 ± 1.305.42 ± 0.6811.63 ± 1.250.48 ± 0.091.76 ± 0.6921.95 ± 1.4849.36 ± 2.360.24FGY2 (crd1Δ)0.17 ± 0.08ND5.57 ± 0.1215.46 ± 1.976.22 ± 0.362.81 ± 0.4919.15 ± 1.5946.99 ± 3.570.33BY4741 (WT)ND7.19 ± 0.035.03 ± 0.5010.49 ± 0.390.65 ± 0.10ND26.13 ± 0.8147.28 ± 0.920.22ydl142c (crd1Δ)NDND5.56 ± 0.4511.98 ± 1.787.92 ± 0.16ND24 ± 3.2347.32 ± 0.650.25 Open table in a new tab CL plays a crucial role in mitochondrial function. Defective CL biosynthesis due to a mutation in the human G4.5 (TAZ) gene leads to the severe genetic disorder known as Barth syndrome (23Bione S. D'Adamo P. Maestrini E. Gedeon A.K. Bolhuis P.A. Toniolo D. Nat. Genet. 1996; 12: 385-389Google Scholar). The severity of symptoms, including cardiomyopathy, skeletal myopathy, and neutropenia, is not strongly correlated with the degree of mutation in the TAZ gene, indicating that the phenotype is dependent on multiple factors that are, to date, not well understood (24Chen R. Tsuji T. Ichida F. Bowles K.R. Yu X. Watanabe S. Hirono K. Tsubata S. Hamamichi Y. Ohta J. Imai Y. Bowles N.E. Miyawaki T. Towbin J.A. Mol. Genet. Metab. 2002; 77: 319-325Google Scholar). An understanding of the function of CL may ultimately help to explain the varying degrees of severity observed in Barth syndrome. Several studies have shown that the yeast crd1Δ mutant, which has no CL, exhibits a variety of mitochondrial and cellular defects, including temperature sensitive growth, loss of mitochondrial DNA, decreased respiratory function and membrane potential, and defective import of proteins into the mitochondria (1Schlame M. Rua D. Greenberg M.L. Prog. Lipid Res. 2000; 39: 257-288Google Scholar, 2Pfeiffer K. Gohil V. Stuart R.A. Hunte C. Brandt U. Greenberg M.L. Schagger H. J. Biol. Chem. 2003; 278: 52873-52880Google Scholar, 3Koshkin V. Greenberg M.L. Biochem. J. 2002; 364: 317-322Google Scholar, 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, 18Koshkin V. Greenberg M.L. Biochem. J. 2000; 347: 687-691Google Scholar). More recently, Zhang et al. (13Zhang M. Su X. Mileykovskaya E. Amoscato A.A. Dowhan W. J. Biol. Chem. 2003; 278: 35204-35210Google Scholar) reported that several phenotypes in the CL-deficient mutant were exacerbated by reduced expression of PET56 and concluded that phenotypes associated with the crd1Δ mutant should be reevaluated. In this report, we examined the role of pet56 in crd1Δ mutant phenotypes and concluded the following. (i) Decreased viability and loss of mitochondrial DNA at elevated temperature are independent of pet56. Episomal expression of CRD1 but not PET56 complements the growth defects and restores mitochondrial DNA stability. (ii) Coupling and oxidative phosphorylation are defective in crd1Δ mutant mitochondria in PET56 and pet56 genetic backgrounds. Oxidative phosphorylation is completely uncoupled in the mutant at 40 °C or in the hypotonic reaction medium. (iii) The crd1Δ mutant exhibits increased PE and decreased phosphatidylcholine, especially in nonfermentable medium. This altered phospholipid profile is apparent in both PET56 and pet56 backgrounds, although it is more pronounced in the latter. In summary, all crd1Δ mutant phenotypes tested are independent of PET56. Yeast “wild type” strains differ greatly with respect to their degree of thermal tolerance. Among the strains used for the current study, wild type strain BY4741 tolerates significantly higher temperatures than does FGY3. Therefore, the temperature sensitivity of crd1Δ in the BY4741 genetic background is not apparent below 39 °C, a temperature that is permissible for growth of the wild type. This may explain why previous studies did not detect temperature sensitivity in the crd1Δ mutant. PET56, as well as other genes, may very well contribute to overall thermal tolerance, in which case the expression of this and other genes may suppress phenotypes associated with thermal sensitivity. The suppression phenotype underscores the importance of comparing mutants with isogenic wild type strains. The phenotypes reported previously for the crd1Δ mutant (1Schlame M. Rua D. Greenberg M.L. Prog. Lipid Res. 2000; 39: 257-288Google Scholar, 2Pfeiffer K. Gohil V. Stuart R.A. Hunte C. Brandt U. Greenberg M.L. Schagger H. J. Biol. Chem. 2003; 278: 52873-52880Google Scholar, 3Koshkin V. Greenberg M.L. Biochem. J. 2002; 364: 317-322Google Scholar, 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, 18Koshkin V. Greenberg M.L. Biochem. J. 2000; 347: 687-691Google Scholar) were all identified by comparison with an isogenic wild type strain, suggesting that the reduced expression of pet56 in these strains had no bearing on crd1Δ-associated phenotypes. The current study confirms this conclusion. We previously showed that PG was only slightly 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). Similar findings were reported by Tuller et al. (22Tuller G. Hrastnik C. Achleitner G. Schiefthaler U. Klein F. Daum G. FEBS Lett. 1998; 421: 15-18Google Scholar), who did not detect any PG in crd1Δ cells grown in YPD medium. In contrast, Chang et al. (25Chang S.C. Heacock P.N. Mileykovskaya E. Voelker D.R. Dowhan W. J. Biol. Chem. 1998; 273: 14933-14941Google Scholar) and Zhang et al. (13Zhang M. Su X. Mileykovskaya E. Amoscato A.A. Dowhan W. J. Biol. Chem. 2003; 278: 35204-35210Google Scholar) showed that PG was elevated in the mutant in YPD medium. In all the above studies, measurements were taken at a single point in the growth phase. However, growth phase is known to regulate phospholipid levels (26Jakovcic S. Getz G.S. Rabinowitz M. Jakob H. Swift H. J. Cell Biol. 1971; 48: 490-502Google Scholar). This effect is specifically important for mitochondrial phospholipids (PG and CL), as mitochondrial development is subject to glucose-mediated repression, resulting in reduced mitochondria-specific phospholipids (22Tuller G. Hrastnik C. Achleitner G. Schiefthaler U. Klein F. Daum G. FEBS Lett. 1998; 421: 15-18Google Scholar, 26Jakovcic S. Getz G.S. Rabinowitz M. Jakob H. Swift H. J. Cell Biol. 1971; 48: 490-502Google Scholar, 27Gaynor P.M. Hubbell S. Schmidt A.J. Lina R.A. Minskoff S.A. Greenberg M.L. J. Bacteriol. 1991; 173: 6124-6131Google Scholar). To resolve this discrepancy, phospholipid analyses were carried out at different growth phases in two different crd1Δ strains by using a sensitive radiolabeling approach. CL and PG levels increased by >2-fold from the early logarithmic to the late stationary phase in YPD medium (Table II), whereas in YPGE medium the levels remained constantly high (Table III), which is consistent with previous reports (22Tuller G. Hrastnik C. Achleitner G. Schiefthaler U. Klein F. Daum G. FEBS Lett. 1998; 421: 15-18Google Scholar, 26Jakovcic S. Getz G.S. Rabinowitz M. Jakob H. Swift H. J. Cell Biol. 1971; 48: 490-502Google Scholar). PG levels in crd1Δ cells were comparable with wild type levels of CL plus PG. However, because the wild type CL levels were almost two times higher in YPGE as compared with the YPD medium in the logarithmic phase, the elevation of PG in crd1Δ cells was more apparent in the nonfermentable medium (Tables II and III). Consistent with the previous report (22Tuller G. Hrastnik C. Achleitner G. Schiefthaler U. Klein F. Daum G. FEBS Lett. 1998; 421: 15-18Google Scholar), the crd1Δ mutation resulted in a significant increase in the non-bilayer-forming lipid PE in the FGY3 (pet56) background and a somewhat smaller increase in the PET56 background (Table III). This result suggests that cells may require a critical amount of non-bilayer-forming phospholipids such CL and PE, and the increase in PE when CL is limiting may reflect a response to this need. In Escherichia coli, the increase in CL that accompanies decreased membrane PE may reflect this need as well (28Rietveld A.G. Killian J.A. Dowhan W. de Kruijff B. J. Biol. Chem. 1993; 268: 12427-12433Google Scholar). This suggests that a mechanism may exist to maintain a critical amount of non-bilayer-forming phospholipids in mitochondria. Molecular characterization of the increased rate of PE biosynthesis in CL mutants may help to elucidate this mechanism. Mutants in different steps of CL biosynthesis differ greatly with respect to temperature sensitivity. The pgs1Δ mutant, which lacks PG and CL, exhibits the most severe growth defects at elevated temperatures. The crd1Δ mutant, which can make PG but not CL, exhibits temperature-sensitive colony formation but can grow at elevated temperatures when cells are patched onto YPD plates. In the taz1Δ mutant, which can synthesize CL, CL levels are less than those in wild type and are lacking in unsaturated fatty acids typical of wild type CL. This mutant has the least severe temperature sensitivity; it grows normally on glucose or glycerol plus ethanol but not on ethanol alone at elevated temperatures (29Gu Z. Valianpour F. Chen S. Vaz F.M. Hakkaart G.A. Wanders R.J. Greenberg M.L. Mol. Microbiol. 2004; 51: 149-158Google Scholar). Although thermal sensitivity of the bioenergetic functions may explain temperature sensitivity in non-fermentable medium, the reason for the loss of viability on glucose is not known. Identification of suppressors of this phenotype or synthetic lethal mutants may shed light on the essential functions of CL. These studies are in progress. We thank Dr. John Lopes for providing BY4741 strain, its isogenic crd1Δ mutant, and the plasmid pPLG SP6. We also thank Guiling Li for providing the plasmid pRS415-CRD1 and Asimur Rahman for preparing the figures." @default.
• W2031189145 created "2016-06-24" @default.
• W2031189145 creator A5013405117 @default.
• W2031189145 creator A5028190348 @default.
• W2031189145 creator A5072552838 @default.
• W2031189145 creator A5088654871 @default.
• W2031189145 date "2004-07-01" @default.
• W2031189145 modified "2023-09-26" @default.
• W2031189145 title "Absence of Cardiolipin Results in Temperature Sensitivity, Respiratory Defects, and Mitochondrial DNA Instability Independent of pet56" @default.
• W2031189145 cites W1517722666 @default.
• W2031189145 cites W1548309530 @default.
• W2031189145 cites W1588347494 @default.
• W2031189145 cites W1598131238 @default.
• W2031189145 cites W1786885738 @default.
• W2031189145 cites W1942537637 @default.
• W2031189145 cites W1972293491 @default.
• W2031189145 cites W1972666591 @default.
• W2031189145 cites W1974145389 @default.
• W2031189145 cites W1976019826 @default.
• W2031189145 cites W1999914003 @default.
• W2031189145 cites W2000181169 @default.
• W2031189145 cites W2004693594 @default.
• W2031189145 cites W2014521800 @default.
• W2031189145 cites W2014716696 @default.
• W2031189145 cites W2024298913 @default.
• W2031189145 cites W2030113968 @default.
• W2031189145 cites W2033050778 @default.
• W2031189145 cites W2036799921 @default.
• W2031189145 cites W2041982853 @default.
• W2031189145 cites W2042183674 @default.
• W2031189145 cites W2051576043 @default.
• W2031189145 cites W2062564411 @default.
• W2031189145 cites W2063398291 @default.
• W2031189145 cites W2069129416 @default.
• W2031189145 cites W2099103099 @default.
• W2031189145 cites W2112727465 @default.
• W2031189145 cites W2116878121 @default.
• W2031189145 cites W2150493533 @default.
• W2031189145 cites W2169333620 @default.
• W2031189145 doi "https://doi.org/10.1074/jbc.m403275200" @default.
• W2031189145 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15169766" @default.
• W2031189145 hasPublicationYear "2004" @default.
• W2031189145 type Work @default.
• W2031189145 sameAs 2031189145 @default.
• W2031189145 citedByCount "95" @default.
• W2031189145 countsByYear W20311891452012 @default.
• W2031189145 countsByYear W20311891452013 @default.
• W2031189145 countsByYear W20311891452014 @default.
• W2031189145 countsByYear W20311891452015 @default.
• W2031189145 countsByYear W20311891452016 @default.
• W2031189145 countsByYear W20311891452017 @default.
• W2031189145 countsByYear W20311891452018 @default.
• W2031189145 countsByYear W20311891452019 @default.
• W2031189145 countsByYear W20311891452020 @default.
• W2031189145 countsByYear W20311891452021 @default.
• W2031189145 countsByYear W20311891452022 @default.
• W2031189145 crossrefType "journal-article" @default.
• W2031189145 hasAuthorship W2031189145A5013405117 @default.
• W2031189145 hasAuthorship W2031189145A5028190348 @default.
• W2031189145 hasAuthorship W2031189145A5072552838 @default.
• W2031189145 hasAuthorship W2031189145A5088654871 @default.
• W2031189145 hasBestOaLocation W20311891451 @default.
• W2031189145 hasConcept C104317684 @default.
• W2031189145 hasConcept C105702510 @default.
• W2031189145 hasConcept C121332964 @default.
• W2031189145 hasConcept C12554922 @default.
• W2031189145 hasConcept C127413603 @default.
• W2031189145 hasConcept C153911025 @default.
• W2031189145 hasConcept C185592680 @default.
• W2031189145 hasConcept C207821765 @default.
• W2031189145 hasConcept C21200559 @default.
• W2031189145 hasConcept C24326235 @default.
• W2031189145 hasConcept C24586158 @default.
• W2031189145 hasConcept C2778918659 @default.
• W2031189145 hasConcept C2781302388 @default.
• W2031189145 hasConcept C41625074 @default.
• W2031189145 hasConcept C534529494 @default.
• W2031189145 hasConcept C552990157 @default.
• W2031189145 hasConcept C55493867 @default.
• W2031189145 hasConcept C57879066 @default.
• W2031189145 hasConcept C86803240 @default.
• W2031189145 hasConceptScore W2031189145C104317684 @default.
• W2031189145 hasConceptScore W2031189145C105702510 @default.
• W2031189145 hasConceptScore W2031189145C121332964 @default.
• W2031189145 hasConceptScore W2031189145C12554922 @default.
• W2031189145 hasConceptScore W2031189145C127413603 @default.
• W2031189145 hasConceptScore W2031189145C153911025 @default.
• W2031189145 hasConceptScore W2031189145C185592680 @default.
• W2031189145 hasConceptScore W2031189145C207821765 @default.
• W2031189145 hasConceptScore W2031189145C21200559 @default.
• W2031189145 hasConceptScore W2031189145C24326235 @default.
• W2031189145 hasConceptScore W2031189145C24586158 @default.
• W2031189145 hasConceptScore W2031189145C2778918659 @default.
• W2031189145 hasConceptScore W2031189145C2781302388 @default.
• W2031189145 hasConceptScore W2031189145C41625074 @default.
• W2031189145 hasConceptScore W2031189145C534529494 @default.
• W2031189145 hasConceptScore W2031189145C552990157 @default.
• W2031189145 hasConceptScore W2031189145C55493867 @default.

• next