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• W2034190304 abstract "In yeast, as in other eukaryotes, phosphatidylcholine (PC) can be synthesized via methylation of phosphatidylethanolamine or from free choline via the CDP-choline pathway. In yeast, PC biosynthesis is required for the repression of the phospholipid biosynthetic genes, including the INO1 gene, in response to inositol. In this study, we analyzed the effect of mutations in genes encoding enzymes involved in PC biosynthesis on the transcriptional regulation of phospholipid biosynthetic genes. We report that repression of INO1 transcription in response to inositol is clearly dependent on ongoing PC biosynthesis, but it is independent of the route of synthesis. Our results also suggest that intermediates in the phosphatidylethanolamine methylation and CDP-choline pathways are not responsible for generating the regulatory signal that results in repression of INO1 and other coregulated genes of phospholipid biosynthesis. Furthermore, repression of INO1 is not tightly correlated to the proportion of PC in the total cellular phospholipids. Rather, we report that when the rate of synthesis of PC becomes growth limiting, the addition of inositol fails to repress the phospholipid biosynthetic genes, but when the rate of PC synthesis is sufficient to sustain normal growth, the addition of inositol to the growth medium has the effect of repressing INO1 and other phospholipid biosynthetic genes. In yeast, as in other eukaryotes, phosphatidylcholine (PC) can be synthesized via methylation of phosphatidylethanolamine or from free choline via the CDP-choline pathway. In yeast, PC biosynthesis is required for the repression of the phospholipid biosynthetic genes, including the INO1 gene, in response to inositol. In this study, we analyzed the effect of mutations in genes encoding enzymes involved in PC biosynthesis on the transcriptional regulation of phospholipid biosynthetic genes. We report that repression of INO1 transcription in response to inositol is clearly dependent on ongoing PC biosynthesis, but it is independent of the route of synthesis. Our results also suggest that intermediates in the phosphatidylethanolamine methylation and CDP-choline pathways are not responsible for generating the regulatory signal that results in repression of INO1 and other coregulated genes of phospholipid biosynthesis. Furthermore, repression of INO1 is not tightly correlated to the proportion of PC in the total cellular phospholipids. Rather, we report that when the rate of synthesis of PC becomes growth limiting, the addition of inositol fails to repress the phospholipid biosynthetic genes, but when the rate of PC synthesis is sufficient to sustain normal growth, the addition of inositol to the growth medium has the effect of repressing INO1 and other phospholipid biosynthetic genes. Phosphatidylcholine is synthesized in eukaryotic cells via two distinct pathways. One pathway involves three sequential methylations of phosphatidylethanolamine (PE) 1The abbreviations used are: PEphosphatidylethanolaminePCphosphatidylcholine.The structural genes used are: INO1inositol-1-phosphate synthaseCHO1phosphatidylserine synthaseCHO2 (PEM1)PE N-methyltransferaseOPI3 (PEM2)phospholipid N-methyltransferaseCKI1choline kinaseCPT1sn-1,2-diacylglycerol choline phosphotransferaseEPT1sn-1,2-diacylglycerol ethanolamine phosphotransferase. (1Bremer J. Greenberg D.M. Biochim. Biophys. Acta. 1960; 37: 173-175Crossref PubMed Scopus (103) Google Scholar). Alternatively, PC can be synthesized from free choline via the CDP-choline (“Kennedy”) pathway (2Kennedy E.P. Weiss S.B. J. Biol. Chem. 1956; 222: 193-214Abstract Full Text PDF PubMed Google Scholar). These two pathways are found in all eukaryotes that have been investigated, including mammals (3Bjornstad P. Bremer J. J. Lipid Res. 1966; 7: 38-45Abstract Full Text PDF PubMed Google Scholar) and yeast (4Steiner M.R. Lester R.L. Biochim. Biophys. Acta. 1972; 260: 222-243Crossref PubMed Scopus (108) Google Scholar, 5Waechter C. Lester R. Arch. Biochem. Biophys. 1973; 158: 401-410Crossref PubMed Scopus (38) Google Scholar). In mammals, synthesis from free choline via the CDP-choline pathway represents the major route of PC biosynthesis. Synthesis of PC via the methylation of PE has been detected only in hepatocytes and brain cells (3Bjornstad P. Bremer J. J. Lipid Res. 1966; 7: 38-45Abstract Full Text PDF PubMed Google Scholar). In yeast, PC can be synthesized de novo via the methylation pathway or, when choline is present in the growth medium, via the CDP-choline pathway (Fig. 1). phosphatidylethanolamine phosphatidylcholine. inositol-1-phosphate synthase phosphatidylserine synthase PE N-methyltransferase phospholipid N-methyltransferase choline kinase sn-1,2-diacylglycerol choline phosphotransferase sn-1,2-diacylglycerol ethanolamine phosphotransferase. Phospholipid biosynthesis is highly regulated in yeast and, curiously, the regulation of inositol- and choline-containing phospholipids is coordinated. Much of this coordinate regulation occurs at the level of gene transcription in response to the soluble precursors inositol and choline. In the presence of inositol, transcription of coregulated biosynthetic genes is repressed. If choline is added to medium in which inositol is already present, the genes are further repressed. However, if choline is present in the growth medium by itself, it has little or no effect on transcription of the coregulated genes. Genes that have been shown to exhibit this pattern of transcriptional regulation include INO1 (inositol-1-phosphate synthase), CKI1 (choline kinase), CPT1 (choline phospho-transferase), CHO1 (phosphatidylserine synthase), CHO2/PEM1 (phosphatidylethanolamine methyltransferase), and OPI3/PEM2 (phospholipid methyltransferase) (Fig. 1). The genes encoding the inositol and choline transporters are also subject to this regulation (6Paltauf F. Kohlwein S. Henry S.A. Broach J. Jones E. Pringle J. Molecular Biology of the Yeast Saccharomyces cerevisiae. Vol II. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 415Google Scholar, 7Carman G.M. Henry S.A. Ann. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar, 8Greenberg M.L. Lopes J.M. Microbiology Review. 1996; Vol. 60: 1-20Crossref Google Scholar). Furthermore, the structural genes that show this coordinated regulation in response to inositol and choline all respond to a single set of regulatory genes, including INO2, INO4, and OPI1 (6Paltauf F. Kohlwein S. Henry S.A. Broach J. Jones E. Pringle J. Molecular Biology of the Yeast Saccharomyces cerevisiae. Vol II. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 415Google Scholar, 7Carman G.M. Henry S.A. Ann. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar, 8Greenberg M.L. Lopes J.M. Microbiology Review. 1996; Vol. 60: 1-20Crossref Google Scholar). Another intriguing aspect of this coordinated regulatory response is its dependence on ongoing PC biosynthesis. Yeast strains carrying mutations in the CHO1, CHO2, or OPI3 structural genes are conditionally defective in PC biosynthesis (see Fig. 1 for the position of each lesion in the pathway). The cho1, cho2, and opi3 mutants also exhibit a conditional overproduction of inositol (Opi−) phenotype (9Greenberg M.L. Klig L.S. Letts V.A. Loewy B.S. Henry S.A. J. Bacteriol. 1983; 153: 791-799Crossref PubMed Google Scholar, 10Hirsch J.P. Henry S.A. Mol. Cell. Biol. 1986; 6: 3320-3328Crossref PubMed Scopus (181) Google Scholar, 11Summers E.F. Letts V.A. McGraw P. Henry S.A. Genetics. 1988; 120: 909-922Crossref PubMed Google Scholar, 12Greenberg M.L. Reiner B. Henry S.A. Genetics. 1982; 100: 19-33Crossref PubMed Google Scholar, 13Letts V.A. Henry S.A. J. Bacteriol. 1985; 163: 560-567Crossref PubMed Google Scholar, 14McGraw P. Henry S.A. Genetics. 1989; 122: 317-330Crossref PubMed Google Scholar). The Opi− phenotype is indicative of overexpression of inositol-1-phosphate synthase due to misregulation of the INO1 gene (10Hirsch J.P. Henry S.A. Mol. Cell. Biol. 1986; 6: 3320-3328Crossref PubMed Scopus (181) Google Scholar, 12Greenberg M.L. Reiner B. Henry S.A. Genetics. 1982; 100: 19-33Crossref PubMed Google Scholar), and the cho1, cho2, and opi3 mutants all have been shown to derepress INO1 in the presence of inositol (9Greenberg M.L. Klig L.S. Letts V.A. Loewy B.S. Henry S.A. J. Bacteriol. 1983; 153: 791-799Crossref PubMed Google Scholar, 10Hirsch J.P. Henry S.A. Mol. Cell. Biol. 1986; 6: 3320-3328Crossref PubMed Scopus (181) Google Scholar, 11Summers E.F. Letts V.A. McGraw P. Henry S.A. Genetics. 1988; 120: 909-922Crossref PubMed Google Scholar, 13Letts V.A. Henry S.A. J. Bacteriol. 1985; 163: 560-567Crossref PubMed Google Scholar, 14McGraw P. Henry S.A. Genetics. 1989; 122: 317-330Crossref PubMed Google Scholar). The other coregulated enzymes, including the CHO1, CHO2, and OPI3 gene products themselves, as well as enzymes of the CDP-choline pathway, show this same pattern of aberrant regulation when PC biosynthesis is interrupted (6Paltauf F. Kohlwein S. Henry S.A. Broach J. Jones E. Pringle J. Molecular Biology of the Yeast Saccharomyces cerevisiae. Vol II. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 415Google Scholar). Normal regulation in response to inositol is restored in these mutants and their Opi− phenotype is eliminated if a metabolite that enters the PC biosynthetic pathway downstream of the genetic block is supplied exogenously. Thus, INO1 regulation is restored in cho1 mutants (which are defective in phosphatidylserine biosynthesis) if ethanolamine, monomethylethanolamine, dimethylethanolamine, or choline is supplied (13Letts V.A. Henry S.A. J. Bacteriol. 1985; 163: 560-567Crossref PubMed Google Scholar). Regulation of INO1 is restored in cho2 mutants (which are defective in PE methylation) in response to each of the three methylated species, but not in response to ethanolamine (11Summers E.F. Letts V.A. McGraw P. Henry S.A. Genetics. 1988; 120: 909-922Crossref PubMed Google Scholar). In opi3 mutants, regulation is restored only in response to dimethylethanolamine or choline (14McGraw P. Henry S.A. Genetics. 1989; 122: 317-330Crossref PubMed Google Scholar). However, the effect of mutations in the CDP-choline pathway for PC biosynthesis on this regulation had not been explored when we commenced this study. In the present study, we have analyzed the role of PC metabolism in regulation of phospholipid biosynthesis by determining the effects of mutations in both routes of PC biosynthesis, alone or in combination. Our results demonstrate that the regulatory response is independent of the route of synthesis of PC. Analysis of the phospholipid content and growth properties of strains carrying mutations in PC biosynthesis suggests that the transcriptional response to inositol is most likely generated by the formation of PC itself and not by the availability of choline or any other intermediate in PC biosynthesis. Sources of materials were: [32P]orthophosphate (carrier free), [α-32P]cytidine 5′-triphosphate (specific activity, 800 Ci/mmol), [methyl-14C]choline chloride (specific activity, 40-60 mCi/mmol), DuPont NEN; SG81 paper, GF/A glass fiber filters, Whatman; nitrocellulose, Schleicher & Schüll; SP6/T7 Transcription Kit, Boehringer Mannheim. All other materials were reagent grade or better. To analyze which steps in PC biosynthesis are required for generation of the signal necessary for proper regulation of the coregulated genes involved in phospholipid biosynthesis, we constructed strains with defects in either one or both routes of PC biosynthesis (i.e. CDP-choline and methylation pathways, as shown in Fig. 1). To block the methylation pathway, we chose the cho2 mutant, which is defective in the first methylation step in the conversion of PE to phosphatidylmonomethylethanolamine. Strains carrying the cho2 gene disruption have grossly altered membrane phospholipid composition (PC content, between 6 and 10% of total phospholipid) and grow considerably slower than wild-type strains in the absence of choline or its methylated precursors (11Summers E.F. Letts V.A. McGraw P. Henry S.A. Genetics. 1988; 120: 909-922Crossref PubMed Google Scholar, 15Summers, E., 1989, Phospholipid Methylation and the Regulation of Phospholipid Biosynthesis in Saccharomyces cerevisiae. Ph.D. thesis, Albert Einstein College of Medicine.Google Scholar). To block the CDP-choline pathway, we used a strain in which the CKI1 gene was disrupted, as well as a double mutant strain carrying CPT1 and EPT1 gene disruptions simultaneously. The CKI1 gene encodes choline kinase, and when this gene is disrupted, yeast cells lose almost all of their choline kinase activity and most of their ethanolamine kinase activity (16Hosaka K. Tsumomu K. Yamashita S. J. Biol. Chem. 1989; 264: 2053-2059Abstract Full Text PDF PubMed Google Scholar). The cpt1 and ept1 mutations block the last steps of the CDP-choline pathway and the CDP-ethanolamine pathway, respectively, which are necessary for the formation of PC and PE from sn-1,2-diacylglycerol and CDP-choline or CDP-ethanolamine. We used a double mutant strain since it has been shown that the CPT1 and EPT1 gene products are both capable of catalyzing the cholinephosphotransferase reaction in vitro (17Hjelmstad R.H. Bell R.M. J. Biol. Chem. 1991; 266: 5094-5103Abstract Full Text PDF PubMed Google Scholar). The contribution of the EPT1 gene product to this reaction is much lower than that of the CPT1 gene product, but it is still noticeable in vivo (18McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 28010-28016Abstract Full Text PDF PubMed Google Scholar). The HJ000 strains (cpt1, ept1) and their congenic wild-type parental strain, DBY746, were generously provided by Dr. Robert Bell (Duke University Medical Center). To construct a CHO2 disruption in the genetic background of cpt1, ept1, the SalI-BglII internal fragment of the CHO2 gene (11Summers E.F. Letts V.A. McGraw P. Henry S.A. Genetics. 1988; 120: 909-922Crossref PubMed Google Scholar, 15Summers, E., 1989, Phospholipid Methylation and the Regulation of Phospholipid Biosynthesis in Saccharomyces cerevisiae. Ph.D. thesis, Albert Einstein College of Medicine.Google Scholar), carried on plasmid pSPT18 (Boehringer Mannheim), was replaced with the TRP1 gene. This construct was used to generate a disruption of the CHO2 gene in HJ000 (cpt1, ept1) and its congenic wild-type parental strain, DBY746, by homologous recombination (19Rothstein R.J. Wu R. Grossman L. Moldave K. Methods in Enzymology: Recombinant DNA. Vol 101C. Academic Press, New York1983: 202Google Scholar). Stable TRP+ transformants were tested for the characteristic cho2 mutant inositol excretion (Opi−) phenotype (11Summers E.F. Letts V.A. McGraw P. Henry S.A. Genetics. 1988; 120: 909-922Crossref PubMed Google Scholar, 12Greenberg M.L. Reiner B. Henry S.A. Genetics. 1982; 100: 19-33Crossref PubMed Google Scholar), and the disruption of a CHO2 gene was confirmed by the absence of CHO2 mRNA on Northern blots. In this way, we obtained a set of isogenic strains with blocks in the methylation pathway, the CDP-choline pathway, or simultaneously in both pathways. The full genotypes of these strains are given in Table I.Table IYeast strainsStrainGenotypeSourceDBY746Matα his3-Δ1, leu2-3,112, ura3-52, trp1-289R. BellDBY746 cho2Matα his3-Δ1, leu2-3,112, ura3-52, trp1-289, cho2::TRP1This studyHJ000Mata his3-Δ1, leu2-3,112, ura3-52, trp1-289, cpt1::LEU2, ept1-Δ1::URA3R. BellHJ000 cho2Mata his3-Δ1, leu2-3,112, ura3-52, trp1-289, cpt1::LEU2, ept1-Δ1::URA3, cho2::TRP1This studyCTY393Mata his3-200, ura3-52, lys2-801, cki1-281::HIS3V. Bankaitisw303-1BMatα ade2-1, can1-100, his3,11-15, leu2-3,-112, trp1-1, ura3-52R. RothsteinDC5Mata his3,11-15, leu2-3,-112J. BroachSH335Matα leu2-3,-112, ade2, ura3, cki1::HIS3This studySH336 (diploid)Mata/Matα cki1::HIS3/CKI1, CHO2/CHO2, his3/his3, leu2/leu2, ade2/ADE2, ura3/URA3This study1-9AMatα ade2, ura3, his3, leu2This study1-9BMata ade2, leu2, his3, cki1::HIS3This study1-9CMata leu2, his3, cki1::HIS3, cho2::LEU2This study1-9DMatα ura3, leu2, his3, cho2::LEU2This study Open table in a new tab Strain CTY393 (cki1 gene disruption mutant) was provided by Dr. Vytas Bankaitis (University of Alabama at Birmingham). Strain CTY393 was crossed to w303-1B and sporulated. Spore SH335 was crossed to DC5 to generate diploid SH336. A CHO2 disruption in the cki1 background was carried out in diploid strain SH336. The CHO2 gene in SH336 was disrupted by LEU2 gene using a construct similar to one used by Summers et al. (11Summers E.F. Letts V.A. McGraw P. Henry S.A. Genetics. 1988; 120: 909-922Crossref PubMed Google Scholar, 15Summers, E., 1989, Phospholipid Methylation and the Regulation of Phospholipid Biosynthesis in Saccharomyces cerevisiae. Ph.D. thesis, Albert Einstein College of Medicine.Google Scholar). Integration of the LEU2 gene in the CHO2 locus was confirmed by PCR. Strain SH336, with a disrupted CHO2 gene, was sporulated and sister spores 1-9A, 1-9B, 1-9C, and 1-9D (genotypes given in Table I) were used in further studies. Yeast strains were maintained on YEPD medium (1% yeast extract, 2% Bactopeptone, 3% glucose). Vitamin-defined synthetic media contained, per liter: 30 g of glucose, 5 g of ammonium sulfate, 1 g of potassium phosphate monobasic, 0.5 g of magnesium sulfate, 0.1 g of sodium chloride, 0.1 g of calcium chloride, 0.5 mg of boric acid, 0.04 mg of cupric sulfate, 0.1 mg of potassium iodide, 0.2 mg of ferric chloride, 0.4 mg of manganese sulfate, 0.2 mg of sodium molybdate, 0.4 mg of zinc sulfate, 20 mg of adenine, 20 mg of arginine, 20 mg of histidine, 60 mg of leucine, 230 mg of lysine, 20 mg of methionine, 300 mg of threonine, 20 mg of tryptophan, 40 mg of uracil, 2 μg of biotin, 400 μg of panthothenate, 2 μg of folic acid, 400 μg of niacin, 200 μg of p-aminobenzoic acid, 400 μg of pyridoxine hydrochloride. Where indicated, media were supplemented with 75 μM inositol (I+) and/or 1 mM choline (C+). All cultures were grown aerobically at 30°C with shaking. Genetic techniques such as mating, sporulation, and tetrad dissection were carried out using standard methodologies (20Sherman F. Fink G.R. Lawrence C.W. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1978Google Scholar). Yeast transformation was done by the lithium acetate method (21Gietz D. St. Jean A. Woods R.A. Schiestl R.H. Nucl. Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2899) Google Scholar) with minor modifications. RNA probes for Northern blot hybridization were synthesized according to manufacturer recommendations for the SP6/T7 Transcription Kit (Boehringer Mannheim) from plasmids described in Hudak et al. (22Hudak K.A. Lopes J.M. Henry S.A. Genetics. 1994; 136: 475-483Crossref PubMed Google Scholar), linearized with a restriction enzyme, and transcribed with a RNA polymerase as follows (plasmid/restriction enzyme/RNA polymerase): pAB309Δ/EcoRI/SP6 (TCM1), pMH203/EcoRI/SP6 (OPI3), pJH310/HindIII/T7 (INO1), pTG109/BamHI/T7 (CHO2). RNA was isolated from yeast using glass bead disruption and hot phenol extraction (23Elion E.A. Warner J.R. Cell. 1984; 39: 663-673Abstract Full Text PDF PubMed Scopus (152) Google Scholar). The TCM1 ribosomal protein gene, expression of which is unaffected by availability of inositol and choline, was used as a standard for RNA loading, as described previously (10Hirsch J.P. Henry S.A. Mol. Cell. Biol. 1986; 6: 3320-3328Crossref PubMed Scopus (181) Google Scholar). Northern hybridization was performed essentially as described by Hirsch and Henry (10Hirsch J.P. Henry S.A. Mol. Cell. Biol. 1986; 6: 3320-3328Crossref PubMed Scopus (181) Google Scholar), and the results were visualized by autoradiography and quantified by an AMBIS 4000 phosphorimager (AMBIS, Inc.). Steady-state labeling with [32P]orthophosphate was performed following the method of Atkinson et al. (24Atkinson K.D. Jensen B. Kolat A.I. Storm E.M. Henry S.A. Fogel S. J. Bacteriol. 1980; 141: 558-564Crossref PubMed Google Scholar). Cells were labeled for at least five generations with 5 μCi of [32P]orthophosphate/ml in synthetic media, as described above, and harvested in the late logarithmic phase of growth (unless otherwise indicated). Labeled lipids were extracted as described by Atkinson et al. (24Atkinson K.D. Jensen B. Kolat A.I. Storm E.M. Henry S.A. Fogel S. J. Bacteriol. 1980; 141: 558-564Crossref PubMed Google Scholar). Two-dimensional paper chromatography on silica-impregnated paper was carried out using the method of Steiner and Lester (4Steiner M.R. Lester R.L. Biochim. Biophys. Acta. 1972; 260: 222-243Crossref PubMed Scopus (108) Google Scholar). Labeled spots corresponding to specific lipids were quantified using an AMBIS 4000 phosphorimager. Yeast cells were grown for at least five to six generations with 0.2 μCi/ml of [methyl-14C]choline chloride (specific activity, 40-60 mCi/mmol) in vitamin-defined synthetic media containing 1 mM of unlabeled choline. Cultures were harvested in late logarithmic phase to early stationary phase of growth. Lipids were extracted as described by Atkinson et al. (24Atkinson K.D. Jensen B. Kolat A.I. Storm E.M. Henry S.A. Fogel S. J. Bacteriol. 1980; 141: 558-564Crossref PubMed Google Scholar) and separated in the first dimension using the method of Steiner and Lester (4Steiner M.R. Lester R.L. Biochim. Biophys. Acta. 1972; 260: 222-243Crossref PubMed Scopus (108) Google Scholar). The position of radioactively labeled PC was determined by autoradiography. Labeled spots were removed and counted by liquid scintillation. Yeast cultures were grown overnight in vitamin-defined synthetic medium containing 75 μM inositol and 1 mM choline (I+C+) to mid-logarithmic phase of growth, when radiolabeled choline was added (1 μCi/ml, 40-60 mCi/mmol [methyl-14C]choline chloride). Uptake was allowed to proceed for 60 min at 30°C with shaking. Samples were taken at indicated time points, and choline uptake was terminated by vacuum filtration of cultures through Whatman GF/A glass fiber filters, which were immediately rinsed with 15 ml of ice-cold 20 mM choline. The filters were allowed to dry, and the associated radiolabel was determined by liquid scintillation counting. The triple mutant strain cpt1, ept1, cho2 (HJ000 cho2) is viable but displayed relatively slow growth regardless of the presence or absence of choline in the medium. The doubling time for this strain was 6.5 h in synthetic medium with or without choline (Table II). Summers et al. (11Summers E.F. Letts V.A. McGraw P. Henry S.A. Genetics. 1988; 120: 909-922Crossref PubMed Google Scholar) reported that when cho2 strains are shifted to choline-free medium, they initially grow at a rate comparable to their rate of growth in choline-supplemented medium (doubling time, 2.5-3.0 h). However, after five to six generations in the absence of choline, the doubling time of cho2 strains slows to 6 h or longer. Thus, the growth rate we observed for the cho2, cpt1, ept1 strain was similar to the reported sustainable growth rate (11Summers E.F. Letts V.A. McGraw P. Henry S.A. Genetics. 1988; 120: 909-922Crossref PubMed Google Scholar) for cho2 strains grown for an extended period of time in the absence of choline. The doubling time of the cho2 strain, DBY746 cho2, in YEPD medium was comparable to the congenic DBY746 wild-type strain (1.5 h). However, the growth rate of the cpt1, ept1, cho2 triple mutant (which is congenic to DBY746 and DBY746 cho2) in YEPD medium was 6.5 h, comparable to its rate of growth in synthetic medium. Moreover, regardless of growth condition, the cells of the triple mutant displayed morphological abnormalities not seen in the DBY746 or DBY746 cho2 strains, including large, elongated cells with multiple buds and a tendency to clump. The triple mutant also generates respiratory-deficient petites at high frequency (25Griac P. Henry S.A. Op den Kamp J.A.F. NATO ASI Series: Molecular Dynamics of Biological Membranes. Springer- Verlag, Berlin, Germany1996: 339Google Scholar); a characteristic not observed in the wild type, the cho2, or the cpt1, ept1 congenic strains. In the absence of choline, the cho2, cki1 strain (1-9C) exhibited a doubling time of 6.5 h, comparable to other cho2 strains and the triple mutant, cpt1, ept1, cho2. However, unlike the cpt1, ept1, cho2 strain, the cho2, cki1 strain doubled every 2.7 h when supplemented with choline (Table II), a doubling time comparable to cho2 single mutants growing in the presence of choline.Table II.Phospholipid compositions of wild-type and mutant strainsStrainGenotypeMediumProportion of phospholipids (%)Doubling timePAPIPSPEPMMEPDMEPCOtherhDBY746Wild typeI+C+2.00.824.78.816.51.32.639.06.3DBY746Wild typeI+C−2.01.028.07.822.30.72.331.56.3DBY746cho2I+C+2.50.521.79.524.6NDaND, not detected.ND37.06.8DBY746 cho2cho2I+C−6.00.930.67.047.9NDND7.46.2HJ000cpt1, ept1I+C+2.01.229.28.121.30.40.934.34.8HJ000cpt1, ept1I+C−2.01.427.57.122.60.72.032.56.2HJ000 cho2cpt1, ept1, cho2I+C+6.51.233.95.146.8NDND9.73.2HJ000 cho2cpt1, ept1, cho2I+C−6.51.432.65.544.8NDND10.75.01-9Dcho2I+C+3.00.821.39.422.9NDND36.98.51-9Dcho2I+C+, stationary phase0.421.810.122.0NDND30.615.11-9Ccho2, cki1I+C+2.71.328.77.145.4NDND11.75.81-9Ccho2, cki1I+C+, stationary phase0.123.09.539.2NDND18.010.2a ND, not detected. Open table in a new tab The overproduction of inositol (Opi−) phenotype (12Greenberg M.L. Reiner B. Henry S.A. Genetics. 1982; 100: 19-33Crossref PubMed Google Scholar) is indicative of overexpression of inositol-1-phosphate synthase due to misregulation of the INO1 gene. Neither of the wild-type strains used in this study (DBY746 and 1-9A) or the strains carrying mutations in the CDP-choline pathway (HJ000 and 1-9B) displayed an Opi− phenotype, regardless of the presence or absence of choline in the medium. However, the cho2 single mutant strains used in this study (DBY746 cho2 and 1-9D) displayed the Opi− phenotype, but only on media lacking choline, consistent with the previously reported phenotype of cho2 strains (11Summers E.F. Letts V.A. McGraw P. Henry S.A. Genetics. 1988; 120: 909-922Crossref PubMed Google Scholar). Those strains with metabolic lesions in both pathways leading to synthesis of PC; i.e. cpt1, ept1, cho2 (HJ000 cho2) and cho2, cki1 (1-9C), displayed an Opi− phenotype regardless of the presence or absence of choline in the medium. However, the size of the Opi− inositol excretion ring was considerably larger for the cpt1, ept1, cho2 strain (HJ000 cho2) compared to the cho2, cki1 strain (1-9C). Mutants with defects in the CDP-choline pathway display lower levels of choline uptake than wild-type cells, but the decreased rate of choline import is apparent only after metabolites of the CDP-choline pathway have accumulated within the cell (18McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 28010-28016Abstract Full Text PDF PubMed Google Scholar). We assessed levels of choline uptake in the cpt1, ept1, cho2 triple mutant, in the cpt1, ept1 double mutant, and in the cho2 mutant and compared them to the congenic wild-type strain, DBY746 (Fig. 2). The rate of choline uptake in the strains containing the cpt1 and ept1 mutations, cpt1, ept1 (HJ000) and cpt1, ept1, cho2 (HJ000 cho2), was decreased by about one-half compared to their CPT1, EPT1 counterparts (wild-type (DBY746) or DBY746 cho2 strains, respectively). In contrast, strains carrying the cho2 mutation in the CPT1, EPT1 background (DBY746 cho2) or in combination with the cpt1 and ept1 mutations (HJ001 cho2) had significantly increased uptake of choline when compared to their CHO2 counterparts (i.e. the wild-type strain, DBY746, or the cpt1, ept1 strain, HJ000, respectively; Fig. 2). To determine whether the cki1 mutation or the combination of cpt1 and ept1 mutations could completely prevent synthesis of PC from 14C-labeled choline, mutant strains were grown in defined medium containing 75 μM inositol and 1 mM choline in the presence of 14C-labeled choline and the lipids were then extracted. Incorporation of the label into PC was analyzed as described under “Experimental Procedures.” Incorporation of labeled choline into PC in the cki1 strain (1-9B) occurred at approximately 10% of the level of incorporation observed in the wild-type strain (data not shown). This result is consistent with previous reports that the cki1 mutant retains some limited ability to incorporate choline into PC via the CDP-choline pathway (16Hosaka K. Tsumomu K. Yamashita S. J. Biol. Chem. 1989; 264: 2053-2059Abstract Full Text PDF PubMed Google Scholar, 25Griac P. Henry S.A. Op den Kamp J.A.F. NATO ASI Series: Molecular Dynamics of Biological Membranes. Springer- Verlag, Berlin, Germany1996: 339Google Scholar). In the cho2, cki (1-9C) strain, incorporation of labeled choline into PC was elevated about 5-fold compared to the congenic cki1 strain and was approximately 50% of the level observed in wild-type cells (data not shown), as previously reported (25Griac P. Henry S.A. Op den Kamp J.A.F. NATO ASI Series: Molecular Dynamics of Biological Membranes. Springer- Verlag, Berlin, Germany1996: 339Google Scholar). Strains carrying a combination of the cpt1 and ept1 mutations (i.e. cpt1, ept1 (HJ000) or cpt1, ept1, cho2 (HJ000 cho2)) were both incapable of incorporating any detectable labeled choline into PC (data not shown), confirming that the block in PC biosynthesis via the CDP choline pathway is complete in the cpt1, ept1 double mutant, as reported previously (17Hjelmstad R.H. Bell R.M. J. Biol. Chem. 1991; 266: 5094-5103Abstract Full Text PDF PubMed Google Scholar, 18McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 28010-28016Abstract Full Text PDF PubMed Google Scholar). Phospholipid composition was determined for the various" @default.
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