Differential Partitioning of Lipids Metabolized by Separate Yeast Glycerol-3-phosphate Acyltransferases Reveals That Phospholipase D Generation of Phosphatidic Acid Mediates Sensitivity to Choline-containing Lysolipids and Drugs
2002; Elsevier BV; Volume: 277; Issue: 41 Linguagem: Inglês
10.1074/jbc.m207753200
ISSN1083-351X
AutoresVanina Zaremberg, Christopher R. McMaster,
Tópico(s)Plant Surface Properties and Treatments
ResumoIn this study we demonstrate that theGAT1 and GAT2 genes encode the major glycerol-3-phosphate acyltransferase activities in Saccharomyces cerevisiae. Genetic inactivation of either GAT1 orGAT2 did not alter cell growth but inactivation of both resulted in growth cessation. Metabolic analyses of gat1and gat2 yeast detected that the major differences were: (i) a 50% increase in the rate of triacylglycerol synthesis ingat1 yeast and a corresponding 50% decrease ingat2 yeast, and (ii) a 5-fold increase in glycerophosphocholine production through deacylation of phosphatidylcholine synthesized through the CDP-choline pathway ingat1 yeast, whereas gat2 yeast displayed a 10-fold decrease. To address why we observed alterations in phospholipid turnover specific to phosphatidylcholine produced through the CDP-choline pathway in gat1 and gat2 yeast we tested their sensitivity to various cytotoxic lysolipids and observed that gat2 cells were more sensitive to lysophosphatidylcholine, but not other lysolipids. To pursue the mechanism we analyzed their sensitivity to choline-containing lysolipids or drugs that could not be deacylated and/or reacylated. Our data showed that gat1 and gat2 yeast were resistant and sensitive to lysoplatelet activating factor, platelet activating factor, and the anti-tumor lipid edelfosine, respectively, indicating that their sensitivity to these compounds was not because of differences in rates of phosphatidylcholine deacylation. As growth of gat2 cells was impaired in the presence of ethanol, a phospholipase D (Spo14p) inhibitor, we inferred that phospholipase D may play important biologic and metabolic roles in phenotypes observed in gat yeast. Genetic inactivation of the SPO14 gene resulted in increased susceptibility, whereas expression of Escherichia coli diacylglycerol kinase relieved growth inhibition, to choline-containing lysolipids and drugs. Our results are consistent with a model whereby phosphatidic acid generated from phosphatidylcholine hydrolysis by Spo14p regulates susceptibility to choline-containing lysolipid analogs and drugs. In this study we demonstrate that theGAT1 and GAT2 genes encode the major glycerol-3-phosphate acyltransferase activities in Saccharomyces cerevisiae. Genetic inactivation of either GAT1 orGAT2 did not alter cell growth but inactivation of both resulted in growth cessation. Metabolic analyses of gat1and gat2 yeast detected that the major differences were: (i) a 50% increase in the rate of triacylglycerol synthesis ingat1 yeast and a corresponding 50% decrease ingat2 yeast, and (ii) a 5-fold increase in glycerophosphocholine production through deacylation of phosphatidylcholine synthesized through the CDP-choline pathway ingat1 yeast, whereas gat2 yeast displayed a 10-fold decrease. To address why we observed alterations in phospholipid turnover specific to phosphatidylcholine produced through the CDP-choline pathway in gat1 and gat2 yeast we tested their sensitivity to various cytotoxic lysolipids and observed that gat2 cells were more sensitive to lysophosphatidylcholine, but not other lysolipids. To pursue the mechanism we analyzed their sensitivity to choline-containing lysolipids or drugs that could not be deacylated and/or reacylated. Our data showed that gat1 and gat2 yeast were resistant and sensitive to lysoplatelet activating factor, platelet activating factor, and the anti-tumor lipid edelfosine, respectively, indicating that their sensitivity to these compounds was not because of differences in rates of phosphatidylcholine deacylation. As growth of gat2 cells was impaired in the presence of ethanol, a phospholipase D (Spo14p) inhibitor, we inferred that phospholipase D may play important biologic and metabolic roles in phenotypes observed in gat yeast. Genetic inactivation of the SPO14 gene resulted in increased susceptibility, whereas expression of Escherichia coli diacylglycerol kinase relieved growth inhibition, to choline-containing lysolipids and drugs. Our results are consistent with a model whereby phosphatidic acid generated from phosphatidylcholine hydrolysis by Spo14p regulates susceptibility to choline-containing lysolipid analogs and drugs. phosphatidic acid glycerol 3-phosphate phosphatidylcholine lysophosphatidylcholine triacylglycerol diacylglycerol platelet activating factor lysoplatelet activating factor Glycerophospholipids are found in cell membranes and are generally comprised of two fatty acid molecules attached to the first two carbons of the glycerol backbone with a phospho-head group attached to the third carbon. Glycerophospholipids provide the physical permeability barrier for cellular and organellar membranes and also serve as a reservoir for numerous second messenger signaling molecules including arachidonic acid, phosphatidic acid (PA),1 diacylglycerol (DAG), and inositol polyphosphates (1Kent C. Annu. Rev. Biochem. 1995; 64: 315-343Crossref PubMed Scopus (311) Google Scholar, 2Murakami M. Nakatani Y. 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Nutr. 2000; 20: 77-103Crossref PubMed Scopus (263) Google Scholar). Glycerolipid synthesis is initiated by glycerol-3-phosphate (Gly-3-P) acyltransferase through the transfer of a fatty acid from fatty acyl-CoA to the sn-1 position of Gly-3-P to form lysophosphatidic acid (8Paulauskis J.D. Sul H.S. J. Biol. Chem. 1988; 263: 7049-7054Abstract Full Text PDF PubMed Google Scholar, 9Dircks L.K., Ke, J. Sul H.S. J. Biol. Chem. 1999; 274: 34728-34734Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 10Tillman T.S. Bell R.M. J. Biol. Chem. 1986; 261: 9144-9149Abstract Full Text PDF PubMed Google Scholar). Lysophosphatidic acid can also be formed from the acylation of dihydroxyacetone phosphate and subsequent reduction of the product (11de Vet E.C. Hilkes Y.H. Fraaije M.W. van den Bosch H. J. Biol. Chem. 2000; 275: 6276-6283Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 12Athenstaedt K. Daum G. J. Biol. Chem. 2000; 275: 235-240Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Lysophosphatidic acid is further fatty acylated by lysophosphatidic acid acyltransferase to form PA. PA can either be: (i) converted to DAG for subsequent incorporation into phosphatidylcholine (PC), phosphatidylethanolamine, or TAG (13Hjelmstad R.H. Bell R.M. J. Biol. Chem. 1991; 266: 4357-4365Abstract Full Text PDF PubMed Google Scholar, 14McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 28010-28016Abstract Full Text PDF PubMed Google Scholar, 15McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 14776-14783Abstract Full Text PDF PubMed Google Scholar, 16Henneberry A.L. Lagace T.A. Ridgway N.D. McMaster C.R. Mol. Biol. Cell. 2001; 12: 511-520Crossref PubMed Scopus (56) Google Scholar, 17Henneberry A.L. McMaster C.R. Biochem. J. 1999; 339: 291-298Crossref PubMed Scopus (91) Google Scholar, 18Henneberry A.L. Wistow G. McMaster C.R. J. Biol. Chem. 2000; 275: 29808-29815Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 19Buhman K.K. Chen H.C. Farese Jr., R.V. J. Biol. Chem. 2001; 276: 40369-40372Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), or (ii) metabolized to CDP-diacylglycerol for the synthesis of phosphatidylinositol in all eukaryotes (20Nikawa J. Kodaki T. Yamashita S. J. Biol. Chem. 1987; 262: 4876-4881Abstract Full Text PDF PubMed Google Scholar, 21Shen H. Dowhan W. J. Biol. Chem. 1997; 272: 11215-11220Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), and phosphatidylserine in yeast (22Birner R. Bürgermeister M. Schneiter R. Daum G. Mol. Biol. Cell. 2001; 12: 997-1007Crossref PubMed Scopus (206) Google Scholar, 23Bae-Lee M.S. Carman G.M. J. Biol. Chem. 1984; 259: 10857-10862Abstract Full Text PDF PubMed Google Scholar). In mammalian cells, two isoforms of Gly-3-P acyltransferase have been identified and are localized to either the mitochondrial or microsomal subcellular compartments (24Sul H.S. Wang D. Annu. Rev. Nutr. 1998; 18: 331-351Crossref PubMed Scopus (236) Google Scholar, 25Athenstaedt K. Weys S. Paltauf F. Daum G. J. Bacteriol. 1999; 181: 1458-1463Crossref PubMed Google Scholar, 26Gonzalez-Baró M.R. Granger D.A. Coleman R.A. J. Biol. Chem. 2001; 276: 43182-43188Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 27Igal R.A. Wang S. Gonzalez-Baró M.R. Coleman R.A. J. Biol. Chem. 2001; 276: 42205-42212Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The mammalian gene corresponding to the mitochondrial isoform has been isolated and was used to generate Chinese hamster ovary cells overexpressing the enzyme. Increased expression of mitochondrial Gly-3-P acyltransferase increased TAG synthesis to 470% and decreased PC synthesis to 70% of controls (27Igal R.A. Wang S. Gonzalez-Baró M.R. Coleman R.A. J. Biol. Chem. 2001; 276: 42205-42212Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). This study concluded that mitochondrial Gly-3-P acyltransferase synthesized downstream lipids required for TAG synthesis and this pool was relatively separate from those that contribute to the synthesis of PC. A gene/cDNA coding for a mammalian microsomal Gly-3-P enzyme has yet to be isolated. The recent identification of two Gly-3-P acyltransferases genes,GAT1 and GAT2, from the yeast Saccharomyces cerevisiae was reported and interestingly neither had a high degree of amino acid similarity to the mammalian mitochondrial Gly-3-P acyltransferase. In vitro substrate specificities were determined and the GAT1 gene product could use both Gly-3-P and dihydroxyacetone phosphate with similar efficiencies and had a broad fatty acyl-CoA specificity profile, whereas the GAT2gene product preferred Gly-3-P over dihydroxyacetone phosphate and had a marked preference for 16-carbon fatty acyl chains (28Zheng Z. Zou J. J. Biol. Chem. 2001; 276: 41710-41716Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The precisein vivo metabolic and biological roles of the Gat1p and Gat2p enzymes are unknown. We provide evidence that yeast Gat1p and Gat2p represent the major Gly-3-P acyltransferases in yeast. The specific genetic inactivation of the GAT1 or GAT2genes allowed for examination of the metabolic role of each Gly-3-P acyltransferase activity in the metabolism of specific glycerolipidsin vivo. Differences in TAG synthesis, and PC synthesis through the CDP-choline pathway and its subsequent turnover were identified between wild type, gat1, and gat2cells. The studies on PC turnover revealed a key role for PA produced from the hydrolysis of PC by phospholipase D in the modulation of susceptibility to choline-containing lysolipid analogues and drugs. Radiolabeled [methyl-14C]choline was purchased from American Radiolabeled Chemicals, and [1-14C]acetate was from PerkinElmer Life Sciences. Lipids were purchased from Avanti Polar Lipids. Edelfosine was the kind gift of Medmark Pharma GmbH. Silica gel thin layer chromatography plates were purchased from Whatman. T7 and V5 antibodies were purchased from Novagen. Standard molecular biology methods, yeast genetic techniques, and transformation methods were used (29Kaiser C. Michaelis S. Mitchell A. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994Google Scholar, 30Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar). Yeast complex medium supplemented to a final concentration of 2% glucose (w/v) (YPD) or galactose (w/v) (YPGal) and synthetic minimal medium using 2% glucose (w/v) (SD), 2% galactose (w/v) (SGal), 3% ethanol (SEtOH), or 3% glycerol (SGly) as carbon source supplemented as required for plasmid maintenance are described (29Kaiser C. Michaelis S. Mitchell A. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994Google Scholar). Yeast strains W303-1a (a ura3-1 his3-11,15 leu2-3,112 trp1-1 ade2-1 can1-100) and W303-1A (αura3-1 his3-11,15 leu2-3,112 trp1-1 ade2-1 can1-100) were the parental strains from which gat1 and gat2yeast were derived. Standard yeast one-step gene disruption cassettes were constructed that replaced the entire open reading frame ofGAT1 or GAT2 and were transformed into yeast to generate strains CMY201 (α ura3-1 his3-11,15 leu2-3,112 trp1-1 ade2-1 can1-100 gat2Δ::HIS3), CMY202 (a ura3-1 his3-11,15 leu2-3,112 trp1-1 ade2-1 can1-100 gat2Δ::HIS3), CMY203 (αura3-1 his3-11,15 leu2-3,112 trp1-1 ade2-1 can1-100 gat1Δ::TRP1), and CMY204 (a ura3-1 his3-11,15 leu2-3,112 trp1-1 ade2-1 can1-100 gat1Δ::TRP1). A plasmid containing theGAT1 open reading frame fused to the coding region for the V5 epitope tag at its 3′ end and under control of the GAL1promoter with URA3 as selectable marker (purchased from Invitrogen) was transformed into CMY201 and CMY204 strains. Ura+ transformants were mated and a diploid strain bearing the plasmid was selected, sporulated, and meiotic progeny were isolated on YPGal plates to generate strain CMY228 (α ura3-1 his3-11,15 leu2-3,112 trp1-1 ade2-1 can1-100 gat1Δ::TRP1 gat2Δ::HIS3[pGAL1::GAT1 URA3]). Strain BY5102 (aura3 his3 leu2 met15 spo14Δ::KanMx)was purchased from ResGen. BY5102 was mated to CMY201 and haploid progeny were generated to create strains CMY240-3A (α ura3 his3 leu2 trp1 gat2::HIS3 spo14::KanMx) and CMY240-3D (a ura3 his3 leu2 trp1 spo14Δ::KanMx).Strain CMY240-3D was transformed with thegat1::TRP1 disruption cassette to generate strain CMY241 (a ura3 his3 leu2 trp1 spo14Δ::KanMx gat1Δ::TRP1). Gene disruption events were confirmed through genomic PCR. The Escherichia coli DAG kinase open reading frame (dgkA) was amplified by PCR using Platinum Hi Fidelity TaqPolymerase (Invitrogen) from the DH5α E. coli strain, TA cloned into the TOPO-pcRII vector, and subcloned into pAH9 (a derivative of p416-GPD that expresses open reading frames using the constitutive glycerol-3-P dehydrogenase promoter) but adds the coding region for the 10-amino acid T7 epitope tag to the 5′ end of the subcloned open reading frame. Plasmids pKR325 (SPO14), pME910(spo14K→H), and pME419 (spo14ΔN) were previously described (31Rudge S.A. Morris A.J. Engebrecht J. J. Cell Biol. 1998; 140: 81-90Crossref PubMed Scopus (119) Google Scholar). Yeast cells growing in mid-log phase were labeled with [14C]choline (10 μm, 1 × 104 dpm/nmol) or [14C]acetate (30 μm, 5 × 104 dpm/nmol) for the indicated time points. Subsequent to incubation with radiolabel, cells were concentrated by centrifugation, washed twice with water, and resuspended in 1 ml of CHCl3/CH3OH (1/1, v/v). Cells were disrupted for 1 min at 4 °C using a BioSpec Multi-Bead Beater containing 0.5 g of 0.5 mm acid-washed glass beads. The beads were washed with 1.5 ml of CHCl3/CH3OH (2/1, v/v) and 1.5 ml of water and 0.5 ml of CHCl3 were added to the combined supernatant to facilitate phase separation. Phospholipids in the organic phase were routinely analyzed by thin layer chromatography on Whatman Silica Gel 60A plates using the solvent system: CHCl3/CH3OH/H2O/CH3COOH (70/30/4/2, v/v/v/v). Neutral lipids were separated using the solvent system: petroleum ether/diethyl ether/acetic acid (80/20/1, v/v/v). Choline containing metabolites in the aqueous phase were separated in a solvent system consisting of CH3OH, 0.6% NaCl, NH4OH (50/50/5, v/v/v). Some plates were sprayed with ENHANCE (PerkinElmer Life Sciences) and exposed to x-ray film, whereas others had their radioactivity located and analyzed using a BioScan radiolabel imaging scanner and the corresponding bands were scraped into vials for scintillation counting. Metabolite identity was based on the mobility of known standards. Yeast were grown to mid-log phase and serial diluted 1/10 starting with an A 600 of 1. The growth of the cells was monitored by spotting 5 μl of each dilution onto solid medium in the absence or presence of the indicated compounds. For the addition of lipids to the plates, the lipids were dried under N2 gas, resuspended in ethanol, and added to the medium. The final ethanol concentration in lipid containing plates never exceeded 0.5%. All compounds were added to the various media after autoclaving and cooling of the media to at least 60 °C. Yeast were grown to mid-log phase in minimal medium supplemented as required to maintain cell growth. Total yeast cellular membranes were prepared as described (32Williams J.G. McMaster C.R. J. Biol. Chem. 1998; 273: 13482-13487Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Gly-3-P acyltransferase and lysophosphatidic acid acyltransferase were assayed in membrane fractions as described (33Mishra S. Kamisaka Y. Biochem. J. 2001; 355: 315-322Crossref PubMed Scopus (7) Google Scholar). DAG kinase activity was measured by the method of Priess et al. (34Preiss J. Loomis C.R. Bishop W.R. Stein R. Niedel J.E. Bell R.M. J. Biol. Chem. 1986; 261: 8597-8600Abstract Full Text PDF PubMed Google Scholar). For Western blots, proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with a V5 monoclonal antibodies coupled directly to horseradish peroxidase, or a T7 monoclonal antibody followed by incubation with a goat anti-mouse secondary antibody coupled to horseradish peroxidase. Proteins were detected using the enhanced chemiluminescence method as directed by the manufacturer (AmershamBiosciences). Lipid phosphorus was determined as described by Ames and Dubin (35Ames B.N. Dubin D.T. J. Biol. Chem. 1960; 235: 769-775Abstract Full Text PDF PubMed Google Scholar), and protein mass using the Lowry protocol (36Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). GAT1 (YKR067w) and GAT2 (YBL011w) genes share 54% similarity and 37% identity over the entire protein sequence including the presence of a putative acyltransferase motif. Other translated open reading frames in the yeast genome contain the same putative acyltransferase motif but lack similarity to Gat1p and Gat2p outside of this motif region. Inactivation of either the GAT1 or GAT2 gene in yeast did not affect cell viability. Mating ofgat1::TRP1 (gat1) andgat2::HIS3 (gat2) mutants and characterization of meiotic progeny by tetrad analysis did not result in the recovery of any progeny that were simultaneously lacking both GAT1 and GAT2 genes indicating that loss of function of GAT1 and GAT2 is synthetically lethal (data not shown and Ref. 28Zheng Z. Zou J. J. Biol. Chem. 2001; 276: 41710-41716Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Synthetic lethality does not necessarily mean that Gat1p and Gat2p are the only Gly-3-P acyltransferases present in S. cerevisiae as there are several other yeast proteins that contain the acyltransferase motif. To address the combined role of Gat1p and Gat2p to total yeast Gly-3-P acyltransferase activity we designed an inducible expression system whereby a yeast strain with genetically inactivated GAT1 andGAT2 genes was maintained by using the GAL1galactose inducible promoter to drive GAT1 expression ingat1::TRP1 gat2::HIS3 yeast. Culturing the yeast on galactose allowed for cell growth, but shifting these cells to glucose containing medium (which suppresses transcription from theGAL1 promoter) resulted in cessation of cell growth after approximately three to four cell divisions. The level of Gat1p after a shift to glucose was monitored by Western blot (Fig.1) and indicated that Gat1p rapidly declined to very low levels after 3 h in glucose and was no longer detected at 12 h. Lipid analysis of the gat1::TRP1 gat2::HIS3 double knockout strain harboring the galactose-inducible GAT1 construct was assessed by labeling with [14C]acetate. Radiolabeled acetate is incorporated into de novo synthesized fatty acids for subsequent lipid acylation. Yeast were grown in galactose (which expressed Gat1p), or shifted to glucose for various time points, and were then labeled with [14C]acetate for 1 h. In cells grown in galactose, radioactivity was associated equally between phospholipids and glycerolipids with each incorporating 50% of the total radiolabel (Figs. 2 and3). Within the phospholipid fraction 37% of the label was associated with PC, 33% with phosphatidylethanolamine, 28% with phosphatidylinositol/phosphatidylserine, and 2% with PA (Fig. 2). In the neutral lipid fraction 47% of the label was in steryl esters, 21% in TAG, 19% in DAG, and 13% in fatty acid (Fig. 3). After the shift to glucose medium radiolabeled acetate incorporation gradually shifted from the acylation of glycerolipids to sterol acylation. This indicates that fatty acids are made but cannot be incorporated into glycerolipids and are instead being stored as steryl esters. After 12 h of growth in glucose the cells took up 70% of the total radiolabeled acetate as compared with cells grown in galactose. Analysis of ratio of radiolabel indicated the label incorporated into phospholipids had decreased to 19% of the total with the neutral lipid fraction now containing 81% of the radiolabeled acetate. Within the phospholipid fraction 41% of the label was associated with PC, 33% with phosphatidylethanolamine, and 26% with phosphatidylinositol/phosphatidylserine. Thus, cells deficient in Gat1p and Gat2p showed a proportional decrease in the labeling of each of these major phospholipid classes (Fig. 3). Almost all of the label that was not incorporated into phospholipids upon inactivation of both Gat1p and Gat2p was now found in steryl esters as they now comprised 87% of the label found in the neutral lipid class, with 11% of the label in TAG, 2% in DAG, and 2% in fatty acid (Fig. 2). The large and generalized decrease in the ability to incorporate fatty acid into glycerolipids upon simultaneous inactivation of GAT1 andGAT2 is consistent with these two genes coding for the main yeast Gly-3-P acyltransferases.Figure 3Acylation of phospholipids subsequent to inactivation of Gat1p expression in gat1 gat2yeast. Yeast strain CMY228 (α ura3-1 his3-11,15 leu2-3,112 trp1-1 ade2-1 can1-100 gat1Δ::TRP1 gat2Δ::HIS3[pGAL1::GAT1 URA3]) was maintained on minimal galactose medium containing the required supplements for cell growth and plasmid maintenance. Early log phase cells were washed with minimal glucose containing medium and maintained in glucose containing medium. Yeast were labeled with [14C]acetate for 1 h at the indicated time points. Lipids were extracted and separated by thin layer chromatography using the solvent system: chloroform/methanol/water/acetic acid (70/30/4/2, v/v/v/v).A, autoradiogram of the lipid separated by thin layer chromatography. Mobility of lipid standards is indicated. B, relative radioactivity as assessed using a radiolabel imaging scanner.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further support that Gat1p and Gat2p represent the major yeast Gly-3-P acyltransferase enzymes, the in vitro enzyme activity of Gly-3-P acyltransferase and the downstream enzyme lysophosphatidic acid acyltransferase were measured in thegat1::TRP1 gat2::HIS3 double knockout yeast harboring the galactose-inducible GAT1 construct after a shift to glucose containing medium. After 12 h in glucose the Gly-3-P acyltransferase activity was reduced to negligible levels while lysophosphatidic acid acyltransferase activity was essentially unaffected (Table I). Our above metabolic and enzymatic data, coupled with the observed synthetic lethality upon simultaneous inactivation of GAT1 and GAT2, indicate that these two genes code for the principal Gly-3-P acyltransferases in S. cerevisiae.Table IGlycerol-3-phosphate and lysophosphatidic acid acyltransferase enzyme activitiesActivity relative to wild type yeast grown on glucoseGlycerol-3-phosphate acyltransferase activityLysophosphatidic acid acyltransferase activity%Wild type grown on glucose1001-aWild type enzyme activities were 2.0 and 21.2 nmol min−1/mg−1 for glycerol-3-phosphate and lysophosphatidic acid acyltransferases, respectively.100gat1∷TRP1 gat2∷HIS3(GAL-GAT1) grown on galactose152135gat1∷TRP1 gat2∷HIS3(GAL-GAT1) grown on glucose for 12 h2115The results are the mean of at least three separate experiments performed in duplicate. Standard errors are less than 15% of the mean for each point.1-a Wild type enzyme activities were 2.0 and 21.2 nmol min−1/mg−1 for glycerol-3-phosphate and lysophosphatidic acid acyltransferases, respectively. Open table in a new tab The results are the mean of at least three separate experiments performed in duplicate. Standard errors are less than 15% of the mean for each point. Although simultaneous inactivation of GAT1 andGAT2 prevented cell growth, inactivation of theGAT1 or GAT2 genes separately did not affect the rate of cell growth on rich or minimal yeast medium. The identical growth rates to wild type yeast allowed for the determination of the role of each Gly-3-P acyltransferases in the synthesis of specific glycerolipids through metabolic labeling analyses. We initially monitored flux through the CDP-choline pathway for synthesis of PC because increased GAT2 expression was previously shown to restore growth to a yeast strain that contained a mutated choline transporter that possessed an increasedK m for choline and relied exclusively on the CDP-choline pathway for PC synthesis (37Matsushita M. Nikawa J. J. Biochem. (Tokyo). 1995; 117: 447-451Crossref PubMed Scopus (19) Google Scholar). In this study,GAT2 (referred to in the previous study as SCT1for suppressor of choline transport) was thought to directly alter the rate of uptake of choline by the choline transporter. However, if increased GAT2 allowed for higher flux through the CDP-choline pathway through an indirect route this could also allow for restoration of cell growth to this yeast strain. We determined the rate of metabolism of radiolabeled choline into PC and its subsequent turnover into glycerophosphocholine ingat1 and gat2 yeast. As shown in Fig.4, gat1 cells produced less radioactive phosphocholine and much higher levels of radioactive glycerophosphocholine than the wild type strain. Conversely, glycerophosphocholine labeling was almost undetectable ingat2 cells, whereas there was an increase in the radioactivity incorporated into phosphocholine. As glycerophosphocholine is an end product of PC deacylation these results indicate that flux through the CDP-choline pathway for subsequent PC deacylation is diminished in the gat2 mutant and is augmented in gat1 cells. The DAG produced downstream of the Gly-3-P acyltransferases could also be used for the synthesis of the neutral lipid TAG. The gat1and gat2 yeast were radiolabeled with acetate and its incorporation into de novo synthesized fatty acid for subsequent esterification into neutral glycerolipids was determined. Acetate labeling of DAG was slightly reduced in gat2 yeast and this was reflected by a 50% decrease in labeling of TAG (Fig.5). In gat1 yeast, DAG labeling was essentially unchanged, whereas TAG labeling was increased by 50%. The metabolism of radiolabeled ethanolamine, serine, and inositol into lipids was unchanged in gat1 and gat2 cells compared with wild type cells (data not shown). Consistent with the inositol metabolic labeling experiments was the observation thatgat1 and gat2 yeast were not inositol auxotrophs indicating that their ability to synthesize inositol and convert it into phosphatidylinositol was unperturbed. In yeast the major PC deacylating phospholipase genes thus far identified are encoded by the PLB1–3 genes (38Merkel O. Fido M. Mayr J.A. Prüger H. Raab F. Zandonella G. Kohlwein S.D. Paltuaf F. J. Biol. Chem. 1999; 274: 28121-28127Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 39Fyrst H. Oskouian B. Kuypers F.A. Saba J.D. Biochemistry. 1999; 38: 5864-5871Crossref PubMed Scopus (45) Google Scholar, 40Lee K.S. Patton J.L. Fido M. Hines L.K. Kohlwein S.D. Paltauf F. Henry S.A. Levin D.E. J. Biol. Chem. 1994; 269: 19725-19730Abstract Full Text PDF PubMed Google Scholar), although a fourth PC deacylating activity whose gene has yet to be identified is known to exist (42Dowd S.R. Bier M.E. Patton-Vogt J.L. J. Biol. Chem. 2001; 276: 3756-3763Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). An increase in the activity of PC/lyso-PC hydrolyzing phospholipase B (Plb2p) has been demonstrated to protect cells versus the cytostatic effects of exogenously added lyso-PC, and ablation of the PLB2 gene resulted in increased lyso-PC susceptibility (38Merkel O. Fido M. Mayr J.A. Prüger H. Raab F. Zandonella G. Kohlwein S.D. Paltuaf F. J. Biol. Chem. 1999; 274: 28121-28127Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 39F
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