Schizosaccharomyces pombe Cells Deficient in Triacylglycerols Synthesis Undergo Apoptosis upon Entry into the Stationary Phase
2003; Elsevier BV; Volume: 278; Issue: 47 Linguagem: Inglês
10.1074/jbc.m306998200
ISSN1083-351X
AutoresQian Zhang, Hai Kee Chieu, Choon Pei Low, Shaochong Zhang, Chew‐Kiat Heng, Hongyuan Yang,
Tópico(s)Microbial Metabolic Engineering and Bioproduction
ResumoTriacylglycerols (TAG) are important energy storage molecules for nearly all eukaryotic organisms. In this study, we found that two gene products (Plh1p and Dga1p) are responsible for the terminal step of TAG synthesis in the fission yeast Schizosaccharomyces pombe through two different mechanisms: Plh1p is a phospholipid diacylglycerol acyltransferase, whereas Dga1p is an acyl-CoA:diacylglycerol acyltransferase. Cells with both dga1+ and plh1+ deleted (DKO cells) lost viability upon entry into the stationary phase and demonstrated prominent apoptotic markers. Exponentially growing DKO cells also underwent dramatic apoptosis when briefly treated with diacylglycerols (DAGs) or free fatty acids. We provide strong evidence suggesting that DAG, not sphingolipids, mediates fatty acids-induced lipoapoptosis in yeast. Lastly, we show that generation of reactive oxygen species is essential to lipoapoptosis. Triacylglycerols (TAG) are important energy storage molecules for nearly all eukaryotic organisms. In this study, we found that two gene products (Plh1p and Dga1p) are responsible for the terminal step of TAG synthesis in the fission yeast Schizosaccharomyces pombe through two different mechanisms: Plh1p is a phospholipid diacylglycerol acyltransferase, whereas Dga1p is an acyl-CoA:diacylglycerol acyltransferase. Cells with both dga1+ and plh1+ deleted (DKO cells) lost viability upon entry into the stationary phase and demonstrated prominent apoptotic markers. Exponentially growing DKO cells also underwent dramatic apoptosis when briefly treated with diacylglycerols (DAGs) or free fatty acids. We provide strong evidence suggesting that DAG, not sphingolipids, mediates fatty acids-induced lipoapoptosis in yeast. Lastly, we show that generation of reactive oxygen species is essential to lipoapoptosis. Triacylglycerols (TAGs) 1The abbreviations used are: TAGtriacylglycerolDAGdiacylglycerolDGATdiacylglycerol acyltransferaseDAPI4′,6-diamidino-2-phenylindoleC2-ceramideN-acetylsphigosinePEphosphatidylethanolamineDiC8 DAG1,2-dioctanoyl-sn-glycerolPBSphosphate-buffered salineTUNELterminal deoxynucleotidyl transferase-mediated dUTP nick-end labelingFITCfluorescein isothiocyanateROSreactive oxygen speciesTMPO3,3,5,5,-tetramethylpyrroline N-oxidePSphosphatidylserinePDATphospholipid DAG acyltransferaseCHOChinese hamster ovaryDGKDAG kinaseDHSdihydrosphingosinePHSphytosphingosinePKCprotein kinase C. are important energy storage molecules that can be found in almost all eukaryotes. In mammals, TAG synthesis plays essential roles in a number of physiological processes, including intestinal fat absorption, energy storage in muscle and adipose tissue, and lactation. It also contributes to pathological conditions such as obesity and hypertriglyceridemia (1Farese Jr., R.V. Cases S. Smith S.J. Curr. Opin. Lipidol. 2000; 11: 229-234Crossref PubMed Scopus (122) Google Scholar). TAG synthesis through both the glycerol-3-phosphate pathway and the monoacylglycerol pathway is acyl-CoA-dependent. The transfer of an acyl group from acyl-CoA to diacylglycerols (DAGs) catalyzed by the enzyme diacylglycerol acyltransferase (DGAT) is regarded as the only committed reaction in TAG synthesis in the glycerolipid pathway, because DAG is diverted from membrane glycerolipid biosynthesis (2Bell R.M. Coleman R.A. Annu. Rev. Biochem. 1980; 49: 459-487Crossref PubMed Scopus (471) Google Scholar). Two distinct mammalian DGAT genes have been identified recently. DGAT1 was cloned based on its sequence homology to genes involved in sterol esterification (3Cases S. Smith S.J. Zheng Y.-W. Myers H.M. Lear S.R. Sande E. Novak S. Collins C. Welch C.B. Lusis A.J. Erickson S.K. Farese Jr., R.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13018-13023Crossref PubMed Scopus (910) Google Scholar, 4Oelkers P. Behari A. Cromley D. Billheimer J.T. Sturley S.L. J. Biol. Chem. 1998; 273: 26765-26771Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). DGAT2 was identified by its homology to a DGAT isolated from the fungus Mortierella rammaniana (5Cases S. Stone S.J. Zhou P. Yen E. Tow B. Lardizabal K.D. Voelker T. Farese Jr., R.V. J. Biol. Chem. 2001; 276: 38870-38876Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar, 6Lardizabal K.D. Mai J.T. Wagner N.W. Wyrick A. Voelker T. Hawkins D.J. J. Biol. Chem. 2001; 276: 38862-38869Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). Other acyl-CoA-dependent TAG-synthesizing enzymes are likely present but are yet to be identified. In addition, acyl-CoA-independent TAG synthesis was also shown to exist in eukaryotes. A DAG transacylase, which synthesizes TAG from two DAGs, was purified from rat intestinal microsomes, and its activity was comparable to that of DGAT (7Lehner R. Kuksis A. J. Biol. Chem. 1993; 268: 8781-8786Abstract Full Text PDF PubMed Google Scholar). triacylglycerol diacylglycerol diacylglycerol acyltransferase 4′,6-diamidino-2-phenylindole N-acetylsphigosine phosphatidylethanolamine 1,2-dioctanoyl-sn-glycerol phosphate-buffered saline terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling fluorescein isothiocyanate reactive oxygen species 3,3,5,5,-tetramethylpyrroline N-oxide phosphatidylserine phospholipid DAG acyltransferase Chinese hamster ovary DAG kinase dihydrosphingosine phytosphingosine protein kinase C. Four genes, i.e. DGA1, LRO1, ARE1, and/or ARE2, have been found to encode proteins capable of synthesizing TAG in the budding yeast Saccharomyces cerevisiae (8Sandager L. Gustavsson M.H. Stahl U. Dahlqvist A. Wiberg E. Banas A. Lenman M. Ronne H. Stymne S J. Biol. Chem. 2002; 277: 6478-6482Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar, 9Oelkers P. Tinkelenberg A. Erdeniz N. Cromley D. Billheimer J.T. Sturley S.L. J. Biol. Chem. 2000; 275: 15609-15612Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 10Oelkers P. Cromley D. Padamsee M. Billheimer J.T. Sturley S.L. J. Biol. Chem. 2002; 277: 8877-8881Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 11Dahlqvist A. Stahl U. Lenman M. Banas A. Lee M. Sandager L. Ronne H. Stymne S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6487-6492Crossref PubMed Scopus (665) Google Scholar). Dga1p is highly homologous to mammalian DGAT2, whereas Lro1p encodes a protein with significant sequence similarity to the mammalian enzyme lecithin cholesterol acyltransferase. Dga1p utilizes acyl-CoA to esterify DAG, whereas Lro1p transfers an acyl group from a phospholipid molecule to the sn-3 position of DAG. Dga1p and Lro1p mediate the bulk of TAG synthesis; however, in their absence, 2–4% of normal TAG synthesis could still be detected. It was later determined that Are1p and Are2p, two acyl-CoA sterol acyltransferases in yeast, are responsible for this residual activity. When all four genes are deleted simultaneously, synthesis of both sterol esters and TAG is completely blocked. However, no obvious growth defects were detected in the budding yeast cells completely free of either TAG or neutral lipids. This is rather surprising, because neutral lipids have long been regarded as a safe depot for polar and potentially toxic lipids such as fatty acids, DAG, or sterols. A recent study has proven that synthesis of TAG prevents fatty acids-induced lipotoxicity in mammalian cells (12Listenberger L.L. Han X. Lewis S.E. Cases S. Farese Jr., R.V. Ory D.S. Schaffer J.E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3077-3082Crossref PubMed Scopus (1482) Google Scholar). The fission yeast S. pombe, similar to the budding yeast, is genetically tractable and equipped with a rich repertoire of molecular tools and a completely sequenced genome (13Forsburg S.L. Trends Genet. 1999; 15: 340-344Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 14Wood V. Gwilliam R. Rajandream M.A. Lyne M. Lyne R. Stewart A. Sgouros J. Peat N. Hayles J. Baker S. Basham D. Bowman S. Brooks K. Brown D. Brown S. Chillingworth T. Churcher C. Collins M. Connor R. Cronin A. Davis P. Feltwell T. Fraser A. Gentles S. Goble A. Hamlin N. Harris D. Hidalgo J. Hodgson G. Holroyd S. Hornsby T. Howarth S. Huckle E.J. Hunt S. Jagels K. James K. Jones L. Jones M. Leather S. McDonald S. McLean J. Mooney P. Moule S. Mungall K. Murphy L. Niblett D. Odell C. Oliver K. O'Neil S. Pearson D. Quail M.A. Rabbinowitsch E. Rutherford K. Rutter S. Saunders D. Seeger K. Sharp S. Skelton J. Simmonds M. Squares R. Squares S. Stevens K. Taylor K. Taylor R.G. Tivey A. Walsh S. Warren T. 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Although the fission and budding yeasts are as divergent from each other as each from the mammals, S. pombe has been shown to have greater similarity to mammals at least in certain steps of cell division and in aspects of stress signaling (15Toone W.M. Jones N. Genes Cells. 1998; 3: 485-498Crossref PubMed Scopus (124) Google Scholar). The enzymes and pathways of lipid metabolism, their physiological significance, and their resemblance to mammalian systems are largely unexplored in S. pombe. In this work, we describe the identification of two genes, plh1+ and dga1+, that encode enzymes that are responsible for the bulk of TAG synthesis in the fission yeast. We provide convincing evidence that fission yeast cells defective in TAG synthesis undergo apoptosis upon entry into the stationary phase. The important role of DAG in the induction of lipoapoptosis is also investigated. Yeast Strains, General Techniques, and Reagents—S. pombe strains MBY257 (h-, his3-D1, ade6-M210, leu1–32, ura4-D18) and MBY266 (h+, his3-D1, ade6-M210, leu1–32, ura4-D18) were used in this study (16Li T. Naqvi N.I. Yang H. Teo T.S. Biochem. Biophys. Res. Commun. 2000; 272: 270-275Crossref PubMed Scopus (58) Google Scholar). Growth media (YES and EMM) and basic genetic, cell, and biochemical techniques were used according to a previous report (17Moreno S. Klar A. Nurse P. Methods Enzymol. 1991; 194: 795-823Crossref PubMed Scopus (3207) Google Scholar). Transformation of yeast was performed with electroporation, followed by prototrophic selection (18Prentice H.L. Nucleic Acids Res. 1992; 20: 621Crossref PubMed Scopus (215) Google Scholar). Yeast extract, Yeast Nitrogen Base, Bacto-peptone, and Bacto-agar were from Difco Laboratories; d-dextrose, d-galactose, and d-raffinose were from Sigma. 3,3,5,5-Tetramethyl-1-1-pyrroline-n-oxide, 1,2-dioctanoyl-sn-glycerol, oleic acid, palmitic acid, 4′,6-diamidino-2-phenylindole (DAPI), and Nile Red were from Sigma. N-Acetylsphingosine (C2-ceramide) was from US Biological. [1-14C]oleoyl-CoA, 1-stearoyl-2-[14C]arachidonyl-sn-glycerol, 1-palmitoyl-2-[1-14C]oleoyl phosphatidylethanolamines (PEs), and [9,10(n)-3H]oleic acid were from Amersham Biosciences. An in situ cell death detection kit and Annexin-v-fluos were from Roche Applied Science. Disruption of plh1+, dga1+, and pca1+—For plh1+ gene disruption, the entire coding region of plh1+ was replaced by the S. pombe his3+ gene. Two pairs of primers: PLH1–55 (GGGGTACCACACCCTATTTGCAACA) and PLH1–53 (CCGCTCGAGGAATTGCTTGAGCAGCAAC), and PLH1–35 (CGGGATCCCGACAAACGAATATGATAAA) and PLH-1–33 (GCTCTAGAGGCTCCATAGAAGGTGAAG), were used to amplify DNA fragments flanking the coding region of plh1+. The PCR products were cloned into a vector containing the his3+ gene to create a gene replacement cassette. For dga1+ gene disruption, the entire coding region was replaced by the S. pombe ura4+ gene. Two pairs of primers: DGA1–55 (GGGGTACCGAATCCATGGGTAGTGAT) and DG11–53 (CCGCTCGAGCCCGTTCTATATAATCGT), and DGA1–35 (CGGGATCCCTTATTGGCCTATGCAATA) and DGA1–33 (GCTCTAGACTGAATGAATATTAGTAACGC), were designed, and a gene replacement cassette containing the ura4+ gene was constructed to disrupt dga1+. For pca1+ gene disruption, the entire coding region was replaced by the kanR marker (19Wach A. Brachat A. Pohlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2258) Google Scholar). Two pairs of primers: pca15 (ATAAGAATGCGGCCGCGGAAGAACTTTGACACGTT) and pca13 (GCTCTAGAGGAAGTTGGATAGTGCTT), and pca25 (CCATCGATGTAGTTCCATCAGATATT) and pca23 (CCGCTCGAGGGTAGGTAGTATAGTTAGA), were used to amplify DNA fragments flanking the coding region of pca1+. The PCR products were cloned into pFA6akanMX4 (19Wach A. Brachat A. Pohlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2258) Google Scholar), flanking kanR. Transformation of Yeast—About 2 μg of gene replacement cassettes was used to transform wild type strains MBY266 and MBY257 by electroporation. Transformed cells were suspended in 200 μl of 1.2 m sorbitol and selected on EMM plates with appropriate amino acid supplements. Clones bearing the individual or double gene deletions were identified by diagnostic PCR with primers in the coding region of ura4+ or his3+ (GAGAAAGAATGCTGAGTAG for ura4+ and GAGTCTTTAATTCATTAC for his3+), and primers in the region outside of the flanking fragment of dga1+ or plh1+ (CGATAGTAGTCAATACCAG and GTATATTAGTATTGCCTAAT accordingly). The DKO strain was constructed by consecutive deletions of plh1+ and dga1+ in MBY266. The TKO strain was generated by deletion of pca1+ from the DKO strain. Expression Plasmids Construction—The entire open reading frame of plh1+ was generated by reverse transcription-PCR using primers PLH5 (ACGCGTCGACCATGGCGTCTTCCCAAGAAGA) and PLH3 (TCCCCCGGGTTAATTTCTAGGTTTATCGAG), whereas the entire coding region of dga1+ was amplified by PCR using the primers DGA1–5 (GGGAATTCCATATGTCAGAAGAAACATAA) and DGA1–3 (TCCCCCGGGTTAGGCTGACAACTTCAAT). The products were digested by SmaI and SalI and cloned into pREP41 or pREP42GFP, downstream of an nmt1 promoter (20Craven R.A. Griffiths D.J. Sheldrick K.S. Randall R.E. Hagan I.M. Carr A.M. Gene (Amst.). 1998; 221: 59-68Crossref PubMed Scopus (200) Google Scholar, 21Basi G. Schmid E. Maundrell K. Gene (Amst.). 1993; 123: 131-136Crossref PubMed Scopus (577) Google Scholar). The open reading frame of DAG kinase was amplified from Escherichia coli genomic DNA by PCR using the primers DGK5 (GGAATTCCATATGGCCAATAATACCACTG) and DGK3 (TCCCCCGGGTTATCCAAAATGCGACCAT) (22Lightner V.A. Bell R.M. Modrich P. J. Biol. Chem. 1983; 258: 10856-10861Abstract Full Text PDF PubMed Google Scholar). The fragment was subcloned into the SmaI and NdeI sites of pREP41. Cell Viability Assay—For cell viability at different growth phases, cells were grown to various densities in YES (determined by A595). The number of viable cells was obtained after cells were diluted properly in distilled water and plated in triplicates on YES agar. Colonies were scored after 3 days of incubation at 30 °C. For cell viability after various treatments, cells were grown to early log phase (A595 = 0.1) before lipids or other chemicals were added. After treatment, cells were collected and viability was analyzed as described above. DAG, Fatty Acids, and Ceramide Treatment—For fatty acids treatment, palmitic acid and oleic acid were dissolved in chloroform as 500 mm stock. Each microliter of fatty acids was dissolved in 12.5 μl of tyloxapol:enthanol (1:1) and added into growth medium. Wild type and DKO strains were grown to early log phase and then incubated in medium containing different concentrations (0.5, 0.8, and 1 mm) of palmitic acid or oleic acid for 0–3 h. Control groups were cultured in the medium added with the same volume of tyloxapol:enthanol without fatty acids. After incubation, cells were analyzed for viability and DNA fragmentation. DiC8 DAG and ceramide were dissolved in Me2SO. The working concentrations for DAG were 0.1, 0.2, or 0.3 mm, whereas for ceramide they were 10 or 20 μm (23Kearns B.G. McGee T.P. Mayinger P. Gedvilaite A. Phillips S.E. Kagiwada S. Bankaitis V.A. Nature. 1997; 387: 101-105Crossref PubMed Scopus (224) Google Scholar, 24Fishbein J.D. Dobrowsky R.T. Bielawska A. Garrett S. Hannun Y.A. J. Biol. Chem. 1993; 268: 9255-9261Abstract Full Text PDF PubMed Google Scholar). Nile Red Staining—Cells were grown to early stationary phase, washed with deionized H2O two times, and incubated with 1 μg/ml Nile Red (1 mg/ml in acetone stock). Fluorescence images were obtained with a Leica DMLB microscope (25Yang H. Bard M. Bruner D.A. Gleeson A. Deckelbaum R.J. Aljinovic G. Pohl T.M. Rothstein R. Sturley S.L. Science. 1996; 272: 1353-1356Crossref PubMed Scopus (232) Google Scholar). Detection of Apoptotic Markers—All assays that follow were performed as previously described (26Madeo F. Frohlich E. Ligr M. Grey M. Sigrist S. Wolf D.H. Frohlich K.U. J. Cell Biol. 1999; 145: 757-767Crossref PubMed Scopus (882) Google Scholar). For 4′,6-diamidino-2-phenylindole (DAPI) staining, cells were fixed with 3.7% formaldehyde for 10 min, washed once with PBS containing 1% Nonidet P-40 and twice with PBS, and then stained with DAPI. Cells were viewed using a Leica DMLB microscope. For terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), cells were fixed with 3.7% formaldehyde for 1 h, digested with zymolase, washed with PBS, incubated in a permeabilization solution (0.1% Triton in 0.1% sodium citrate) for 2 min on ice, washed twice with PBS, and incubated with 50-μl TUNEL mixtures for 1 h at 37 °C. Cells were washed with PBS twice and then viewed using a Leica DMLB microscope. For annexin V staining, cells were washed in sorbitol buffer (1.2 m sorbitol, 0.5 mm MgCl2, potassium phosphate, pH 6.8), digested with zymolase for 2 h at room temperature, harvested, washed in binding buffer (10 mm HEPES/NaOH, 140 mm NaCl, 2.5 mm CaCl2, 1.2 m sorbitol), pelleted, and resuspended in binding buffer. 2 μl of annexin-FITC and 2 μl of propidium iodide were added to a 38-μl cell suspension and then incubated for 20 minutes at room temperature. The cells were harvested, suspended in binding buffer, and applied to microscopic slides. Production of reactive oxygen species (ROS) was detected by dihydroethidium (Sigma), which was used at 5 μg/ml cell culture. After incubation for 10 min, cells were viewed under a Leica DMLB microscope through a Texas Red filter. The free radical spin trap reagent 3,3,5,5,-tetramethylpyrroline N-oxide (TMPO) was used at 125 μg/ml cell culture. Cells were pretreated with TMPO for 2 h before lipids were added. In Vivo Assay of Oleate Incorporation—The incorporation of [3H]oleate into TAG was used as a measurement of DAG esterification essentially as described previously (9Oelkers P. Tinkelenberg A. Erdeniz N. Cromley D. Billheimer J.T. Sturley S.L. J. Biol. Chem. 2000; 275: 15609-15612Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Briefly, Cells were cultured in YES or EMM without appropriate nutrients for plasmid maintenance when necessary. Approximately 5 ml of cells at logarithmic (log) phase (A595 = 0.55–0.80) were pulsed with 5 μCi of [3H]oleate at 30 °C for 30 min with shaking. Cells were washed twice with 0.5% Nonidet P-40, once with dH2O, and lyophilized. The dried cell pellets were resuspended in 50 μl of lyticase stock solution (1700 units/ml in 10% glycerol, 0.02% sodium azide) and incubated at -70 °C for 1 h and at 30 °C for 15 min. Lipids were extracted by hexane and analyzed by TLC. The plates were developed in hexane:diethyl ether:acetic acid (70:30:1) and stained with iodine vapor. Incorporation of label into lipids was determined after scintillation counting and normalization to a [14C]cholesterol internal standard and cell dry weight. For each assay, at least three independent strains of each genotype were used. Statistical analysis was performed using a paired t test. Analysis of DAG Accumulation by Steady-state Labeling—Cells were grown for 18–25 h to mid-log phase or early stationary phase in media containing 1 μCi/ml [3H]oleate (9Oelkers P. Tinkelenberg A. Erdeniz N. Cromley D. Billheimer J.T. Sturley S.L. J. Biol. Chem. 2000; 275: 15609-15612Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Cells were harvested, and lipids were extracted, separated, visualized, and quantified as described above. Isolation of Microsomes—Microsomes were isolated as described (27Zinser E. Daum G. Yeast. 1995; 11: 493-536Crossref PubMed Scopus (307) Google Scholar). Briefly, wild type and mutant strains were cultivated overnight in 1 liter of YES medium at 30 °C to log phase. Cells were collected through centrifugation. The pellets were washed with dH2O, resuspended, and incubated at 0.5 g wet wt/ml in 0.1 m Tris SO4 (with 10 mm dithiothreitol) at room temperature for 10 min. Cells were harvested, washed once with 1.2 m sorbitol, and resuspended at 0.15 g wet wt/ml in 1.2 m sorbitol (with 20 mm K3PO4 and 0.5 mg/ml lyticase), pH 7.2. Spheroplasts were formed after a 90-min incubation at 30 °C. Cells were washed twice with 1.2 m sorbitol, resuspended, and disrupted with 20 strokes in a Dounce homogenizer using a tight fitting pestle at 4 °C. Homogenates were spun at 20,000 × g for 30 min. The pellets were discarded. The supernatants were collected and spun at 100,000 × g for 45 min. The final pellets containing microsomes were resuspended in 10 mm Tris-HCl (pH 7.4). Protein concentrations were determined by using a Bradford assay kit from Bio-Rad. In Vitro (Microsomal) Assay of DAG Esterification—Enzyme activity was determined by the incorporation of [1-14C]oleoyl-CoA, 1-stearoyl-2-[14C]arachidonyl-sn-glycerol or l-3-phosphatidylethanolamines, 1-palmitoyl-2-[1-14C]linoleoyl into TAG. Each standard assay was performed in triplicate in 150 mm Tris-HCl, pH 7.8, and the final volume was 200 μl, containing 80 μg of microsomal proteins, 15 μm bovine serum albumin, 150 μm DAG, 8 mm MgCl2,150 μm phosphatidylserine (PS)/phosphatidylethanolamines (PEs) liposomes (1:1 molar ratio), and 50 μm oleoyl-CoA. All the assays were conducted at room temperature for 25 min. For PDAT assay, oleoyl-CoA was omitted while [14C]phosphatidylethanolamines were added in liposomes at different concentration (0, 15, 30, 45, and 60 μm). For DGAT assay, [14C]oleoyl-CoA was added at different concentrations (0, 5, 20, 25, and 50 μm). In the diacylglycerol transacylase assay, [14C]DAG was added at 0, 7.5, 15, 35, and 70 μm, whereas MgCl2 and bovine serum albumin were omitted. In control assays, all components were the same except microsomes were removed. Reactions were stopped by the addition of 6 ml of chloroform/methanol (2:1). Phase separation was induced by the addition of 1.2 ml of water. 1 μl of [3H]cholesterol and 15 μg of triolein were added as an internal standard and carrier, respectively. The lipid-containing phase was dried with nitrogen, and the lipids were dissolved in 100 μl of chloroform for spotting on TLC plates. The plates were developed in hexane:diethyl ether:acetic acid (70:30:1), and TAG was quantified by scintillation counting Diacylglycerol Kinase Assay—The assay was conducted as described in the Biotrak assay reagents system (Amersham Biosciences). Wild type and DKO yeast cells were grown in YES medium to mid-log phase and then treated with medium containing 0.8 mm palmitate or oleate. Cells were collected at different time points (0, 30, 60, and 120 min). DAG was extracted with other lipids and quantified through a phosphorylation reaction catalyzed by a bacterial DAG kinase. Identification of plh1+and dga1+in S. pombe—We searched the fission yeast genome data base for homologous sequences to human DGAT1 (hDGAT1), human DGAT2 (hDGAT2), and the budding yeast LRO1 using tBLASTX. A sequence with significant homology to hDGAT2 (40% identity at protein level) was identified and named dga1+ (GeneDB systematic name: SPCC1235.15). In addition, as previously reported, an open reading frame highly homologous (45% identity at protein level) to the budding yeast LRO1 was found in the fission yeast genome and named plh1+ (for Pombe LRO1 Homolog 1, GeneDB systematic name: SPBC776.14) in this study. A few open reading frames showing limited homology to DGAT1 were also found, but they are unlikely to play a role in TAG synthesis as suggested by a previous report (9Oelkers P. Tinkelenberg A. Erdeniz N. Cromley D. Billheimer J.T. Sturley S.L. J. Biol. Chem. 2000; 275: 15609-15612Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). plh1+ predicts a protein of 623 amino acids, with a putative transmembrane domain near its N terminus. Plh1p also has a conserved serine lipase motif HS(M/L)G between amino acids 292 and 296. dga1+ encodes a 349-residue protein with at least one transmembrane domain. The region of the putative glycerol phospholipid domain in hDGAT2 was also found to be conserved in Dga1p (45% identity over 80 amino acids). Deletion of plh1+and dga1+Resulted in a Viable Yeast Cell without Detectable TAG—To determine whether Plh1p and Dga1p are involved in TAG synthesis in the fission yeast, we generated Δplh1, Δdga1 single and Δplh1Δdga1 double deletion (referred to as the DKO strain thereafter) mutants by homologous recombination. All mutants were viable at 16 °C, 30 °C, and 37 °C on rich or minimal media and on different carbon sources (data not shown). We were also unable to observe any obvious morphological changes in the DKO cells under light microscope. To investigate whether cellular TAG mass was affected in these strains, cells were grown to mid-log phase, and lipids were extracted, separated by TLC, and stained by iodine vapor. Although the TAG mass in each single deletion mutant was visually indistinguishable from that of the wild type cells, virtually no TAG mass could be seen for DKO cells (data not shown). The sterol ester mass was clearly visible for all mutants, ruling out a lipid extraction error for the DKO strain. To further examine the ability of these strains to synthesize TAG, cells in log phase were pulse-labeled with [3H]oleate, and its incorporation into TAG was measured (Fig. 1A). No significant differences in oleate incorporation into TAG were detected between wild type and the Δdga1 mutant. However, TAG synthesis decreased by nearly 50% due to the loss of Plh1p. Most notably, the double mutant was almost totally deficient in TAG synthesis. In contrast, sterol ester biosynthesis was normal in all mutants (data not shown). In the budding yeast, it has been confirmed that Are1p and/or Are2p are responsible for the residual TAG synthesis activity (about 3% of the wild type level). In S. pombe, there are two proteins (GeneDB systematic names: SPAC13G7.05 and SPCP1E11.05) that share strong homology with Are1p and Are2p. We have determined that these two homologs catalyze sterol esterification in S. pombe (data not shown); however, whether these proteins have a role in TAG synthesis remains to be examined. To confirm that either Plh1p or Dga1p was sufficient for TAG synthesis, we overexpressed plh1+ and dga1+ in wild type and DKO strains. Both genes were placed under the control of a modified nmt1 promoter (21Basi G. Schmid E. Maundrell K. Gene (Amst.). 1993; 123: 131-136Crossref PubMed Scopus (577) Google Scholar), and each gene was able to complement the TAG synthesis defect in the DKO mutant, indicating an overlapping function of these two genes (Fig. 1B). Overexpression of plh+ and dga1+ also caused a significant increase in TAG synthesis in WT and mutant strains, suggesting these genes could be regulated at transcription level. These results imply that TAG synthesis is mediated by two gene products in fission yeast, whereas Plh1p plays a major role at log phase. To further confirm the absence of TAG in the DKO strain, we treated yeast cells with Nile Red, a fluorescent dye with strong and specific affinity for neutral lipids (28Greenspan P. Mayer E.P. Fowler S.D. J. Cell Biol. 1985; 100: 965-973Crossref PubMed Scopus (1932) Google Scholar). In both of the wild type and DKO cells, cytoplasmic fluorescent droplets could be seen in early stationary phase cultures. However, the number and intensity of the droplets observed in DKO cells was significantly less than those in wild type strains (Fig. 1C). In Vitro Microsomal Assays of DAG Esterification—The results described above demonstrated the essential roles of Plh1p and Dga1p in TAG synthesis; however, they did not reveal the exact molecular function of these two proteins. Based on sequence homology and experimental data from previous studies (8Sandager L. Gustavsson M.H. Stahl U. Dahlqvist A. Wiberg E. Banas A. Lenman M. Ronne H. Stymne S J. Biol. Chem. 2002; 277: 6478-6482Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar, 9Oelkers P. Tinkelenberg A. Erdeniz N. Cromley D. Billheimer J.T. Sturley S.L. J. Biol. Chem. 2000; 275: 15609-15612Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 10Oelkers P. Cromley D. Padamsee M. Billheimer J.T. Sturley S.L. J. Biol. Chem. 2002; 277: 8877-8881Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 11Dahlqvist A. Stahl U. Lenman M. Banas A. Lee M. Sandager L. Ronne H. Stymne S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6487-6492Crossref PubMed Scopus (665) Google Scholar), it is highly likely that both of Plh1p and Dga1p carry out DAG esterification, with Dga1p functioning as an acyl-CoA DAG acyltransferase (DGAT), whereas Plh1p functioning as a phospholipid DA
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