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• W2092034105 abstract "Phospholipids are major components of cellular membranes that participate in a range of cellular processes. Phosphatidic acid (PA) is a key molecule in the phospholipid biosynthetic pathway. In Saccharomyces cerevisiae, SLC1 has been identified as the gene encoding lysophosphatidic acid acyltransferase, which catalyzes PA synthesis. However, despite the importance of PA, disruption of SLC1 does not affect cell viability (Nagiec, M. M., Wells, G. B., Lester, R. L., and Dickson, R. C. (1993) J. Biol. Chem. 268, 22156–22163). We originally aimed to identify the acetyl-CoA:lyso platelet-activating factor acetyltransferase (lysoPAF AT) gene in yeast. Screening of a complete set of yeast deletion clones (4741 homozygous diploid clones) revealed a single mutant strain, YOR175c, with a defect in lysoPAF AT activity. YOR175c has been predicted to be a member of the membrane-bound O-acyltransferase superfamily, and we designated the gene LPT1. An Lpt1-green fluorescent protein fusion protein localized at the endoplasmic reticulum. Other than lysoPAF AT activity, Lpt1 catalyzed acyltransferase activity with a wide variety of lysophospholipids as acceptors, including lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, and lysophosphatidylserine. A liquid chromatography-mass spectrometry analysis indicated that lysophosphatidylcholine and lysophosphatidylethanolamine accumulated in the Δlpt1 mutant strain. Although the Δlpt1 mutant strain did not show other detectable defects, the Δlpt1 Δslc1 double mutant strain had a synthetic lethal phenotype. These results indicate that, in concert with Slc1, Lpt1 plays a central role in PA biosynthesis, which is essential for cell viability. Phospholipids are major components of cellular membranes that participate in a range of cellular processes. Phosphatidic acid (PA) is a key molecule in the phospholipid biosynthetic pathway. In Saccharomyces cerevisiae, SLC1 has been identified as the gene encoding lysophosphatidic acid acyltransferase, which catalyzes PA synthesis. However, despite the importance of PA, disruption of SLC1 does not affect cell viability (Nagiec, M. M., Wells, G. B., Lester, R. L., and Dickson, R. C. (1993) J. Biol. Chem. 268, 22156–22163). We originally aimed to identify the acetyl-CoA:lyso platelet-activating factor acetyltransferase (lysoPAF AT) gene in yeast. Screening of a complete set of yeast deletion clones (4741 homozygous diploid clones) revealed a single mutant strain, YOR175c, with a defect in lysoPAF AT activity. YOR175c has been predicted to be a member of the membrane-bound O-acyltransferase superfamily, and we designated the gene LPT1. An Lpt1-green fluorescent protein fusion protein localized at the endoplasmic reticulum. Other than lysoPAF AT activity, Lpt1 catalyzed acyltransferase activity with a wide variety of lysophospholipids as acceptors, including lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, and lysophosphatidylserine. A liquid chromatography-mass spectrometry analysis indicated that lysophosphatidylcholine and lysophosphatidylethanolamine accumulated in the Δlpt1 mutant strain. Although the Δlpt1 mutant strain did not show other detectable defects, the Δlpt1 Δslc1 double mutant strain had a synthetic lethal phenotype. These results indicate that, in concert with Slc1, Lpt1 plays a central role in PA biosynthesis, which is essential for cell viability. The cell membrane is a semipermeable lipid bilayer found in all living cells that physically separates the cytoplasm of the cell from the extracellular environment. Glycerophospholipids and sphingophospholipids are the major components of most cell membranes. Phosphatidic acid (PA) 2The abbreviations used are: PA, phosphatidic acid; PAF, platelet-activating factor; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LPS, lysophosphatidylserine; LPG, lysophosphatidylglycerol; LPI, lysophosphatidylinositol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PG, phosphatidylglycerol; PI, phosphatidylinositol; CL, cardiolipin; G-3-P, glycerol 3-phosphate; DHAP, dihydroxyacetone phosphate; lysoPAF AT, acetyl-CoA:lysoPAF acetyltransferase; MBOAT, membrane-bound O-acyltransferase; LC/MS, liquid chromatography/mass spectrometry; HA, hemagglutinin; ORF, open reading frame; GFP, green fluorescent protein; ER, endoplasmic reticulum; aa, amino acid(s); CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; LPAAT, lysophosphatidic acid acyltransferase. is a key intermediate in the biosynthesis of glycerophospholipids. PA is synthesized by two major de novo biosynthetic pathways that utilize either glycerol 3-phosphate (G-3-P) or dihydroxyacetone phosphate (DHAP) as precursors (1Carman G.M. Zeimetz G.M. J. Biol. Chem. 1996; 271: 13293-13296Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 2Sorger D. Daum G. Appl. Microbiol. Biotechnol. 2003; 61: 289-299Crossref PubMed Scopus (120) Google Scholar). G-3-P is acylated by G-3-P acyltransferase at the sn-1 position to form lysophosphatidic acid (LPA). DHAP is acylated at the sn-1 position by DHAP acyltransferase to produce 1-acyl-DHAP, which is reduced by 1-acyl-DHAP reductase to form LPA. LPA produced by these two different pathways is further acylated by LPA acyltransferase in the sn-2 position to yield PA. In mammals, several LPA acyltransferase genes have been cloned, and their gene products have been characterized (3West J. Tompkins C.K. Balantac N. Nudelman E. Meengs B. White T. Bursten S. Coleman J. Kumar A. Singer J.W. Leung D.W. DNA Cell Biol. 1997; 16: 691-701Crossref PubMed Scopus (112) Google Scholar, 4Eberhardt C. Gray P.W. Tjoelker L.W. J. Biol. Chem. 1997; 272: 20299-20305Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 5Stamps A.C. Elmore M.A. Hill M.E. Kelly K. Makda A.A. Finnen M.J. Biochem. J. 1997; 326: 455-461Crossref PubMed Scopus (43) Google Scholar, 6Aguado B. Campbell R.D. Biochem. Soc. Transact. 1997; 25: S597Crossref PubMed Scopus (2) Google Scholar, 7Aguado B. Campbell R.D. J. Biol. Chem. 1998; 273: 4096-4105Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 8Dircks L. Sul H.S. Prog. Lipid Res. 1999; 38: 461-479Crossref PubMed Scopus (59) Google Scholar, 9Li D. Yu L. Wu H. Shan Y. Guo J. Dang Y. Wei Y. Zhao S. J. Hum. Genet. 2003; 48: 438-442Crossref PubMed Scopus (40) Google Scholar, 10Lu B. Jiang Y.J. Zhou Y. Xu F.Y. Hatch G.M. Choy P.C. Biochem. J. 2005; 385: 469-477Crossref PubMed Scopus (91) Google Scholar, 11Ye G.M. Chen C. Huang S. Han D.D. Guo J.H. Wan B. Yu L. DNA Seq. 2005; 16: 386-390Crossref PubMed Scopus (34) Google Scholar, 12Yamashita A. Kawagishi N. Miyashita T. Nagatsuka T. Sugiura T. Kume K. Shimizu T. Waku K. J. Biol. Chem. 2001; 276: 26745-26752Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 13Kume K. Shimizu T. Biochem. Biophys. Res. Commun. 1997; 237: 663-666Crossref PubMed Scopus (49) Google Scholar). In yeast, SLC1, originally obtained as a gene suppressing a defect in the biosynthesis of the sphingolipid long chain base (1Carman G.M. Zeimetz G.M. J. Biol. Chem. 1996; 271: 13293-13296Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 14Nagiec M.M. Wells G.B. Lester R.L. Dickson R.C. J. Biol. Chem. 1993; 268: 22156-22163Abstract Full Text PDF PubMed Google Scholar), was identified as an LPA acyltransferase gene (15Athenstaedt K. Daum G. J. Bacteriol. 1997; 179: 7611-7616Crossref PubMed Scopus (96) Google Scholar). Glycerophospholipids, including phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC), are synthesized from PA through the cytidinediphosphodiacylglycerol pathway. Cytidinediphosphodiacylglycerol is also used as a precursor for the synthesis of phosphatidylinositol and cardiolipin. Alternatively, PE and PC are also synthesized via the CDP-ethanolamine and CDP-choline, respectively (Kennedy pathway) (1Carman G.M. Zeimetz G.M. J. Biol. Chem. 1996; 271: 13293-13296Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 16Kennedy E.P. Fed. Proc. 1961; 20: 934-940PubMed Google Scholar). Glycerophospholipids can also be generated by remodeling (also known as the Lands' cycle (17Lands W.E. J. Biol. Chem. 1960; 235: 2233-2237Abstract Full Text PDF PubMed Google Scholar, 18Lands W.E. Merkl I. J. Biol. Chem. 1963; 238: 898-904Abstract Full Text PDF PubMed Google Scholar, 19Merkl I. Lands W.E. J. Biol. Chem. 1963; 238: 905-906Abstract Full Text PDF PubMed Google Scholar), in which the rapid turnover of the sn-2 acyl moiety of glycerophospholipids is carried out by phospholipases and lysophospholipid acyltransferases, but the specific enzyme involved in the remodeling of glycerophospholipids had not been identified. Very recently, a gene for mouse lysophosphatidylcholine (LPC) acyltransferase was cloned by two different research groups and designated LPCAT1 (20Nakanishi H. Shindou H. Hishikawa D. Harayama T. Ogasawara R. Suwabe A. Taguchi R. Shimizu T. J. Biol. Chem. 2006; 281: 20140-20147Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 21Chen X. Hyatt B.A. Mucenski M.L. Mason R.J. Shannon J.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 11724-11729Crossref PubMed Scopus (151) Google Scholar). LPCAT1 is highly expressed in alveolar type II cells and is thought to be involved in the production of surfactant lipids. A related gene, LPCAT2, which encodes an enzyme having both acetyl-CoA:lyso-platelet-activating factor acetyltransferase (lysoPAF AT) activity and LPC acyltransferase activity, has also been reported (22Shindou H. Hishikawa D. Nakanishi H. Harayama T. Ishii S. Taguchi R. Shimizu T. J. Biol. Chem. 2007; 282: 6532-6539Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). In this study, we searched for lysoPAF AT genes in yeast by measuring the enzyme activity of each strain in a complete set of yeast deletion clones. By this brute force approach, we identified a novel lysoPAF AT gene that did not show any homology with LPCAT2, and which we designated LPT1 (lysoPAF AT 1). LPT1 gene products possess lysophospholipid acyltransferase activity with a wide variety of substrate acceptors, including LPC, LPE, LPA, LPI, LPS, and LPG. We also characterized in detail the enzymatic activity and cellular role of Lpt1. Strains and Media—The complete set of Yeast Deletion Clones (Homozygous Diploid) and the parental strain BY4743 were purchased from Invitrogen. Other yeast strains used in this study are shown in Table 1. Strains were grown on either YPD medium (1% yeast extract, 2% Bacto-peptone, and 2% glucose), synthetic dextrose (0.67% yeast nitrogen base without amino acids (Difco), 2% glucose, and appropriate supplements) or synthetic complete (SC) medium (SD medium, with dropout powder) prepared as previously described (23Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2543) Google Scholar). Selection for integration of deletion cassettes containing loxP-kanMX-loxP was performed on YPD plates containing 200 μg/ml G418.TABLE 1Yeast strains used in this studyStrainRelevant genotypeSource or referenceBY4743aStrain is congenic with S288CMATa/α his3delta1/his3delta1 leu2delta0/leu2delta0 lys2delta0/Lys2 met15delta0/MET15 ura3delta0/ura3delta0InvitrogenYOR175caStrain is congenic with S288CMATa/α YOR175c::kanMX/YOR175c::kanMXInvitrogenW303-1AbStrain is congenic with W303-1AMATa ade2-1 his3–11,15 leu2–3,112 trp1-1 ura3-1(61Wallis J.W. Chrebet G. Brodsky G. Rolfe M. Rothstein R. Cell. 1989; 58: 409-419Abstract Full Text PDF PubMed Scopus (453) Google Scholar)HTY210bStrain is congenic with W303-1AMATa lpt1::kanMXThis studyHTY228bStrain is congenic with W303-1AMATα slc1::kanMXThis studyHTY229bStrain is congenic with W303-1AMATa/α lpt1::kanMX/LPT1 slc1::kanMX/SLC1This studyHTY231bStrain is congenic with W303-1AMATα taz1::kanMXThis studyHTY232bStrain is congenic with W303-1AMATa /α lpt1::kanMX/LPT1 taz1::kanMX/TAZ1This studyHTY233bStrain is congenic with W303-1AMATa lpt1::kanMX taz1::kanMXThis studyMLY40MATα ura3–52(62Lorenz M.C. Heitman J. EMBO J. 1997; 16: 7008-7018Crossref PubMed Google Scholar)HTY212cStrain is congenic with Σ1278bMATα lpt1::kanMXThis studya Strain is congenic with S288Cb Strain is congenic with W303-1Ac Strain is congenic with Σ1278b Open table in a new tab Plasmid Construction—YEplac112 (24Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2521) Google Scholar) was digested with AatII, blunted, and BAP treated, and then the BsaB1-BsaA1 fragment containing the rop region from pBR322 was ligated to it to create YEp(rop). The LPT1 gene with its promoter region was amplified by PCR using the primers 5′-GAACACGAGAATTTACGCTTGT-3′ and 5′-CCGGACGACTTCTAGTTGC-3′ and ligated into the SmaI site of plasmid pUC19 to form LPT1/pUC19. The open reading frame (ORF) of LPT1 was amplified by PCR using primers (5′-GGTCGACTCATGTACAATCCTGTGGACGCTG-3′ and 5′-TTGAGCTCTACTAGCGGCCGCCCTCTTCCTTTTTTGAAATAGGC-3′) to introduce a SalI site before the first ATG codon and NotI and SacI sites just before and after the stop codon, respectively. The resultant fragment was ligated into pMW119 (Nippongene, Japan), and a NotI DNA fragment containing the triple hemagglutinin (HA) epitope coding sequence or a NotI-SacI DNA fragment containing the green fluorescent protein (GFP) coding sequence was inserted to yield LPT1-ORF(HA)/pMW119 or LPT1-ORF(GFP)/pMW119. A 2.7-kb SalI-SacI fragment of LPT1/pUC19 was inserted into pMW119 (HindIII−), in which the HindIII site is disrupted. Then the plasmid was cleaved with HindIII and SacI, and the 3′-region of the LPT1 gene was replaced with the corresponding region of LPT1-ORF(HA)/pMW119 or LPT1-ORF(GFP)/pMW119 to create LPT1-HA/pMW119(HindIII−) or LPT1-GFP/pMW119(HindIII−). Finally, the SalI-SacI fragment containing LPT1-HA and LPT1-GFP was inserted into YEp(rop) to form LPT1-HA/YEp(rop) and LPT1-GFP/YEp(rop), respectively. His-382, a potential active site residue, was changed to asparagine by using the QuikChange site-Directed mutagenesis kit (Stratagene). Site-directed mutagenesis was performed by using LPT1-HA/YEp(rop) as a template with the following primers: 5′-CTTCCGCATTTTGGAATGGTACCAGACCTGGG-3′ and 5′-AGTACCCAGGTCTGGTACCATTCCAAAATGCGGAAGTTAGG-3′ (altered nucleotides are underlined). The ORF of the PLB1 gene was amplified by PCR using primers (5′-AGTCGACATGAAGTTGCAGAGTTTGTTGG-3′ and 5′-TGCGGCCGCCAATTAGACCGAAGACGGCACTAATG-3′) to introduce a SalI site before the first ATG codon and a NotI site just before the stop codon, and the ORF was ligated into the SmaI site of plasmid pUC19 to form PLB1/pUC19. A SalI-NotI fragment containing the PLB1 ORF was inserted into YEGAp (25Hong J. Tamaki H. Akiba S. Yamamoto K. Kumagai H. J. Biosci. Bioeng. 2001; 92: 434-441Crossref PubMed Scopus (72) Google Scholar) to form PLB1/YEGAp, in which the PLB1 gene was expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. Strain Construction—The LPT1 allele was deleted in strains W303-1A and Σ1278b by using a PCR-derived loxP-kanMX-loxP cassette containing the kanamycin resistance gene (26Guldener U. Heck S. Fielder T. Beinhauer J. Hegemann J.H. Nucleic Acids Res. 1996; 24: 2519-2524Crossref PubMed Scopus (1361) Google Scholar) with the following primers: 5′-CGCCAAGACAAACCGTGGTGATTTAATTCTGCTGCTGATCGCTTCCAGCTGAAGCTTCGTACGC-3′ and 5′-GACAACAAGACTGTGACTTCCACACGCATCTGTCGTTTTTGGCCAGCATAGGCCACTAGTGGATCTG-3′. The deletion of the SLC1 allele in strain W303-1A was made by transforming the cells with the loxP-kanMX-loxP cassette derived by PCR with the following primers: 5′-TTCAATAGAGAAGTTTAGTGGTTTCCCTCCGTCAGTGAATTCGAGCAGCTGAAGCTTCGTACGC-3′ and 5′-CAGTTTTTGGGTCTATATACTACTCTAAAAATGTGGTGGTGGCTTGCATAGGCCACTAGTGGATCTG-3′. Haploid strains with single gene mutations were crossed, sporulated, and dissected to try to obtain double mutant strains. Enzyme Preparation for Screening—Each strain of the complete set of Yeast Deletion Clones were grown in 5 ml of YPD medium containing 200 μg/ml G418 at 30 °C overnight. Cells were collected by centrifugation and suspended in 2 volumes of 50 mm Tris-HCl buffer (pH 8) containing 1 mm 2-mercaptoethanol and 2 mm phenylmethylsulfonyl fluoride. The cells were disrupted with glass beads by vortexing at 4 °C, and then subjected to an enzyme assay as described below, except that the reaction was performed at 30 °C. Preparation of Microsomal Fractions—Yeast cells were suspended in 2 volumes of 50 mm Tris-HCl buffer (pH 7.5) containing 5 mm 2-mercaptoethanol and 2 mm phenylmethylsulfonyl fluoride, and disrupted with glass beads by vortexing at 4 °C. The cell debris was removed by centrifugation at 18,800 × g for 15 min at 4 °C to obtain the crude enzyme fraction. The microsomal fraction was pelleted by centrifugation of the crude enzyme fraction at 100,000 × g for 60 min at 4 °C, and the pellet was resuspended in a small amount of the same buffer. Alternatively, cells were suspended in 5 volumes of spheroplasting buffer (20 mm Tris-HCl (pH 7.5), 1.2 m sorbitol, 50 mm potassium acetate, and 1 mm 2-mercaptoethanol); Zymolyase 100T (Seikagaku, Japan) was added to a concentration of 1 mg/ml, and the cells were incubated at 30 °C for 10–30 min. The spheroplasts were washed with spheroplasting buffer and lysed in ice-cold lysis buffer (20 mm Tris-HCl (pH 7.5), 100 mm sorbitol, 50 mm potassium acetate, 1 mm 2-mercaptoethanol, and 2 mm phenylmethylsulfonyl fluoride) using a Teflon homogenizer. The cell lysate was further subjected to centrifugation and ultracentrifugation to obtain the microsome fraction as described above. We performed centrifugation at 18,800 × g before ultracentrifugation to avoid the degradation of Lpt1-HA. When we examined the enzyme properties of samples obtained at lower centrifugation speeds before ultracentrifugation, the reaction was not linear, and quick enzyme degradation was observed during the assay even in the presence of protease inhibitors. We found that the microsome fraction prepared after performing centrifugation at 18,800 × g was more stable and performed better in the enzyme assay even if the procedure resulted in the removal of a large amount of nuclei and nucleus-attached ER. Enzyme and Protein Assay—lysoPAF AT activity was measured by a modification of the procedure of Gomez-Cambronero et al. (27Gomez-Cambronero J. Velasco S. Sanchez-Crespo M. Vivanco F. Mato J.M. Biochem. J. 1986; 237: 439-445Crossref PubMed Scopus (13) Google Scholar). The reaction mixture (0.4 ml) contained 50 mm Tris-HCl buffer (pH 7.5), 50 μm lysoPAF C-16 (Cayman Chemical Co.), 3 μm of [14C]acetyl-CoA (185 GBq/mmol, Moravek Biochemicals, Inc.), 60 μm unlabeled acetyl-CoA, 2 mm phenylmethylsulfonyl fluoride, 5 mm 2-mercaptoethanol, and the enzyme solution. The reaction was performed at 0–15 °C for 20 min, after which total lipid was extracted by the method of Bligh and Dyer (28Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42855) Google Scholar) and subjected to TLC. Samples were applied to DC-Alufolien Kieselgel 60 TLC plates (Merck) and developed with chloroform/methanol/water (65:25:4, v/v). The TLC plate was exposed to an imaging plate, and synthesized [14C]PAF was visualized and quantified with a BAS-2500 Bio-imaging analyzer (Fujifilm). The authentic [3H]PAF (6.0 Ci/mmol, PerkinElmer Life Sciences) was detected by fluorography using EN3HANCE Spray (PerkinElmer Life Sciences). To investigate the substrate specificities of this enzyme, various lysophospholipids and acyl-CoAs (Sigma or Avanti Polar Lipids, Alabaster AL) were used in place of either lysoPAF or acetyl-CoA. The protein concentration was determined by the method of Bradford (29Bradford M.M. Analyt. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216334) Google Scholar). Western Blot Analysis—Microsome fraction (2 μg of total protein) was electrophoresed on a 10% SDS-PAGE and then electroblotted onto polyvinylidene difluoride membranes. The polyvinylidene difluoride membranes were blocked with TBS-T (Tris-buffered saline, pH 7.6, 0.1% Tween 20) containing 5% skim milk for 1 h at room temperature and then incubated with 0.1 μg/ml of 12CA5 monoclonal antibody or GFP polyclonal antibody for 1 h at room temperature. After washing with TBS-T, the membranes were incubated with a 1:5000 dilution of anti-mouse IgG peroxidase-linked whole antibody (from sheep) (Amersham Biosciences) or goat anti-rabbit IgG-horseradish peroxidase for 1 h at room temperature. After washing with TBS-T, HA- or GST-tagged protein was detected by the Super Signal West Pico detection reagent (Pierce). Platelet Aggregation Assay—Total lipids were extracted by the method of Bligh and Dyer and applied to an alumina column. The column was washed with chloroform, and the PAF fraction was eluted with chloroform-methanol (1:1). The eluted fraction was further loaded onto a paper fiber-made TLC sheet (Chromato sheet, Wako) and developed with chloroform/methanol/water (65:25:4). The area containing PAF was cut off, and the PAF fraction was eluted with chloroform/methanol (1:1), dried under N2 gas, and dissolved in 100 μl of bovine serum albumin/saline. Washed rabbit platelets were prepared by the method of Pincard et al. (30Pinckard R.N. Farr R.S. Hanahan D.J. J. Immunol. 1979; 123: 1847-1857PubMed Google Scholar). Tyrode's solution (160 μl, pH 7.2, containing 1.3 mm Ca2+) was mixed with 40 μl of washed rabbit platelets and preincubated at 37 °C for 1 min. Then 10 μl of sample lipid was added. Aggregation was assayed by monitoring the change in light transmittance with a Hematracer PAT-4A (Nikko, Japan). Confocal Microscopy—The Δlpt1 mutant strain transformed with LPT1-GFP/YEp(rop) was grown in SC(Trp−) liquid medium to early log phase, and the cells were analyzed using a confocal microscope (FV1000, Olympus) equipped with a 100 × oil-immersion objective. Liquid Chromatography/Mass Spectrometric Analysis—LC/MS analysis was performed in the positive mode on an LCMS-2010 mass spectrometer (Shimadzu, Kyoto, Japan) equipped with an electrospray ion source. High performance liquid chromatography separation was carried out on a normalphase column (Develosil 60, 2.0 × 150 mm, Nomura Chemicals, Seto, Japan). The column was eluted with solvent A (acetonitrile-methanol (2:1) containing 0.1% ammonium formate (pH 6.4)) and solvent B (methanol-H2O (2:1) containing 0.1% ammonium formate (pH 6.4)) at a flow rate of 0.2 ml/min. The gradient separation was carried out using the following conditions: isocratic elution with 100% solvent A for 5 min followed by a linear gradient of 100% to 70% solvent A over 40 min. Spectra were obtained over a mass range from m/z 400–1200 with a scan time of 1 s. Identification of lysoPAF AT in the Yeast S. cerevisiae—We previously reported that Saccharomyces cerevisiae produce platelet-activating factor (PAF) (31Nakayama R. Kumagai H. Saito K. Biochim. Biophys. Acta. 1994; 1199: 137-142Crossref PubMed Scopus (16) Google Scholar). To identify the gene for lysoPAF AT, we screened a complete set of homozygous diploid yeast deletion clones as described under “Experimental Procedures.” Using [14C]acetyl-CoA and lysoPAF, we detected lysoPAF AT activity in both crude cell-free extracts and microsome fractions (100,000 × g pellets) of yeast. In the 4741 strains screened, we found one clone that did not produce [14C]PAF. This clone lacked ORF YOR175c, an as-yet-unidentified gene encoding a putative membrane-bound O-acyltransferase. We designated this gene LPT1. We deleted the corresponding gene in several yeast strains with different genetic backgrounds. All strains tested possessed lysoPAF AT activity, and when the LPT1 gene was deleted, the activity completely disappeared (Fig. 1A). In contrast, the enzyme activity increased when the LPT1 gene was introduced with a multicopy vector (Fig. 1A). We also prepared microsome fractions from the Δlpt1 strain transformed with the LPT1 overexpression vector or a control vector and used these fractions for PAF synthesis with lysoPAF and acetyl-CoA as the substrates. The lipids were extracted from the enzyme reaction products and analyzed by LC/MS. Synthesized PAF was identified by comparing their retention time in the column and m/z 524.4 with those of standard C16: PAF. The reaction with microsomes obtained from the Δlpt1 strain transformed with the LPT1 overexpression vector produced a time-dependent increase of C16:PAF production; this increase was not observed in the Δlpt1 strain harboring the control vector (data not shown), suggesting that LPT1 is required for PAF synthesis. The lipid fractions were further purified, and the PAF fractions were subjected to a platelet aggregation assay. The PAF fraction from the LPT1-overexpressing strain showed strong platelet aggregation activity, which was hardly observed when the PAF fraction from the Δlpt1 strain with the control vector was used (Fig. 1B) or when lysoPAF was omitted from the reaction mixture. The platelet aggregation activity was also strongly inhibited in the presence of 10−6 m WEB2086, a PAF antagonist (Fig. 1B). From these results we identified LPT1 as a lysoPAF AT gene in the yeast S. cerevisiae. Lpt1 Is a Member of the MBOAT Superfamily—YOR175c (LPT1) has been reported to be a member of the membrane bound O-acyltransferase (MBOAT) superfamily (32Hofmann K. Trends Biochem. Sci. 2000; 25: 111-112Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). The MBOAT superfamily proteins harbor several membrane-spanning regions and share sequence similarity regions. Four other MBOAT genes are present in S. cerevisiae. ARE1 and ARE2 were identified as genes encoding ergosterol acyltransferases (33Yang 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 (225) Google Scholar), and the Gup1 and Gup2 proteins were initially reported to be involved in glycerol uptake (34Holst B. Lunde C. Lages F. Oliveira R. Lucas C. Kielland-Brandt M.C. Mol. Microbiol. 2000; 37: 108-124Crossref PubMed Scopus (86) Google Scholar). Gup1 was recently proposed to be an acyltransferase involved in the remodeling of glycosylphosphatidylinositol anchors (35Bosson R. Jaquenoud M. Conzelmann A. Mol. Biol. Cell. 2006; 17: 2636-2645Crossref PubMed Scopus (113) Google Scholar); however, Lpt1 showed low similarity to Gup1 (15%/260 aa), Gup2 (14%/93 aa), Are1 (18.5%/108 aa), and Are2 (16.7%/234 aa). A search of databases for homologues of yeast Lpt1 identified several MBOATs, including SPBC16A3.10 in the fission yeast Schizosaccharomyces pombe, OACT1 and -2 in both human and mouse (Fig. 2), and C54G7.2 and C08F8.4 in Caenorhabditis elegans. The predicted amino acid sequence of LPT1 shared 27.5% identity with OACT1 (per 459 aa) and 28.8% identity with OACT2 (per 458 aa). It was predicted that a conserved histidine residue within a long hydrophobic region might be a candidate for the active-site residue (32Hofmann K. Trends Biochem. Sci. 2000; 25: 111-112Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). The corresponding histidine residue was found at position of 382 in Lpt1 (Fig. 2). To test whether His-382 is involved in the catalytic center, we constructed an HA-tagged mutant Lpt1 expression vector in which His-382 was replaced by asparagine. Although we confirmed the expression of both native and mutant Lpt1-HA in the microsomal fraction of the Δlpt1 mutant strain by Western blot analysis, we did not detect lysoPAF AT activity in the mutant enzyme fraction (see Fig. 6, B and C). From this result, we concluded that His-382 is critical for the catalytic reaction and is most likely an active-site residue.FIGURE 6Characterization of tagged and mutant Lpt1. A, cellular localization of Lpt1-GFP. Δlpt1 cells expressing the Lpt1-GFP fusion protein were grown in SC(Trp−) medium and examined under a fluorescence microscope as described under “Experimental Procedures.” Differential interference contrast image (right). Lpt1-GFP image (left). B, Western blot analysis. The HA-tagged mutant (H382N) and wild-type Lpt1 expression vectors were transformed into a Δlpt1 mutant strain. Both mutant (H382N) and wild-type Lpt1-HA proteins were detected in the microsomal fraction using 12CA5 monoclonal antibody (left, middle). Lpt1-GFP protein was detected by using anti-GFP antibody (right). C, enzyme assay. lysoPAF-AT (top) and LPC acyltransferase assays (bottom) were performed as described under “Experimental Procedures” with [14C]acetyl-CoA (2:0-CoA) and [1-14C]arachidonoyl-CoA (20:4-CoA) using lysoPAF and palmitoyl-LPC, respectively. Microsome fractions from Δlpt1 cells harboring the native, mutant, or tagged Lpt1 expression vector were examined.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Substrate Selectivity of Lpt1—We tried to solubilize the enzyme from the microsomal fraction using various surfactants, including CHAPS and octylglucoside, but the enzyme activity was considerably decreased by solubilization (data not shown). We therefore used the microsome fraction from the Lpt1-overproducing strain to characterize the enzyme. Although the enzyme reaction was performed at 30 °C during the screening, we noticed that the enzyme activity decreased rapidly at this reaction temperature because the Lpt1 protein degraded even in the presence of various protease inhibitors (data not shown). For these reasons, the reaction was performed at 0–15" @default.
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• W2092034105 date "2007-11-01" @default.
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• W2092034105 title "LPT1 Encodes a Membrane-bound O-Acyltransferase Involved in the Acylation of Lysophospholipids in the Yeast Saccharomyces cerevisiae" @default.
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