FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2058885206> ?p ?o ?g. }

• W2058885206 endingPage "36117" @default.
• W2058885206 startingPage "36110" @default.
• W2058885206 abstract "Glycerophosphocholine is formed via the deacylation of the phospholipid phosphatidylcholine. The protein encoded by Saccharomyces cerevisiae open reading frame YPL110c effects glycerophosphocholine metabolism in vivo, most likely by acting as a glycerophosphocholine phosphodiesterase. Deletion of YPL110c causes an accumulation of glycerophosphocholine in cells prelabeled with [14C]choline. Correspondingly, overexpression of YPL110c results in reduced intracellular glycerophosphocholine in cells prelabeled with [14C]choline. Glycerophospho[3H]choline supplied in the growth medium accumulates to a much greater extent in the intracellular fraction of a YPL110Δ strain than in a wild type strain. Furthermore, glycerophospho[3H]choline accumulation requires the transporter encoded by GIT1, a known glycerophosphoinositol transporter. Growth on glycerophosphocholine as the sole phosphate source requires YPL110c and the Git1p permease. In contrast to glycerophosphocholine, glycerophosphoinositol metabolism is unaffected by deletion of YPL110c. The open reading frame YPL110c has been termed GDE1. Glycerophosphocholine is formed via the deacylation of the phospholipid phosphatidylcholine. The protein encoded by Saccharomyces cerevisiae open reading frame YPL110c effects glycerophosphocholine metabolism in vivo, most likely by acting as a glycerophosphocholine phosphodiesterase. Deletion of YPL110c causes an accumulation of glycerophosphocholine in cells prelabeled with [14C]choline. Correspondingly, overexpression of YPL110c results in reduced intracellular glycerophosphocholine in cells prelabeled with [14C]choline. Glycerophospho[3H]choline supplied in the growth medium accumulates to a much greater extent in the intracellular fraction of a YPL110Δ strain than in a wild type strain. Furthermore, glycerophospho[3H]choline accumulation requires the transporter encoded by GIT1, a known glycerophosphoinositol transporter. Growth on glycerophosphocholine as the sole phosphate source requires YPL110c and the Git1p permease. In contrast to glycerophosphocholine, glycerophosphoinositol metabolism is unaffected by deletion of YPL110c. The open reading frame YPL110c has been termed GDE1. The yeast Saccharomyces cerevisiae synthesizes and degrades the major glycerophospholipids via pathways that are very similar to those employed by higher eukaryotes (1Carman G.M. Kersting M.C. Biochem. Cell Biol. 2004; 82: 62-70Crossref PubMed Scopus (43) Google Scholar, 2Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (261) Google Scholar). One pathway of phospholipid degradation in yeast as well as higher eukaryotes is deacylation, which results in the formation of water-soluble glycerophosphodiesters. S. cerevisiae cells growing in medium containing nonlimiting amounts of Pi and inositol deacylate phosphatidylinositol via the action of phospholipases of the B type, Plb3p and Plb1p, to produce glycerophosphoinositol (GroPIns) 2The abbreviations used are:GroPInsglycerophosphoinositolPCphosphatidylcholineGroPChoglycerophosphocholineGPDEglycerophosphodiester phosphodiesteraseGSTglutathione S-transferaseHPLChigh pressure liquid chromatographyMES4-morpholineethanesulfonic acid 2The abbreviations used are:GroPInsglycerophosphoinositolPCphosphatidylcholineGroPChoglycerophosphocholineGPDEglycerophosphodiester phosphodiesteraseGSTglutathione S-transferaseHPLChigh pressure liquid chromatographyMES4-morpholineethanesulfonic acid (3Angus W.W. Lester R.L. Arch. Biochem. Biophys. 1972; 151: 483-495Crossref PubMed Scopus (68) Google Scholar, 4Patton J.L. Pessoa-Brandao L. Henry S.A. J. Bacteriol. 1995; 177: 3379-3385Crossref PubMed Google Scholar, 5Merkel O. Fido M. Mayr J.A. Prüger H. Raab F. Zandonella G. Kohlwein S.D. Paltauf F. J. Biol. Chem. 1999; 274: 28121-28127Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Much of the GroPIns produced is excreted into the medium, and external GroPIns can be transported into the yeast cell in times of nutritional stress (phosphate or inositol limitation) via the permease encoded by GIT1 (see Fig. 1) (6Patton-Vogt J.L. Henry S.A. Genetics. 1998; 149: 1707-1715Crossref PubMed Google Scholar, 7Almaguer C. Cheng W. Nolder C. Patton-Vogt J. J. Biol. Chem. 2004; 279: 31937-31942Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Git1p, a member of the major facilitator superfamily of transport proteins (8Nelissen B. De Wachter R. Goffeau A. FEMS Microbiol. Rev. 1997; 21: 113-134Crossref PubMed Google Scholar), was originally isolated based upon its ability to confer growth to an inositol auxotroph supplied with GroPIns as its inositol source (6Patton-Vogt J.L. Henry S.A. Genetics. 1998; 149: 1707-1715Crossref PubMed Google Scholar). Subsequent studies have shown that although inositol limitation up-regulates Git1p transport activity, phosphate limitation does so to a much greater extent and GroPIns can be used as the sole source of phosphate for the cell (7Almaguer C. Cheng W. Nolder C. Patton-Vogt J. J. Biol. Chem. 2004; 279: 31937-31942Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The enzyme(s) required for GroPIns catabolism remain uncharacterized.Phosphatidylcholine (PC) deacylation also occurs in S. cerevisiae, resulting in the formation of intracellular and extracellular glycerophosphocholine (GroPCho) (Fig. 1). In general, S. cerevisiae produces more internal than external GroPCho (9Dowd 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). However, external GroPCho production increases as the pH of the medium is raised above 5 (11Merkel O. Oskolkova O.V. Raab F. El-Toukhy R. Paltauf F. Biochem. J. 2005; 387: 489-496Crossref PubMed Scopus (28) Google Scholar). Similarly, internal GroPCho production increases upon increased flux through the CDP-choline pathway for PC synthesis as a consequence of temperature elevation or choline supplementation (9Dowd 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). S. cerevisiae Nte1p, a phospholipase B and homolog of human neuropathy target esterase, is responsible for the production of intracellular GroPCho via PC deacylation (10Zaccheo O. Dinsdale D. Meacock P.A. Glynn P. J. Biol. Chem. 2004; 279: 24024-24033Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Plb1p (11Merkel O. Oskolkova O.V. Raab F. El-Toukhy R. Paltauf F. Biochem. J. 2005; 387: 489-496Crossref PubMed Scopus (28) Google Scholar) is thought to be primarily responsible for the formation of extracellular GroPCho (Fig. 1).As is true for S. cerevisiae, various mammalian cells respond to an increase in PC synthesis by increasing PC deacylation (12Baburina I. Jackowski S. J. Biol. Chem. 1999; 274: 9400-9408Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 13Barbour S.E. Kapur A. Deal C.L. Biochim. Biophys. Acta. 1999; 1439: 77-88Crossref PubMed Scopus (77) Google Scholar, 14Walkey C.J. Kalmar G.B. Cornell R.B. J. Biol. Chem. 1994; 269: 5742-5749Abstract Full Text PDF PubMed Google Scholar), suggesting a conserved role for this degradative pathway in maintaining PC homeostasis. The build-up of GroPCho in the cell has been associated with a number of disease processes, including cancer (15Glunde K. Jie C. Bhujwalla Z.M. Cancer Res. 2004; 64: 4270-4276Crossref PubMed Scopus (314) Google Scholar) and Alzheimer disease (16Walter A. Korth U. Hilgert M. Hartmann J. Weichel O. Fassbender K. Schmitt A. Klein J. Neurobiol. Aging. 2004; 25: 1299-1303Crossref PubMed Scopus (114) Google Scholar). In addition, GroPCho has been implicated in diverse cellular functions such as maintenance of renal osmolarity (17Bauernschmitt H.G. Kinne R.K. Biochim. Biophys. Acta. 1993; 1150: 25-34Crossref PubMed Scopus (23) Google Scholar), inhibition of lysophospholipase activity (18Fallbrook A. Turenne S.D. Mamalias N. Kish S.J. Ross B.M. Brain Res. 1999; 834: 207-210Crossref PubMed Scopus (20) Google Scholar), and inhibition of phosphatidylinositol transfer protein alpha (19Komatsu H. Westerman J. Snoek G.T. Taraschi T.F. Janes N. Biochim. Biophys. Acta. 2003; 1635: 67-74Crossref PubMed Scopus (8) Google Scholar). Clearly, cell physiology impacts, and is impacted by, GroPCho levels. In turn, the level of GroPCho in the cell is a function of both its formation via PC deacylation and its degradation via glycerophosphodiesterases. Glycerophosphodiesterase encoding genes and glycerophosphodiesterase activities acting upon GroPCho have been reported for several cell types, including Escherichia coli (20Tommassen J. Eiglmeier K. Cole S.T. Overduin P. Larson T.J. Boos W. Mol. Gen. Genet. 1991; 226: 321-327Crossref PubMed Scopus (71) Google Scholar), Haemophilus influenzae (21Munson Jr., R.S. Sasaki K. J. Bacteriol. 1993; 175: 4569-4571Crossref PubMed Google Scholar), carrot cell wall (22Van Der Rest B. Rolland N. Boisson A.M. Ferro M. Bligny R. Douce R. Biochem. J. 2004; 379: 601-607Crossref PubMed Google Scholar), kidney (23Kwon E.D. Zablocki K. Peters E.M. Jung K.Y. Garcia-Perez A. Burg M.B. Am. J. Physiol. 1996; 270: C200-C207Crossref PubMed Google Scholar), and brain (24Sok D.E. Neurochem. Res. 1998; 23: 1061-1067Crossref PubMed Scopus (7) Google Scholar). GroPIns-specific glycerophosphodiesterase activities have been observed in various rat tissues (25Dawson R.M. Hemington N. Richards D.E. Irvine R.F. Biochem. J. 1979; 182: 39-49Crossref PubMed Scopus (5) Google Scholar, 26Dawson R.M. Hemington N. Biochem. J. 1977; 162: 241-245Crossref PubMed Scopus (8) Google Scholar), and a gene encoding a glycerophosphoinositol glycerophosphodiesterase (GDE1/MIR16) has been cloned from rat (27Zheng B. Berrie C.P. Corda D. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1745-1750Crossref PubMed Scopus (69) Google Scholar).The use of S. cerevisiae as a powerful model for studying phospholipid metabolism is well established (1Carman G.M. Kersting M.C. Biochem. Cell Biol. 2004; 82: 62-70Crossref PubMed Scopus (43) Google Scholar, 2Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (261) Google Scholar). An understudied aspect of this metabolism is that of the glycerophosphodiesters, such as GroPCho, produced through phospholipid deacylation. Thus, these studies were undertaken to further our knowledge of glycerophosphodiester metabolism in this important model organism. We demonstrate that the protein encoded by YPL110c (here named GDE1) affects glycerophosphocholine levels in the cell, most probably by acting as a glycerophosphodiester phosphodiesterase. Furthermore, we report that GroPCho is transported intact into the cell in a manner dependent upon the GroPIns permease, Git1p.MATERIALS AND METHODSStrains and Media—Strains were maintained on YEPD medium (1% yeast extract, 2% Bactopeptone, 2% glucose). The base medium for experiments was chemically defined synthetic media (4Patton J.L. Pessoa-Brandao L. Henry S.A. J. Bacteriol. 1995; 177: 3379-3385Crossref PubMed Google Scholar) lacking inositol (I–). Where indicated, I– medium was supplemented with 75 μm inositol to make I+ medium. For experiments involving the overexpression of GDE1 from the GAL1 promoter (see Fig. 4), the strains were grown in I– medium lacking dextrose but containing 2% raffinose and 2% galactose. For experiments involving the addition of exogenous GroPIns or GroPCho (Sigma; catalog number G4007), the base medium was altered by substituting 1g of KCl for 1g of KH2PO4/liter and adding KH2PO4 to a final concentration of 0.2 mm (low Pi) or 10 mm (high Pi) (7Almaguer C. Cheng W. Nolder C. Patton-Vogt J. J. Biol. Chem. 2004; 279: 31937-31942Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Strains obtained from Research Genetics (JPV125 and JPV126) were checked by PCR to confirm the expected gene deletion. Strain JPV131 was isolated following tetrad dissection of diploid JPV126. To make the gde1Δ ypl206cΔ (JPV431) double mutant, the KanMX marker of strain JPV125 was exchanged with HIS3 using the marker swap plasmid M4754 (28Voth W.P. Jiang Y.W. Stillman D.J. Yeast. 2003; 20: 985-993Crossref PubMed Scopus (71) Google Scholar). Plasmid M4754 was digested with NotI, and the released fragment containing HIS3 flanked by regions of the KanMX gene was used to transform JPV125. His+ colonies were checked by PCR to verify integration at the correct location, and the resulting strain was named JPV433. Strain JPV431 was isolated following tetrad dissection of the diploid formed by crossing JPV433 with JPV131 (TABLE ONE).FIGURE 4Overexpression of GDE1 results in reduced intracellular GroPCho upon labeling with [14C]choline. Wild type (wt) and a wild type strain bearing pGAL-GST-GDEl (pGAL-GDEl) were grown to logarithmic phase on 2% raffinose, 2% galactose medium containing 18 μm [14C]choline. Wild type containing multicopy GDE1 (Wt-M) or empty vector (Wt-E) were grown to logarithmic phase in I– medium containing 18 μm [14C]choline. A, the distribution of counts between the trichloroacetic acid (TCA)-soluble and membrane fractions of the cell was determined. The values represent the mean s± S.E. of triplicate determinations derived from two independent experiments. B, the identity of the water-soluble counts was determined for a single experiment of A. The values represent the mean s± S.E. of duplicate determinations. C, Western blot of cell lysates obtained from a wild type strain (wt) and a strain bearing pGAL-GST-GDEl (pGAL-GDE1*) grown in raffinose/galactose medium as in A. Middle lane, strain bearing pGAL-GST-GDEl (pGAL-GDE1) grown under noninducing (2% glucose) conditions.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE ONEYeast strainsStrainRelevant genotypeGenotypeSourceJPV125gde1Δhis3Δ1, leu2Δ0, met15Δ0, ura3Δ0, YPL110c::KanMX, MATaResearch GeneticsJPV126YPL206cΔ/YPL206chis3Δ1/his3Δ, leu2Δ0/leu2Δ0, met15Δ0/MET15, lys2Δ0/LYS2, ura3Δ0/ ura3Δ0, YPL206c::KanMX/YPL206c, MATa/αResearch GeneticsJPV131YPL206cΔhis3Δ1, leu2Δ0, ura3Δ0, YPL206c::KanMX, MATαThis studyJPV203wild typehis3Δ1, leu2Δ0, met15Δ0, ura3Δ0, MATaResearch GeneticsJPV431gde1Δ YPL206cΔhis3Δ1, leu2Δ0, met15Δ0, ura3Δ0, YPL110c::HIS3, YPL206c::KanMX, MATaThis studyJPV433gde1Δhis3Δ1, leu2Δ0, met15Δ0, ura3Δ0, YPL110c::HIS3, MATaThis studyJPV436pGAL-GST-YPL110chis3Δ1, leu2Δ0, met15Δ0, ura3Δ0, YPL110c::KanMX-pGAL-GST-YPL110c, MATaThis study Open table in a new tab Construction of pGAL-GST-GDE1 Allele—Plasmid pFA6a-kanMX6-PGAL-GST (29Longtine M.S. McKenzie 3rd, A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4110) Google Scholar) was used as template to amplify a module for insertion into the genome at the 5′ end of GDE1. The 5′ ends of the forward and reverse primers bore 40 nucleotides homologous to the target gene sequences, followed by 20 nucleotides homologous to the plasmid. The underlined sequences are homologous to the target genes: forward primer, 5′-TAA TTG CGA CTT CCA CGT GTT CGC AGG TGG AGC AAT GTT ACA GAA TTC GAG CTC GTT TAA AC-3′, and reverse primer, 5′-CTC TGG AAT GCG ATG ATT GGC AAA GGT TTT TCC GAA CTT CAT ACG CGG AAC CAG ATC CGA TT-3′. The PCR product was transformed (30Gietz D. St Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2878) Google Scholar) into JPV203, and G418-resistant colonies were selected (31Wach A. Brachat A. Pohlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2225) Google Scholar). To verify integration at the correct location, genomic DNA was isolated and used as template in PCR using forward primer, 5′-TTC TTT CTT TTT ATG CAT CTT-3′, and reverse primer, 5′-CAA CGA CTT GTA GCC AAC ATA-3′. Integration of the PCR module resulted in the following changes in the genomic DNA: (i) deletion of ∼500 bp immediately upstream of the native GDE1 start codon and replacement with the GAL1 promoter and (ii) fusion of the GST gene to the 5′ end of GDE1. The resulting strain was named pGAL-GST-GDE1 (JPV436).Construction of Plasmid B94 Containing Multicopy GDE1—Cosmid 70990 (ATCC) was digested with XbaI and HindIII to release a 5617-bp fragment containing open reading frame YPL110c and a neighboring open reading frame (CAR1). This fragment was ligated into vector YCplac33 (32Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2506) Google Scholar) that had been digested with XbaI and HindIII, creating plasmid B88. Plasmid B88 was digested with XbaI and BglIII to release only open reading frame YPL110c and flanking sequences (900 bp upstream and 488 bp downstream). The released fragment was ligated into the XbaI and BamHI sites of YEplac195 (32Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2506) Google Scholar) to create plasmid B94.[14C]Choline Labeling and Metabolite Analysis—Strains were uniformly labeled by growing in I– medium containing 1 μCi/ml [14C]choline at a concentration of 18 μm. For some experiments, a chase was performed by washing the cells free of label and reinoculating them into I– medium containing 1 mm nonradiolabeled choline. Prior to HPLC analysis of the [14C]choline-labeled metabolites (27Zheng B. Berrie C.P. Corda D. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1745-1750Crossref PubMed Scopus (69) Google Scholar), water-soluble fractions were processed by extracting three times with water-saturated ether to remove trichloroacetic acid, drying down, and resuspending in solvent A (27Zheng B. Berrie C.P. Corda D. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1745-1750Crossref PubMed Scopus (69) Google Scholar).[3H]Choline-GroPCho Labeling and Metabolite Analysis—Strains were grown in I+ low Pi medium containing 10 μm [3H]choline-GroPCho to the late logarithmic/early stationary phase of growth (A600 = 1–1.5). The counts associated with the membranous and water-soluble fractions were determined through trichloroacetic acid extraction as described previously (9Dowd 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). Ion exchange chromatography was employed for the separation of [3H]choline-containing metabolites (33Cook S.J. Wakelam M.J. Biochem. J. 1989; 263: 581-587Crossref PubMed Scopus (153) Google Scholar).Analysis of GroPIns Metabolism—Strains were grown for several generations in I– medium containing either low Pi or no Pi (7Almaguer C. Cheng W. Nolder C. Patton-Vogt J. J. Biol. Chem. 2004; 279: 31937-31942Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) and supplemented with 5–25 μm [3H]GroPIns. Counts associated with the membranous and water-soluble fractions were determined through trichloroacetic acid extraction as described previously (9Dowd 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). The water-soluble counts were separated by anion exchange chromatography (4Patton J.L. Pessoa-Brandao L. Henry S.A. J. Bacteriol. 1995; 177: 3379-3385Crossref PubMed Google Scholar).Phospholipid Composition—Wild type and gde1Δ mutant strains were grown to logarithmic phase in I– or I+ media containing 10 μCi/ml [32P]orthophosphate. Labeled lipids were extracted (34Atkinson 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 individual phospholipid species were resolved (35Steiner M.R. Lester R.L. Biochim. Biophys. Acta. 1972; 260: 222-243Crossref PubMed Scopus (108) Google Scholar) by two-dimensional chromatography.Western Blot Analysis—Western analysis was performed as described previously (7Almaguer C. Cheng W. Nolder C. Patton-Vogt J. J. Biol. Chem. 2004; 279: 31937-31942Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) using a monoclonal antibody against GST (Covance Inc.; catalog number MMS-112P) as the primary antibody.Cell Disruption and in Vitro Assays of Phosphodiesterase Activity—Several growth conditions, three methods of cell disruption, and multiple assay conditions were employed in attempts to detect in vitro glycerophosphodiester phosphodiesterase activity in wild type and GIT1 overexpressing strains of S. cerevisiae. The strains were grown in medium in which both inositol and phosphate concentrations were varied (I+ low Pi,I– low Pi,I+ high Pi, and I– high Pi). Disruptions were performed using either glass bead breakage (36Beck T. Schmidt A. Hall M.N. J. Cell Biol. 1999; 146: 1227-1238Crossref PubMed Scopus (249) Google Scholar), detergent lysis using celLytic Y reagent (Sigma), or enzymatic lysis using a celLytic Y plus reagent kit (Sigma) in the presence of 1 mm phenylmethylsulfonyl fluoride or yeast protease inhibitor mixture (Sigma; catalog number P8215). Following disruption, the lysates were centrifuged for either 90,000 × g or 20,000 × g for 1 h. In each experiment, lysates, resuspended pellets, and supernatants were assayed for activity. The basic assay mix contained 55 mm Tris-HCl, pH 7.4, 10 mm MgCl2, 0.5 mm [3H]GroPCho, and 40–100 μg of protein in a total volume of 100 μl, and the reactions were allowed to proceed for 1 h at 30°C. Variations on this assay included substitution of ZnCl or CaCl for MgCl, the addition of 5 mm dithiothreitol, the addition of various concentrations of detergents (Triton X-100, Nonidet P-40, and SDS), changing the pH of the buffer to 8.5 or 6.0 (50 mm MES), and allowing the reaction to proceed for several hours. The reactions were stopped by the addition of 300μl of cold methanol, followed by the addition of 300 μl of chloroform to produce two phases. The upper layer was removed, dried down, and suspended in solvent A for subsequent HPLC analysis. In some cases, the reaction products were analyzed by ion exchange chromatography (33Cook S.J. Wakelam M.J. Biochem. J. 1989; 263: 581-587Crossref PubMed Scopus (153) Google Scholar).Production of Radiolabeled GroPIns and GroPCho—Tritium-labeled GroPIns ([3H]GroPIns) and GroPCho ([3H]GroPCho) were produced through the deacylation of phosphatidyl-myo-[2-3H]inositol and phosphatidylcholine-methyl-[3H], respectively (American Radiolabeled Chemicals), as described (37Hama H. Takemoto J.Y. DeWald D.B. Methods. 2000; 20: 465-473Crossref PubMed Scopus (34) Google Scholar).RESULTSGde1p (YPL110cp) and YPL206c Contain Glycerophosphodiester Phosphodiesterase (GPDE) Motifs—A thorough understanding of the importance of phospholipid deacylation to cellular physiology requires knowledge about all aspects of the metabolism, including the fate of the resulting glycerophosphodiesters. Proteins containing GPDE motifs (Pfam accession number PF03009) as described by Pfam (38Bateman A. Coin L. Durbin R. Finn R.D. Hollich V. Griffiths-Jones S. Khanna A. Marshall M. Moxon S. Sonnhammer E.L. Studholme D.J. Yeats C. Eddy S.R. Nucleic Acids Res. 2004; 32: D138-D141Crossref PubMed Google Scholar) are wide-spread in nature, with over 500 proteins containing the motif being reported by InterPro (www.ebi.ac.uk/interpro/) (39Mulder N.J. Apweiler R. Attwood T.K. Bairoch A. Bateman A. Binns D. Bradley P. Bork P. Bucher P. Cerutti L. Copley R. Courcelle E. Das U. Durbin R. Fleischmann W. Gough J. Haft D. Harte N. Hulo N. Kahn D. Kanapin A. Krestyaninova M. Lonsdale D. Lopez R. Letunic I. Madera M. Maslen J. McDowall J. Mitchell A. Nikolskaya A.N. Orchard S. Pagni M. Ponting C.P. Quevillon E. Selengut J. Sigrist C.J. Silventoinen V. Studholme D.J. Vaughan R. Wu C.H. Nucleic Acids Res. 2005; 33: D201-D205Crossref PubMed Scopus (454) Google Scholar). Two S. cerevisiae gene products, Gde1p and YPL206c, contain GPDE motifs that could potentially be involved in the hydrolysis of GroPCho and/or GroPIns (Fig. 2). The focus of this work, Gde1p (Fig. 2), also contains ankyrin repeats (cd00204) and an SPX domain (pfam03105), as predicted by the NCBI CDART tool (db.yeastgenome.org/cgi-bin/protein/getDomain) (40Wheeler D.L. Church D.M. Federhen S. Lash A.E. Madden T.L. Pontius J.U. Schuler G.D. Schriml L.M. Sequeira E. Tatusova T.A. Wagner L. Nucleic Acids Res. 2003; 31: 28-33Crossref PubMed Scopus (738) Google Scholar, 41Geer L.Y. Domrachev M. Lipman D.J. Bryant S.H. Genome Res. 2002; 12: 1619-1623Crossref PubMed Scopus (536) Google Scholar). Ankyrin repeats mediate protein-protein interactions in a variety of proteins (42Sedgwick S.G. Smerdon S.J. Trends Biochem. Sci. 1999; 24: 311-316Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar). SPX domains (named after yeast Syg1P and Pho81P and human XPR1) have been found in yeast proteins associated with G-protein signaling (Syg1p) (43Spain B.H. Koo D. Ramakrishnan M. Dzudzor B. Colicelli J. J. Biol. Chem. 1995; 270: 25435-25444Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) and phosphate sensing and transport (Pho81p, Pho90p, and Pho91p) (44Pinson B. Merle M. Franconi J.M. Daignan-Fornier B. J. Biol. Chem. 2004; 279: 35273-35280Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). GDE1 encodes a protein of 1223 amino acids with a predicted molecular mass of 138 kDa. The UniProt accession number for GDE1, located on the left arm of chromosome 16, is Q02979.FIGURE 2The occurrence of GPDE motifs in S. cerevisiae. The NCBI CDART tool (38Bateman A. Coin L. Durbin R. Finn R.D. Hollich V. Griffiths-Jones S. Khanna A. Marshall M. Moxon S. Sonnhammer E.L. Studholme D.J. Yeats C. Eddy S.R. Nucleic Acids Res. 2004; 32: D138-D141Crossref PubMed Google Scholar) predicts SPX, ankyrin repeat (ANK), and GPDE motifs in Gde1p and a GPDE motif in YPL206c.View Large Image Figure ViewerDownload Hi-res image Download (PPT)A gde1Δ Mutant Accumulates [14C]Choline-GroPCho upon Labeling with [14C]Choline—We began our studies by analyzing the distribution of choline-containing metabolites in strains bearing deletions in YPL110c and YPL206c. The cells were radiolabeled to steady state with [14C]choline and the distribution of radiolabel upon chasing with nonradiolabeled choline for four h was analyzed. In wild type yeast, exogenous [14C]choline becomes incorporated into phosphatidylcholine. Label that appears later in the trichloroacetic acid-soluble fraction of the cell is mostly in the form of GroPCho (9Dowd 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). Deletion of GDE1 resulted in an increase in water-soluble associated [14C]choline-labeled compounds at the expense of membrane-associated counts (Fig. 3A). The distribution of [14C]choline counts in the YPL206cΔ strain, in contrast, was very similar to that of the wild type strain (Fig. 3A). All of the membrane-associated [14C]choline counts were found to reside in PC (data not shown). HPLC analysis of the trichloroacetic acid-soluble metabolites indicated that in both strains, the majority of trichloroacetic acid-soluble counts (Fig. 3B) were [14C]choline-GroPCho, with the remaining counts being [14C]choline.FIGURE 3A gde1Δ mutant accumulates [14C]choline-GroPCho upon labeling with [14C]choline. Strains grown to logarithmic phase in I– medium containing 18 μm [14C]choline were chased for 4 h in fresh medium containing 1 mm nonradioactive choline. A, the distribution of counts between the trichloroacetic acid (TCA)-soluble and membrane fractions of the cell at the end of the chase was determined for each strain. The values represent the means ± S.E. of two independent experiments performed in duplicate. B, the identity of the water-soluble counts was determined by HPLC for wild type (wt) and gde1Δ. The values represent the means ± S.E. of triplicate determinations derived from two independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Overexpression of GDE1 Decreases Intracellular GroPCho upon Labeling with [14C]Choline—The labeling pattern of [14C]choline-associated counts was analyzed in a strain bearing a N-terminal GST fusion allele of GDE1 under the control of the GAL1 promoter (pGal-GST-GDE1). In cells grown to uniform [14C]choline labeling on galactose, there was roughly 2-fold fewer counts associated with the trichloroacetic acid-soluble portion of the cell in the GDE1 overexpressing strain as compared with the wild type (Fig. 4A) and roughly 4-fold fewer [14C]choline-GroPCho counts (Fig. 4B). Western analysis employing antibodies against the GST tag confirmed that the fusion protein was expressed under inducing conditions (Fig. 4C). Similar results were obtained when GDE1 was overexpressed from a multicopy plasmid, B94, under the control of its own promoter. The wild type strain containing GDE1 on a multicopy plasmid contained fewer trichloroacetic acid-soluble [14C]choline-associated counts as compared with a wild type strain containing an empty vector (Fig. 4A), which translated into roughly 2-fold less internal GroPCho (Fig. 4B).Because our [14C]choline labeling studies with GDE1 delete and GDE1 overexpressing strains were consistent with the assignment of GDE1 as a GroPC phosphodiesterase encoding gene, we attempted to detect in vitro enzymatic activity. Despite growing the cells under a variety of conditions, employ" @default.
• W2058885206 created "2016-06-24" @default.
• W2058885206 creator A5027897996 @default.
• W2058885206 creator A5035089234 @default.
• W2058885206 creator A5046387281 @default.
• W2058885206 creator A5082357799 @default.
• W2058885206 creator A5088448170 @default.
• W2058885206 date "2005-10-01" @default.
• W2058885206 modified "2023-10-18" @default.
• W2058885206 title "Glycerophosphocholine-dependent Growth Requires Gde1p (YPL110c) and Git1p in Saccharomyces cerevisiae" @default.
• W2058885206 cites W111456367 @default.
• W2058885206 cites W1487585902 @default.
• W2058885206 cites W1487624404 @default.
• W2058885206 cites W1569932645 @default.
• W2058885206 cites W1726251904 @default.
• W2058885206 cites W1782411768 @default.
• W2058885206 cites W1934992854 @default.
• W2058885206 cites W1964045660 @default.
• W2058885206 cites W1966072209 @default.
• W2058885206 cites W1967390330 @default.
• W2058885206 cites W1970748888 @default.
• W2058885206 cites W1978492851 @default.
• W2058885206 cites W1981388125 @default.
• W2058885206 cites W1987433777 @default.
• W2058885206 cites W1987492472 @default.
• W2058885206 cites W1991443411 @default.
• W2058885206 cites W1991578658 @default.
• W2058885206 cites W1994010162 @default.
• W2058885206 cites W2018995110 @default.
• W2058885206 cites W2019100689 @default.
• W2058885206 cites W2021438758 @default.
• W2058885206 cites W2022781070 @default.
• W2058885206 cites W2023658753 @default.
• W2058885206 cites W2030630765 @default.
• W2058885206 cites W2034162523 @default.
• W2058885206 cites W2045839655 @default.
• W2058885206 cites W2055184294 @default.
• W2058885206 cites W2055425310 @default.
• W2058885206 cites W2059347808 @default.
• W2058885206 cites W2059606380 @default.
• W2058885206 cites W2074872956 @default.
• W2058885206 cites W2086660631 @default.
• W2058885206 cites W2088112537 @default.
• W2058885206 cites W2092881720 @default.
• W2058885206 cites W2100057937 @default.
• W2058885206 cites W2114049512 @default.
• W2058885206 cites W2119195082 @default.
• W2058885206 cites W2122268504 @default.
• W2058885206 cites W2134671651 @default.
• W2058885206 cites W2140472853 @default.
• W2058885206 cites W2140873658 @default.
• W2058885206 cites W2153931772 @default.
• W2058885206 cites W2157888844 @default.
• W2058885206 cites W2164031736 @default.
• W2058885206 cites W2169805130 @default.
• W2058885206 cites W2401621255 @default.
• W2058885206 cites W29795379 @default.
• W2058885206 cites W38089431 @default.
• W2058885206 cites W4210400672 @default.
• W2058885206 cites W4297851984 @default.
• W2058885206 doi "https://doi.org/10.1074/jbc.m507051200" @default.
• W2058885206 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16141200" @default.
• W2058885206 hasPublicationYear "2005" @default.
• W2058885206 type Work @default.
• W2058885206 sameAs 2058885206 @default.
• W2058885206 citedByCount "68" @default.
• W2058885206 countsByYear W20588852062012 @default.
• W2058885206 countsByYear W20588852062013 @default.
• W2058885206 countsByYear W20588852062014 @default.
• W2058885206 countsByYear W20588852062015 @default.
• W2058885206 countsByYear W20588852062016 @default.
• W2058885206 countsByYear W20588852062017 @default.
• W2058885206 countsByYear W20588852062018 @default.
• W2058885206 countsByYear W20588852062019 @default.
• W2058885206 countsByYear W20588852062020 @default.
• W2058885206 countsByYear W20588852062021 @default.
• W2058885206 countsByYear W20588852062023 @default.
• W2058885206 crossrefType "journal-article" @default.
• W2058885206 hasAuthorship W2058885206A5027897996 @default.
• W2058885206 hasAuthorship W2058885206A5035089234 @default.
• W2058885206 hasAuthorship W2058885206A5046387281 @default.
• W2058885206 hasAuthorship W2058885206A5082357799 @default.
• W2058885206 hasAuthorship W2058885206A5088448170 @default.
• W2058885206 hasBestOaLocation W20588852061 @default.
• W2058885206 hasConcept C185592680 @default.
• W2058885206 hasConcept C2777576037 @default.
• W2058885206 hasConcept C2779222958 @default.
• W2058885206 hasConcept C55493867 @default.
• W2058885206 hasConcept C86803240 @default.
• W2058885206 hasConceptScore W2058885206C185592680 @default.
• W2058885206 hasConceptScore W2058885206C2777576037 @default.
• W2058885206 hasConceptScore W2058885206C2779222958 @default.
• W2058885206 hasConceptScore W2058885206C55493867 @default.
• W2058885206 hasConceptScore W2058885206C86803240 @default.
• W2058885206 hasIssue "43" @default.
• W2058885206 hasLocation W20588852061 @default.
• W2058885206 hasLocation W20588852062 @default.
• W2058885206 hasOpenAccess W2058885206 @default.

• next