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• W2035508001 abstract "Phosphatidylcholine is the most abundant phospholipid in eukaryotic cells, comprising 50% of total cellular phospholipid, and thus plays a major role in cellular and organellar biogenesis. In this study, we have used both nutritional deprivation as well as a conditional temperature sensitive allele of PCT1(CTP:phosphocholine cytidylyltransferase) coupled with an inactivated phosphatidylethanolamine methylation pathway to determine how cells respond to inactivation of phosphatidylcholine synthesis. Metabolic studies determined that phosphatidylcholine biosynthesis decreased to negligible levels within 1 h upon shift to the nonpermissive temperature for the temperature-sensitivePCT1 allele. Phosphatidylcholine mass decreased to negligible levels upon removal of choline from the medium or growth at the nonpermissive temperature, with the levels of the other major phospholipids increasing slightly. Cell growth rate visibly slowed upon cessation of phosphatidylcholine synthesis. Cells remained viable for 7–8 h after phosphatidylcholine synthesis was prevented; however, at time points beyond 8 h, viability was significantly reduced but only if the cells had been previously grown at 37 °C and not 25 °C. The inhibition of phosphatidylcholine synthesis at 37 °C did not alter Golgi-derived vesicle transport to the vacuole as monitored by carboxypeptidase Y processing or to the plasma membrane as determined by invertase secretion. Immunofluorescence microscopy localized Pct1p to the nucleus and nuclear membrane. Pct1p activity is regulated by Sec14p, a cytoplasm/Golgi localized phosphatidylcholine/phosphatidylinositol binding protein that regulates Golgi-derived vesicle transport partially through its ligand-dependent regulation of PCT1 derived enzyme activity. Our nuclear localization of Pct1p indicates that the regulation of Pct1p by Sec14p is indirect. Phosphatidylcholine is the most abundant phospholipid in eukaryotic cells, comprising 50% of total cellular phospholipid, and thus plays a major role in cellular and organellar biogenesis. In this study, we have used both nutritional deprivation as well as a conditional temperature sensitive allele of PCT1(CTP:phosphocholine cytidylyltransferase) coupled with an inactivated phosphatidylethanolamine methylation pathway to determine how cells respond to inactivation of phosphatidylcholine synthesis. Metabolic studies determined that phosphatidylcholine biosynthesis decreased to negligible levels within 1 h upon shift to the nonpermissive temperature for the temperature-sensitivePCT1 allele. Phosphatidylcholine mass decreased to negligible levels upon removal of choline from the medium or growth at the nonpermissive temperature, with the levels of the other major phospholipids increasing slightly. Cell growth rate visibly slowed upon cessation of phosphatidylcholine synthesis. Cells remained viable for 7–8 h after phosphatidylcholine synthesis was prevented; however, at time points beyond 8 h, viability was significantly reduced but only if the cells had been previously grown at 37 °C and not 25 °C. The inhibition of phosphatidylcholine synthesis at 37 °C did not alter Golgi-derived vesicle transport to the vacuole as monitored by carboxypeptidase Y processing or to the plasma membrane as determined by invertase secretion. Immunofluorescence microscopy localized Pct1p to the nucleus and nuclear membrane. Pct1p activity is regulated by Sec14p, a cytoplasm/Golgi localized phosphatidylcholine/phosphatidylinositol binding protein that regulates Golgi-derived vesicle transport partially through its ligand-dependent regulation of PCT1 derived enzyme activity. Our nuclear localization of Pct1p indicates that the regulation of Pct1p by Sec14p is indirect. Phosphatidylcholine (PC) 1The abbreviations used are: PC, phosphatidylcholine; PE, phosphatidylethanolamine; CPY, carboxypeptidase Y; YPD, yeast peptone dextrose; HA, hemagglutinin; DAPI, 4′,6-diamidino-2-phenylindole 1The abbreviations used are: PC, phosphatidylcholine; PE, phosphatidylethanolamine; CPY, carboxypeptidase Y; YPD, yeast peptone dextrose; HA, hemagglutinin; DAPI, 4′,6-diamidino-2-phenylindoleis the most abundant phospholipid in eukaryotic cells comprising 50% of cellular phospholipid mass (1Kent C. Annu. Rev. Biochem. 1995; 64: 315-343Crossref PubMed Scopus (306) Google Scholar). In eukaryotic cells, PC can be synthesizedde novo through either the CDP-choline or phosphatidylethanolamine (PE) methylation pathway. The CDP-choline pathway is found in all eukaryotic cell types and synthesizes PC by (i) phosphorylation of choline by choline kinase (CKI1) to produce phosphocholine and (ii) the rate-limiting transfer of a CMP moiety from CTP to phosphocholine by CTP:phosphocholine cytidylyltransferase (PCT1) to produce CDP-choline, followed by (iii) the transfer of phosphocholine from CDP-choline to diacylglycerol by a cholinephosphotransferase reaction (CPT1or EPT1) to produce PC (2Howe A.G. McMaster C.R. Biochim. Biphys. Acta. 2001; 1534: 65-77Crossref PubMed Scopus (26) Google Scholar, 3Hosaka K. Kodaki T. Yamashita S. J. Biol. Chem. 1989; 264: 2053-2059Abstract Full Text PDF PubMed Google Scholar, 4Kim K.H. Voelker D.R. Flocco M.T. Carman G.M. J. Biol. Chem. 1998; 273: 6844-6852Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 5Tsukagoshi Y. Nikawa J. Hosaka K. Yamashita S. J. Bacteriol. 1991; 173: 2134-2136Crossref PubMed Google Scholar, 6Hjelmstad R.H. Bell R.M. J. Biol. Chem. 1991; 266: 4357-4365Abstract Full Text PDF PubMed Google Scholar, 7McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 28010-28016Abstract Full Text PDF PubMed Google Scholar, 8McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 14776-14783Abstract Full Text PDF PubMed Google Scholar). The rate-limiting CTP:phosphocholine cytidylyltransferase is an amphitropic protein that exists in an inactive soluble form that requires translocation to membranes to become active, and this translocation event is believed to be the main form of regulation of this enzyme (9Cornell R.B. Northwood I.C. Trends Biochem. Sci. 2000; 25: 441-447Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 10Johnson J.E. Kalmar G.B. Sohal P.S. Walkey C.J. Yamashita S. Cornell R.B. Biochem. J. 1992; 285: 815-820Crossref PubMed Scopus (45) Google Scholar, 11Lykidis A. Jackson P. Jackowski S. Biochemistry. 2001; 40: 494-503Crossref PubMed Scopus (39) Google Scholar, 12Attard G.S. Templer R.H. Smith W.S. Hunt A.N. Jackowski S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9032-9036Crossref PubMed Scopus (222) Google Scholar). The PE methylation pathway for the synthesis of PC is found in mammalian hepatocytes and yeast cells (13Cui Z. Vance J.E. Chen M.H. Voelker D.E. Vance D.E. J. Biol. Chem. 1993; 268: 16655-16663Abstract Full Text PDF PubMed Google Scholar, 14Walkey C.J., Yu, L. Agellon L.B. Vance D.E. J. Biol. Chem. 1998; 273: 27043-27046Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 15Kodaki T. Yamashita S. J. Biol. Chem. 1987; 262: 15428-15435Abstract Full Text PDF PubMed Google Scholar, 16McGraw P. Henry S.A. Genetics. 1989; 122: 317-330Crossref PubMed Google Scholar), and PC is synthesized through this route by three successive methylations of the ethanolamine head group of PE (encoded by the CHO2 and OPI3genes in yeast). The CHO2 gene product methylates PE once, and the OPI3 gene product transfers the final two methyl groups to form PC (15Kodaki T. Yamashita S. J. Biol. Chem. 1987; 262: 15428-15435Abstract Full Text PDF PubMed Google Scholar, 16McGraw P. Henry S.A. Genetics. 1989; 122: 317-330Crossref PubMed Google Scholar). The contribution of the two pathways to PC synthesis in yeast is dependent on exogenous choline concentration with higher levels of choline favoring synthesis through the CDP-choline pathway (8McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 14776-14783Abstract Full Text PDF PubMed Google Scholar). As the major component of biological membranes, PC would be predicted to participate in organellar and cellular biogenesis as well as the formation of vesicles for the transport of proteins and lipids within cells. A role for PC in vesicle transport has been established through work on the Saccharomyces cerevisiae PC/phosphatidylinositol transfer protein Sec14p. Loss of function of Sec14p results in cell death through cessation of Golgi-derived vesicle transport. Inactivation of PC synthesis through the CDP-choline pathway completely rescues cell growth and vesicle transport defects in the absence of functional Sec14p (17Cleves A.E. McGee T.P. Whitters E.A. Champion K.M. Aitken J.R. Dowhan W. Goebl M. Bankaitis V.A. Cell. 1991; 64: 789-800Abstract Full Text PDF PubMed Scopus (284) Google Scholar, 18Xie Z. Fang M. Rivas M.P. Faulkner A.J. Sternweis P.C. Engebrecht J. Bankaitis V.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12346-12351Crossref PubMed Scopus (145) Google Scholar, 19Bankaitis V.A. Aitken J.F. Cleves A.E. Dowhan W. Nature. 1990; 347: 561-562Crossref PubMed Scopus (434) Google Scholar, 20McGee T.P. Skinner H.B. Whitters E.A. Henry S.A. Bankaitis V.A. J. Cell Biol. 1994; 124: 273-287Crossref PubMed Scopus (152) Google Scholar). When bound to PC, Sec14p is an effective inhibitor of Pct1p (Fig. 1) and is believed to act as a PC sensor with Sec14p-mediated adjustment of the rate of PC synthesis and turnover as a requirement for the regulation of vesicle transport from the Golgi (21Skinner H.B. McGee T.P. McMaster C.R. Fry M.R. Bell R.M. Bankaitis V.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 112-116Crossref PubMed Scopus (135) Google Scholar). In this study, we constructed a yeast strain with an inactivated PE methylation pathway and a conditional temperature-sensitive allele ofPCT1. This allowed us to simultaneously inactivate both routes for PC either by removing choline from the medium or by shifting cells to the nonpermissive temperature for function of thePCT1 temperature-sensitive allele, to address the role of decreased PC synthesis on cell growth and viability. Yeast and E. coli media were from Difco. Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs. AdvanTaq polymerase was a product of CLONTECH. Oligonucleotides were from Invitrogen. Lipids were purchased from Avanti Polar Lipids. [14C]Choline, [3H]methionine, and phosphorus-32 were purchased from American Radiolabeled Chemicals. S. cerevisiae strain D319–8A (α leu2 his4 pct1 ts) (22Tsukagoshi Y. Nikawa J. Yamashita S. Eur. J. Biochem. 1987; 169: 477-486Crossref PubMed Scopus (73) Google Scholar) was mated with strain CTY410 (a his3–200 leu2–9 cho2::LEU2), and diploids were sporulated. Haploid strains that grew poorly on medium lacking choline at 25 °C and did not grow at 37 °C were isolated and mated twice with strain W303-1A (a ura3–1 his3–11 leu2–3,112 trp1–1 ade2–1) to make strain CMY134 (αura his3 leu2 trp1 ade2 cho2::LEU2 pct1 ts). CMY134 was used to assess the role of PC in cell growth and viability. Strain CTY471 (α ura his3 leu2 trp1 ade2 pct1::URA3) was used for expression of wild type and tagged derivatives of PCT1 for metabolic and biochemical analyses. Yeasts were grown on rich yeast peptone dextrose (YPD) medium, or synthetic dextrose minimal medium supplemented as required for plasmid maintenance (23Kaiser C. Michaelis S. Mitchell A. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994Google Scholar). The PCT1 gene was amplified from isolated W303-1A yeast genomic DNA using AdvanTaq polymerase and TA cloned into pCR2.1 Topo (Invitrogen). ThePCT1 gene was subsequently subcloned into the yeast low copy CEN/ARS shuttle vector pRS413 (24Christianson T.W. Sikorski R.S. Dante M. Shero J.H. Hieter P. Gene (Amst.). 1992; 110: 112-119Crossref Scopus (1418) Google Scholar) to create plasmid pMM4. The pMM4 plasmid was digested with BlpI to liberate the majority of the PCT1 open reading frame, and the linearized plasmid DNA was transformed into W303-1A yeast to allow for gap repair of thePCT1 open reading frame from yeast genomic DNA to create plasmid pMM5. Plasmid pMM5 was isolated from yeast, amplified in DH5αE. coli, and sequenced in its entirety to ensure polymerase and gap repair fidelity. An AgeI site was inserted at the most 3′-end of thePCT1 open reading frame using the Morph site-directed mutagenesis kit (5 Prime → 3 Prime, Inc., Boulder, CO), and a 3-fold repeat of the HA epitope tag was inserted into this site by PCR amplification of the 3-fold hemagglutinin epitope repeat from plasmid pGTEP. The wild type and HA-tagged versions of PCT1 were subcloned into pRS413. The potential temperature-sensitive allele of PCT1(pct1 ts) was recovered from yeast strain CMY134 genomic DNA by digesting pRS413 containing the PCT1 gene with Blp1 to liberate the majority of the PCT1 open reading frame from the plasmid. The linearized plasmid DNA was transformed into CMY134 yeast to allow for gap repair of the PCT1 open reading frame from yeast genomic DNA into the plasmid. To ensure that the temperature-sensitive mutation was carried within thePCT1 gene isolated from strain CMY134, the PCT1gene was recovered from strain CMY134 by gap repair and resubcloned into pRS413. Total yeast cell membranes were prepared from midlog phase yeast as described (25Williams J.G. McMaster C.R. J. Biol. Chem. 1998; 273: 13482-13487Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). CTY471 (pct1::URA) containing pMM7 (Myc-taggedPCT1 under control of the inducible GAL1promoter) or pESC-TRP vector control was grown in selective medium with galactose to log phase. Cultures were fixed by adding formaldehyde to the medium to a final concentration of 3.7% and incubating at room temperature for 2 h. Cells were washed twice with phosphate-buffered saline, and the cell wall was digested with zymolyase. The resulting spheroplasts were mounted on slides treated with polylysine and incubated successively in ice-cold methanol, ice-cold acetone and then rehydrated with room temperature phosphate-buffered saline. Cells were incubated in blocking buffer (phosphate buffered saline plus 3% bovine serum albumin) in a humid chamber for 30 min. Mouse anti-c-Myc antibody (1:250 dilution in blocking buffer) was added in a humid chamber for 1 h. Slides were washed three times with blocking buffer and incubated with goat anti-mouse Texas Red-conjugated antibody (1:5000 dilution in blocking buffer) for 1 h in a humid chamber in the dark and then washed three times with blocking buffer. DAPI (1 μg/ml) was added to slides for 1 min, slides were washed three times with phosphate-buffered saline, and 20 μl of 90% glycerol, 10% phosphate-buffered saline was placed on the slides. Coverslips were added and sealed. Fluorescence microscopy was performed using a Zeiss axiophot microscope. Texas Red was visualized with Zeiss filter number 15, which excites at 546/560 nm and emits at 590 nm, whereas the UV filter was used to visualize DAPI-stained cells. For actin staining, cultures were incubated with 0.66 μmAlexaFluor 568 Phalloidin (Molecular Probes, Inc., Eugene, OR) in phosphate-buffered saline for 1 h in the dark. Cells were washed four times with phosphate-buffered saline and mounted on polylysine-treated slides in 90% glycerol, 10% phosphate-buffered saline and visualized with Zeiss filter number 15. Logarithmic phase yeast cells were grown in synthetic minimal medium and labeled with [14C]choline or [3H]methionine for 1 h, or phosphorus-32 for 18 h. Lipids were extracted, lipid and lipid precursors were separated by thin layer chromatography, and radiolabel was quantitated by scintillation counting as described (25Williams J.G. McMaster C.R. J. Biol. Chem. 1998; 273: 13482-13487Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). All of the epitope-tagged versions of Pct1p used in this study were capable of reconstituting PC synthesis to levels similar to those observed for nontagged Pct1p as assessed by the rate of [14C]choline labeling of PC in a yeast strain containing a genetically inactivatedPCT1 gene (CTY471 α ura his3 leu2 trp1 ade2 pct1::URA3). Exponential cultures of cells growing at 25 °C in supplemented minimal medium lacking methionine and cysteine were concentrated and resuspended in fresh medium and incubated at 25 °C for 1 h. Cells were then pulse-labeled for 10 min with [35S]methionine/cysteine (Expre35S35S protein labeling mix; PerkinElmer Life Sciences) and then chased with the subsequent addition of methionine and cysteine at a final concentration of 0.5% each at 37 °C. Aliquots of cells (A 600 = 3) were taken at different times and transferred to tubes containing ice-cold 10 mm NaF/NaN3 in 1 m sorbitol. Cells were disrupted, CPY was immunoprecipitated as described (28Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar), proteins were resolved using 8% SDS-PAGE, and the gel was exposed to x-ray film for subsequent development. To measure invertase secretion, yeast cells were grown to midlog phase at 25 °C in rich (YPD) medium containing the normal level of 2% glucose and centrifuged at 750 rpm for 5 min to pellet the cells. Pellets were washed twice with 5 ml of sterile water and resuspended in 5 ml of YPD containing only 0.1% glucose to induce invertase. These cultures were grown at 37 °C for 2 h, and invertase secretion was measured using the method described (26Goldstein A. Lampen J.O. Methods Enzymol. 1975; 42: 504-511Crossref PubMed Scopus (298) Google Scholar, 27Cleves A.E. Novick P.J. Bankaitis V.A. J. Cell Biol. 1989; 109: 2939-2950Crossref PubMed Scopus (189) Google Scholar). The invertase secretion index of each sample was determined by dividing external invertase (−) by total invertase (+). Protein was measured by the method of Lowry et al. (28Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Lipid phosphorus was determined using the method of Ames and Dubin (29Ames B.N. Dubin D.T. J. Biol. Chem. 1960; 235: 769-775Abstract Full Text PDF PubMed Google Scholar), and diacylglycerol mass was determined by the method of Priess et al. (30Preiss J. Loomis C.R. Bishop W.R. Stein R. Niedel J.E. Bell R.M. J. Biol. Chem. 1986; 261: 8597-8600Abstract Full Text PDF PubMed Google Scholar). Yeast strain CMY134 (cho2::LEU2 pct1 ts) or wild type yeast were maintained at 25 °C and while in log phase were labeled with [14C]choline for 1 h at 25 or 37 °C. As has been recently observed (31Dowd 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), there was an increase in PC synthesis upon shifting the wild type yeast to 37 °C; however, PC labeling was reduced to less than 5% of wild type in thepct1 ts-containing cells. Analysis of the metabolites of the CDP-choline pathway indicated a large rise in the labeling of phosphocholine accompanied the decrease in PC synthesis consistent with a block at the Pct1p step (Fig. 2). To ensure that the temperature sensitivity observed at the Pct1p step was due to a mutation within the PCT1 structural gene within strain CMY134 (and not a regulator of Pct1p), the PCT1 gene was recovered from CMY134 genomic DNA by allele rescue through transformation of CMY134 yeast with a BlpI digest of a CEN/ARS plasmid containing the wild type PCT1 gene. This digestion liberates the majority of the PCT1 open reading frame, and the linearized plasmid DNA was isolated by gel electrophoresis and transformed into CMY134 yeast to allow for allele rescue of the PCT1 open reading frame from yeast genomic DNA. The PCT1 gene recovered from CMY134 by allele rescue was subcloned into the low copy yeast vector pRS413 and transformed into the yeast CTY471 (pct1::URA3). The CTY471 strain is wild type except for genetic inactivation of its chromosomalPCT1 gene, and thus the ability of the PCT1allele recovered from strain CMY134 can be compared with the wild type gene by monitoring the ability to synthesize PC from radiolabeled choline. The CTY471 yeasts were maintained at 25 °C, and the synthesis of PC from radiolabeled choline was monitored in CTY471 containing the PCT1 gene recovered from the genome of CMY134 or the wild type PCT1 gene for 1 h after a shift in the growth temperature from 25 to 37 °C. Upon shifting the yeast to 37 °C, the rate of PC synthesis in the cells containing thePCT1 allele rescued from the CMY134 genome was less than 2% that of the same yeast strain containing the wild type gene (Fig.3 A). This proves that it is the PCT1 gene in strain CMY134 that contains a mutation that renders the Pct1p enzyme activity temperature-sensitive. To ensure that the cho2::LEU2 gene inactivation was preventing PC synthesis through the PE methylation pathway, CMY134 yeasts were labeled with [3H]methionine for 1 h and compared with wild type yeast. As expected, PC synthesis was reduced to 5% of wild type upon inactivation of theCHO2 gene (Fig. 3 B). The levels of yeast phospholipids were determined by labeling CMY134 cells, inoculated at the same stage of early log phase growth, to steady state with 32 phosphorous for 18 h. The relative levels of total phospholipid were drastically different depending on the growth temperature, the presence or absence of choline, and the presence or absence of a functional PCT1 gene (Fig.4 A). Cells grown at 25 °C in the presence of choline contained similar amounts of total phospholipid regardless of the presence of the plasmid-derivedPCT1 gene due to the pct1 ts allele being functional at this temperature. The removal of choline from the medium at 25 °C reduced total recoverable phospholipid to approximately one-third that of cells grown in the presence of choline, indicating that cell growth, phospholipid synthesis, or the amount of phospholipid per cell was dramatically reduced. At the pct1 tsnonpermissive temperature of 37 °C, cells containing plasmid-derivedPCT1 contained 40% of the level of phospholipid as the same strain grown at 25 °C, once again implying cell growth, phospholipid synthesis, or the amount of phospholipid per cell was dramatically reduced. The removal of choline at 37 °C reduced phospholipid levels in the PCT1-containing yeast a further 4-fold. Cells grown at 37 °C without a functional PCT1gene resulted in the recovery of barely detectable levels of lipid phosphorus, implying that there were very few viable cells. Identical dpm from each of the strains from which phosphorus-32-labeled phospholipid was efficiently recovered were separated by two-dimensional thin layer chromatography to determine the relative levels of each phospholipid class. In each case, 40–45% of phospholipid was composed of PC when choline was present in the medium; however, upon the removal of choline, there was a total loss of PC (Fig. 4, B and C). Upon the removal of choline, the bulk of the phospholipid mass of PC was by in large replaced by two phospholipids, with PE increasing from 19–22% total phospholipid to 30–35% and phosphatidylserine increasing from 12–15 to 28–32%. Increases in the proportion of other phospholipids were observed in the absence of PC, with phosphatidylglycerol increasing slightly from 7–10 to 12–15% and phosphatidylinositol increasing from 5–10 to 15–18%. The glycerol backbone and fatty acyl chains required for the synthesis of PC through the CDP-choline pathway are obtained from diacylglycerol. We measured the mass of diacylglycerol in cells that could no longer consume this lipid due to inactivation of the CDP-choline pathway through choline deprivation, growth at the nonpermissive temperature for the pct1 ts allele, or both. At 2 h, there was very little change in the level of diacylglycerol mass under any of the growth conditions; however, after 18 h, the proportion of diacylglycerol compared with total phospholipid increased 3–4-fold at 37 °C but only when PC could not be made (due to choline deprivation and/or growth at the nonpermissive temperature for thepct1 ts allele) (Fig.5). This increase in diacylglycerol mass was not observed when PC synthesis was prevented at 25 °C. CMY134 (cho2::LEU2 pct1 ts) yeasts carrying the wild type yeast PCT1 gene on a low copy plasmid or empty vector were grown at 25 °C in the presence of choline to early log phase and then grown with or without choline at either 25 or 37 °C, and the cell growth rate was monitored (Fig.6 A). In the presence of choline, the cells containing the vector control ceased growth within 5 h, whereas the cells containing the PCT1 plasmid continued to grow through to early stationary phase at a rate similar to cells grown at 25 °C (the permissive temperature for thepct1 ts allele). Cells grown without choline did not continue to grow at either 25 or 37 °C with a more pronounced cessation of cell growth for cells grown at 37 °C regardless of the presence or absence of a functional Pct1p. The cessation of cell growth in the absence of choline was similar to that observed for cells grown in the presence of choline but without a functional Pct1p when grown at the nonpermissive temperature for the pct1 tsallele. We then tested whether cell growth inhibition in cells unable to synthesize PC was due to growth cessation or resulted in a loss of cell viability. Equivalent numbers of cells from CMY134 yeast containing thePCT1 gene or empty vector were serial diluted 8 or 23 h after with or without choline and/or growth at either 25 or 37 °C and then tested for growth on solid medium at 25 °C that was replete with choline. Cells maintained viability up to ∼8 h, regardless of the choline content or growth temperature. However, at longer time points, cells that were unable to synthesize PC through the CDP-choline pathway due to genetic inactivation of the pct1 tsallele or choline deprivation resulted in a dramatic loss of cell viability but only for cells grown at 37 °C (Fig. 6 B). Cells grown at 25 °C remained viable upon the removal of choline from the medium, although their PC mass was dramatically reduced and their growth rates had dropped to levels almost as low as those observed for cells grown at 37 °C. Microscopic visualization of the above cells after 23 h under the indicated growth conditions resulted in the observation that the cells that were compromised for their ability to synthesize PC tended to contain a much larger proportion of small to medium size buds compared with cells that could readily synthesize PC (Fig.7). In addition, the cells that were grown at 37 °C that were unable to restore growth upon return to the permissive temperature contained fewer cells, and those that were visible appeared to possess abnormal internal membranes. Staining these cells with DAPI or phalloidin did not reveal any gross differences in the localization of chromosomal DNA or cortical actin, respectively, when compared with cells that were able to restore cell growth upon return to choline-replete medium at the permissive growth temperature (data not shown). The appearance of what appear to be odd membrane configurations in the cells unable to synthesize PC, with the end result being cell death, implied that there might be alterations in cellular membrane transport. PC synthesis through the CDP-choline pathway is intimately associated with the regulation of vesicle transport through the action of Sec14p. Sec14p is a PC/phosphatidylinositol transfer protein whose essential cell growth and Golgi-derived vesicle transport defects can be rescued if the CDP-choline pathway for PC synthesis is inactivated (17Cleves A.E. McGee T.P. Whitters E.A. Champion K.M. Aitken J.R. Dowhan W. Goebl M. Bankaitis V.A. Cell. 1991; 64: 789-800Abstract Full Text PDF PubMed Scopus (284) Google Scholar, 18Xie Z. Fang M. Rivas M.P. Faulkner A.J. Sternweis P.C. Engebrecht J. Bankaitis V.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12346-12351Crossref PubMed Scopus (145) Google Scholar, 19Bankaitis V.A. Aitken J.F. Cleves A.E. Dowhan W. Nature. 1990; 347: 561-562Crossref PubMed Scopus (434) Google Scholar, 20McGee T.P. Skinner H.B. Whitters E.A. Henry S.A. Bankaitis V.A. J. Cell Biol. 1994; 124: 273-287Crossref PubMed Scopus (152) Google Scholar). In addition, it has been observed that the PC-bound form of Sec14p inhibits the CDP-choline pathway by inhibiting Pct1p activity, although the precise mechanism for this inhibition has yet to be established (21Skinner H.B. McGee T.P. McMaster C.R. Fry M.R. Bell R.M. Bankaitis V.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 112-116Crossref PubMed Scopus (135) Google Scholar). It is currently hypothesized that Sec14p acts as a sensor for the rate of PC synthesis through the CDP-choline pathway with increasing PC synthesis resulting in increased PC bound Sec14p and a titrating down of the rate of PC synthesis through Sec14p inhibition of Pct1p (32Xie Z. Fang M. Bankaitis V.A. Mol. Biol. Cell. 2001; 12: 1117-1129Crossref PubMed Scopus (53) Google Scholar, 33Henneberry A.L. Lagace T.A. Ridgway N.D. McMaster C.R. Mol. Biol. Cell. 2001; 12: 511-520Crossref PubMed Scopus (56) Google Scholar, 34Kearns B.G. McGee T.P. Mayinger P. Gedvilaite A. Phillips S.E. Kagiwada S. Bankaitis V.A. Nature. 1997; 387: 101-105Crossref PubMed Scopus (222) Google Scholar). The decreased PC synthesis results in less PC-bound Sec14p and thereby relieves the inhibition of PC synthesis (Fig. 1). Alterations in the rate of PC synthesis and turnover are believed to be major mechanisms regulating Sec14p-dependent Golgi-derived vesicle transport. Our observation that the inhibition of PC synthesis resulted in rapid cell growth cessation and that this was coupled to the appearance of odd membranous structures in cells destined to die implied that decreased PC synthesis might alter membrane transport from the sites of PC synthesis in the endoplasmic reticulum/Golgi t" @default.
• W2035508001 created "2016-06-24" @default.
• W2035508001 creator A5045631153 @default.
• W2035508001 creator A5060869877 @default.
• W2035508001 creator A5061166792 @default.
• W2035508001 date "2002-11-01" @default.
• W2035508001 modified "2023-09-29" @default.
• W2035508001 title "Cessation of Growth to Prevent Cell Death Due to Inhibition of Phosphatidylcholine Synthesis Is Impaired at 37 °C inSaccharomyces cerevisiae" @default.
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