Inositol Induces a Profound Alteration in the Pattern and Rate of Synthesis and Turnover of Membrane Lipids in Saccharomyces cerevisiae
2006; Elsevier BV; Volume: 281; Issue: 32 Linguagem: Inglês
10.1074/jbc.m603548200
ISSN1083-351X
AutoresMaría L. Gaspar, Manuel Aregullín, Stephen A. Jesch, Susan A. Henry,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoThe addition of inositol to actively growing yeast cultures causes a rapid increase in the rate of synthesis of phosphatidylinositol and, simultaneously, triggers changes in the expression of hundreds of genes. We now demonstrate that the addition of inositol to yeast cells growing in the presence of choline leads to a dramatic reprogramming of cellular lipid synthesis and turnover. The response to inositol includes a 5-6-fold increase in cellular phosphatidylinositol content within a period of 30 min. The increase in phosphatidylinositol content appears to be dependent upon fatty acid synthesis. Phosphatidylcholine turnover increased rapidly following inositol addition, a response that requires the participation of Nte1p, an endoplasmic reticulum-localized phospholipase B. Mass spectrometry revealed that the acyl species composition of phosphatidylinositol is relatively constant regardless of supplementation with inositol or choline, whereas phosphatidylcholine acyl species composition is influenced by both inositol and choline. In medium containing inositol, but lacking choline, high levels of dimyristoylphosphatidylcholine were detected. Within 60 min following the addition of inositol, dimyristoylphosphatidylcholine levels had decreased from ∼40% of total phosphatidylcholine to a basal level of less than 5%. nte1Δ cells grown in the absence of inositol and in the presence of choline exhibited lower levels of dimyristoylphosphatidylcholine than wild type cells grown under these same conditions, but these levels remained largely constant after the addition of inositol. These results are discussed in relationship to transcriptional regulation known to be linked to lipid metabolism in yeast. The addition of inositol to actively growing yeast cultures causes a rapid increase in the rate of synthesis of phosphatidylinositol and, simultaneously, triggers changes in the expression of hundreds of genes. We now demonstrate that the addition of inositol to yeast cells growing in the presence of choline leads to a dramatic reprogramming of cellular lipid synthesis and turnover. The response to inositol includes a 5-6-fold increase in cellular phosphatidylinositol content within a period of 30 min. The increase in phosphatidylinositol content appears to be dependent upon fatty acid synthesis. Phosphatidylcholine turnover increased rapidly following inositol addition, a response that requires the participation of Nte1p, an endoplasmic reticulum-localized phospholipase B. Mass spectrometry revealed that the acyl species composition of phosphatidylinositol is relatively constant regardless of supplementation with inositol or choline, whereas phosphatidylcholine acyl species composition is influenced by both inositol and choline. In medium containing inositol, but lacking choline, high levels of dimyristoylphosphatidylcholine were detected. Within 60 min following the addition of inositol, dimyristoylphosphatidylcholine levels had decreased from ∼40% of total phosphatidylcholine to a basal level of less than 5%. nte1Δ cells grown in the absence of inositol and in the presence of choline exhibited lower levels of dimyristoylphosphatidylcholine than wild type cells grown under these same conditions, but these levels remained largely constant after the addition of inositol. These results are discussed in relationship to transcriptional regulation known to be linked to lipid metabolism in yeast. The addition of the phospholipid precursor, inositol, to the growth medium of yeast leads to increased phosphatidylinositol (PI) 2The abbreviations used are: PI, phosphatidylinositol; PA, phosphatidic acid; ER, endoplasmic reticulum; DAG, 1,2-diacylglycerol; PS, phosphatidyl-serine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DMG, 1,2-dimyristoyl-sn-glycerol; UPR, unfolded protein response pathway; LORE, low oxygen response element; I, inositol; C, choline; FFA, free fatty acid(s); TAG, triacylglycerol(s). synthesis and increased consumption of the immediate precursors of PI, phosphatidic acid (PA), and CDP-diacylglycerol (CDP-DAG) (Fig. 1) (1Kelley M.J. Bailis A.M. Henry S.A. Carman G.M. J. Biol. Chem. 1988; 263: 18078-18085Abstract Full Text PDF PubMed Google Scholar, 2Loewen C.J. Gaspar M.L. Jesch S.A. Delon C. Ktistakis N.T. Henry S.A. Levine T.P. Science. 2004; 304: 1644-1647Crossref PubMed Scopus (371) Google Scholar). Recent genome-wide analyses revealed that growth in the presence of inositol also affects the steady-state transcript abundance of over 100 genes (3Santiago T.C. Mamoun C.B. J. Biol. Chem. 2003; 278: 38723-38730Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 4Jesch S.A. Zhao X. Wells M.T. Henry S.A. J. Biol. Chem. 2005; 280: 9106-9118Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Many of these genes encode enzymes involved in lipid metabolism and contain the conserved UASINO element in their promoters. INO1, which encodes inositol-3-phosphate synthase, is the most highly regulated of the UASINO-containing genes (5Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (264) Google Scholar). INO1 is repressed some 50-fold if inositol is present and 150-fold if choline, precursor to phosphatidylcholine (PC) (Fig. 1), is present in addition to inositol (4Jesch S.A. Zhao X. Wells M.T. Henry S.A. J. Biol. Chem. 2005; 280: 9106-9118Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Activation of INO1 and other UASINO-containing genes requires the Ino2p and Ino4p transcription factors, which bind as a heterodimer directly to UASINO (6Ambroziak J. Henry S.A. J. Biol. Chem. 1994; 269: 15344-15349Abstract Full Text PDF PubMed Google Scholar, 7Lopes J.M. Henry S.A. Nucleic Acids Res. 1991; 19: 3987-3994Crossref PubMed Scopus (66) Google Scholar, 8Schwank S. Ebbert R. Rautenstrauss K. Schweizer E. Schuller H.J. Nucleic Acids Res. 1995; 23: 230-237Crossref PubMed Scopus (109) Google Scholar). In certain genetic backgrounds, increased phospholipase D-mediated turnover of PC also results in derepression of the INO1 gene (9Patton-Vogt J.L. Griac P. Sreenivas A. Bruno V. Dowd S. Swede M.J. Henry S.A. J. Biol. Chem. 1997; 272: 20873-20883Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 10Sreenivas A. Patton-Vogt J.L. Bruno V. Griac P. Henry S.A. J. Biol. Chem. 1998; 273: 16635-16638Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Likewise, mutations affecting the synthesis of PC via methylation of phosphatidylethanolamine (PE) (Fig. 1) result in abnormal patterns of phospholipid accumulation and misregulation of UASINO-containing genes (11Griac P. Swede M.J. Henry S.A. J. Biol. Chem. 1996; 271: 25692-25698Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 12Summers E.F. Letts V.A. McGraw P. Henry S.A. Genetics. 1988; 120: 909-922Crossref PubMed Google Scholar, 13McGraw P. Henry S.A. Genetics. 1989; 122: 317-330Crossref PubMed Google Scholar). These and related observations led to the hypothesis that a signal or signals generated by ongoing lipid metabolism are involved in the transcriptional regulation of UASINO-containing genes (14Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acids Res. Mol. Biol. 1998; 61: 133-179Crossref PubMed Google Scholar). Recently, the mechanism by which a signal from lipid metabolism is sensed and subsequently influences the expression of the INO1 gene was elucidated. The negative regulator, Opi1p, required for repression of the UASINO-containing genes (15Greenberg M.L. Reiner B. Henry S.A. Genetics. 1982; 100: 19-33Crossref PubMed Google Scholar, 16White M.J. Lopes J.M. Henry S.A. Adv. Microb. Physiol. 1991; 32: 1-51Crossref PubMed Google Scholar), was shown to reside in the endoplasmic reticulum (ER) as a part of a protein complex that also contains the membrane-spanning protein Scs2p (17Gavin A.C. Bosche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Hofert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Nature. 2002; 415: 141-147Crossref PubMed Scopus (4010) Google Scholar, 18Loewen C.J. Roy A. Levine T.P. EMBO J. 2003; 22: 2025-2035Crossref PubMed Scopus (448) Google Scholar). Opi1p interacts with Scs2p via a domain called FFAT, and this interaction is required for retention of Opi1p in the ER (2Loewen C.J. Gaspar M.L. Jesch S.A. Delon C. Ktistakis N.T. Henry S.A. Levine T.P. Science. 2004; 304: 1644-1647Crossref PubMed Scopus (371) Google Scholar, 18Loewen C.J. Roy A. Levine T.P. EMBO J. 2003; 22: 2025-2035Crossref PubMed Scopus (448) Google Scholar). However, Opi1p also binds PA, which accumulates as an intermediate in the absence of inositol (2Loewen C.J. Gaspar M.L. Jesch S.A. Delon C. Ktistakis N.T. Henry S.A. Levine T.P. Science. 2004; 304: 1644-1647Crossref PubMed Scopus (371) Google Scholar), and this binding is required in addition to the interaction with Scs2p in order for Opi1p to remain in the ER. Upon the addition of inositol, PA levels in the ER drop as a consequence of increased PI synthesis, resulting in the release of Opi1p from the ER and its subsequent translocation to the nucleus, where it acts to repress INO1 and co-regulated genes (2Loewen C.J. Gaspar M.L. Jesch S.A. Delon C. Ktistakis N.T. Henry S.A. Levine T.P. Science. 2004; 304: 1644-1647Crossref PubMed Scopus (371) Google Scholar). Analysis of the kinetics of the transcriptional responses to inositol genome-wide revealed that over 700 genes show a significant change in expression in at least one time point over a 2-h time course following the addition of inositol to medium already containing choline. Many of these changes occur in the first 15-30 min following the addition of inositol. The transcript abundance of many of these genes is affected only transiently, returning to basal levels within 60-120 min after inositol addition. This explains, in part, the fact that larger numbers of genes were detected in the kinetic analysis of the response to inositol (19Jesch S.A. Liu P.Z.X. Wells M.T. Henry S.A. J. Biol. Chem. June 15, 2006; 10.1074/jbc.M604541200Google Scholar), in comparison with steady-state analysis of transcript abundance (3Santiago T.C. Mamoun C.B. J. Biol. Chem. 2003; 278: 38723-38730Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 4Jesch S.A. Zhao X. Wells M.T. Henry S.A. J. Biol. Chem. 2005; 280: 9106-9118Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Whereas many of the genes regulated in response to inositol contain the UASINO element, the majority of the genes that respond to inositol and choline fall into other categories, including those that respond to the unfolded protein response (UPR) pathway and the low oxygen response element (LORE) (4Jesch S.A. Zhao X. Wells M.T. Henry S.A. J. Biol. Chem. 2005; 280: 9106-9118Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 19Jesch S.A. Liu P.Z.X. Wells M.T. Henry S.A. J. Biol. Chem. June 15, 2006; 10.1074/jbc.M604541200Google Scholar). We now report on an analysis of phospholipid biosynthesis and turnover employing an identical time course and growth conditions that permit direct comparison of changes in potential signaling molecules produced in the course of lipid metabolism with the kinetics of the transcriptional response to inositol reported by Jesch et al. (19Jesch S.A. Liu P.Z.X. Wells M.T. Henry S.A. J. Biol. Chem. June 15, 2006; 10.1074/jbc.M604541200Google Scholar). We report that the addition of inositol to the growth medium induces not only a rapid increase in the rate of synthesis of PI and an almost equally rapid increase in the total cellular content of PI, but also a concurrent decline in PC content brought about by the combined effects of increased turnover and diminished synthesis. We also show that the increased turnover of PC in response to inositol requires Nte1p, a phospholipase B that resides in the ER (20Zaccheo O. Dinsdale D. Meacock P.A. Glynn P. J. Biol. Chem. 2004; 279: 24024-24033Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) and has previously been shown to respond to the presence of choline and increased growth temperature (21Dowd S.R. Bier M.E. Patton-Vogt J.L. J. Biol. Chem. 2001; 276: 3756-3763Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 22Glynn P. Biochim. Biophys. Acta. 2005; 1736: 87-93Crossref PubMed Scopus (62) Google Scholar). The relationships among these changes in lipid metabolism and the correlated changes in transcription will be discussed. Strains and Media—Saccharomyces cerevisiae strains "wild type" BY4742 (MATα his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0) and nte1Δ (MATα nte1::KanMX, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0) are derived from the S288C genetic background (23Brachmann C.B. Davies A. Cost G.J. Caputo E. Li J. Hieter P. Boeke J.D. Yeast. 1998; 14: 115-132Crossref PubMed Scopus (2646) Google Scholar) and were purchased from Research Genetics. Cultures were maintained on YEPD (1% yeast extract, 2% peptone, 2% glucose, 2% agar) medium plates. All experiments were conducted using cultures grown to midlogarithmic phase at 30 °C on a rotary shaker (New Brunswick Scientific Co., Inc.) at 200 rpm using chemically defined synthetic media, as described by Jesch et al. (4Jesch S.A. Zhao X. Wells M.T. Henry S.A. J. Biol. Chem. 2005; 280: 9106-9118Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), containing 1 g of potassium phosphate/liter. Cells were grown in 50-ml batches of complete synthetic media with (I+) or without (I−) inositol (75 μm) with (C+) or without (C−) choline (1 mm) as indicated. Materials—All chemicals were reagent grade. Phospholipid standards were purchased from Avanti Polar Lipids, Inc. Neutral lipid standards and cerulenin were purchased from Sigma. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was purchased from Avanti Polar Lipids, Inc. Radiochemicals were purchased from Amersham Biosciences, and scintillation-counting supplies were purchased from National Diagnostics. Silica gel-loaded SG81 chromatography paper was purchased from Whatman, Inc., and HPTLC plates were from Merck. Growth medium supplies were purchased from Difco. Electrospray Ionization Mass Spectrometry Analysis of Phospholipids—Electrospray ionization tandem mass spectrometry analysis of phospholipid mixtures was carried out utilizing a Bruker Esquire LC Mass Spectrometer (Hewlett-Packard, Inc.) equipped with a standard orthogonal electrospray source and a three-dimensional ion trap. The samples were introduced into the mass spectrometer at a rate of infusion of 120 μl/h. The capillary voltage was set at +3500 or −3500 V for positive or negative modes, respectively. Phospholipid samples for mass spectrometry were isolated from 3-liter cultures (in duplicate) grown to midlogarithmic phase in the specified synthetic media (i.e. no inositol or choline, inositol alone, no inositol with choline, and both choline and inositol) at 30 °C. In general, cells were harvested by rapid filtration through Durapore® membrane filters (0.65-μm polyvinylidene fluoride, Millipore Corp.) and immediately transferred to an Erlenmeyer flask containing a 45 mm aqueous solution of potassium cyanide. The cells were then centrifuged at 5000 rpm at 4 °C for 15 min, and the cell pellet was washed twice with water and lysed with 10% trichloroacetic acid in an ice bath for 25 min. The cell membranes were spun down, and the supernatant was discarded. The cell membrane phospholipids were extracted with a solvent mixture consisting of ethanol, water, ethyl ether, pyridine, and ammonium hydroxide (45:45: 15:3:0.003, v/v/v/v/v). The extract was centrifuged, and the supernatant was recovered and partitioned twice with 50 ml of a chloroform/methanol (2:1, v/v) mixture and 5 ml of a saturated aqueous sodium chloride solution in a separatory funnel. The lower organic phase was collected, pooled, and taken to dryness with a gentle stream of N2. Phospholipid extracts isolated from S. cerevisiae cells were retaken in a mixture of chloroform and methanol (1:2, v/v). Samples for analysis in the negative mode were added to 2.5% of triethylamine or left untreated for analysis in the positive mode (chemical species were detected as sodiated ions). Mass Spectrometry Determination of PI and PC Species Composition at Steady State and Turnover following Inositol Addition—Wild type cultures were grown overnight under four different growth conditions: no inositol or choline (I−C−), inositol alone (I+C−), no inositol with choline (I−C+), and both choline and inositol (I+C+). For steady-state studies, 3-liter cultures of wild type cells were grown at 30 °C for eight generations and rapidly harvested and processed for phospholipid analysis by mass spectrometry as described above. For turnover studies of PI and PC species following inositol addition, 3-liter cultures of wild type or nte1Δ cells were grown in I−C+ medium to midlogarithmic phase. 21 ml of a 10 mm inositol solution were added, and the cells were allowed to continue their growth for 0, 5, 15, 30, and 60 min before they were harvested and processed for PI and PC species composition analysis by electrospray ionization mass spectrometry as described above. Analysis of Fatty Acid Composition—Total fatty acid analysis was carried out in a Hewlett Packard 5890 gas chromatograph (splitless mode) coupled to an HP 5970 B Mass Selective Detector (DB-1 capillary column, 30 × 0.25-mm inner diameter, 0.25-mm film thickness, J & W Scientific, Folsum, CA). The oven temperature was held at 100 °C for 2 min, raised at 10 °C/min to 200 °C, held for 10 min, and raised at 3 °C/min to 300 °C. Fatty acid samples for gas chromatography analysis were isolated from 50-ml yeast cell cultures (in duplicate) grown to midlogarithmic phase in the specified synthetic media (i.e. I−C−, I+C−, I−C+, and I+C+) at 30 °C. The cells were spun down, washed twice with water, and transferred to a 1.5-ml Eppendorf tube and lysed with 1 ml of trichloroacetic acid (10%) for 25 min in an ice bath. The Eppendorf tubes were centrifuged for 5 min at 7500 rpm, and the supernatant was discarded. The cells were washed twice with sterile water and extracted with 1 ml of a solvent mixture consisting of ethanol, water, ethyl ether, pyridine, and ammonium hydroxide (45:45: 15:3:0.003, v/v/v/v/v). The extract was centrifuged, and the supernatant was transferred to a 15-ml glass test tube and partitioned with 1 ml of chloroform/methanol (2:1, v/v) and three drops of a saturated solution of sodium chloride. The lower phase was collected and taken to dryness with a gentle stream of nitrogen. The residue was extracted twice with a total of 1 ml of boron trifluoride in methanol (14%; Sigma) and transferred to 1.5-ml glass vials, capped, and heated to 100 °C in a sand bath for 30 min. The reaction mixture was allowed to cool down to room temperature and extracted with 1 ml of hexane. The hexane extract was collected and washed with water and taken to dryness under a nitrogen stream. Three drops of hexane were added to the residue for analysis by gas chromatography coupled with mass spectrometry. Determination of Steady-state Phospholipid Composition by 32P Labeling—Wild type cultures were grown overnight under the four different growth conditions: I−C−, I+C−, I−C+, and I+C+. Cells were labeled to steady state by the addition of 20 μCi/ml [32P]orthophosphate (specific activity of isotope was 2.7 mCi/mmol phosphate). To analyze the steady-state lipid composition, cells were grown at 30 °C for eight generations in the presence of the label. Labeling for additional generations produced no change in the percentage distribution of the label into lipid classes (data not shown), indicating that a steady-state labeling condition was achieved. 5-ml samples were mixed with 0.5 ml of 50% trichloroacetic acid and allowed to stand on ice for 20 min. The samples were washed twice with distilled water. Labeled lipids were extracted as previously described (24Atkinson K. Fogel S. Henry S.A. J. Biol. Chem. 1980; 255: 6653-6661Abstract Full Text PDF PubMed Google Scholar). The individual phospholipids species were resolved by two-dimensional paper chromatography (25Steiner M.R. Lester R.L. Biochim. Biophys. Acta. 1972; 260: 222-243Crossref PubMed Scopus (108) Google Scholar). Phospholipid identity was based on the mobility of known standards and quantified on a STORM 860 PhosphorImager (Amersham Biosciences). Kinetic Analysis of Changes in Phospholipid Composition following Inositol Addition—To determine changes in the composition of the phospholipids over a set time course following the addition of inositol, cells were first grown in I−C+ medium, and phospholipids were labeled with 20 μCi/ml [32P]orthophosphate to steady state. When the cells reached the midlogarithmic phase of growth (A600 = 0.5), inositol to a final concentration of 75 μm, was added, and aliquots were taken at 2, 5, 15, 30, and 60 min. A control sample (0 min) was taken just prior to the addition of inositol. Cells were harvested, and lipids were extracted and analyzed as described above. Analysis of Phospholipid Turnover following Inositol Addition— To follow the turnover of phospholipids after the addition of inositol, wild type and nte1Δ cells were grown, as described above, to steady state in the presence of 20 μCi/ml [32P]orthophosphate in I−C+ medium. When the cells reached A600 = 0.5, they were quickly collected by filtration, washed with prewarmed I−C+ medium at 30 °C, and placed in unlabeled fresh I+C+ medium prewarmed to 30 °C. Samples were taken at 0, 5, 15, 30, and 60 min and following filtration and transfer to unlabeled medium, and lipid extraction and analysis was performed as described above. As a control, cells were also transferred to fresh unlabeled I−C+ medium, and samples were taken over the same time course as those in I+C+ medium. Short Term Labeling of Phospholipids—To analyze new synthesis of glycerophospholipids following the addition of inositol, wild type and nte1Δ cells were pregrown to A600 = 0.5 in I−C+ medium, and 100 μCi/ml [32P]orthophosphate (specific activity of isotope was 13.51 mCi/mmol of phosphate) was added. Samples were taken 10 and 20 min following the addition of [32P]orthophosphate. At 20 min, inositol was added to a final concentration of 75 μm, and cells were sampled at 10-min intervals until 50 min following 32P introduction (i.e. 30, 40, and 50 min following 32P introduction). Lipids were extracted and quantified by two-dimensional chromatography, as described above. The design of this experiment is very similar to that reported previously in Loewen et al. (2Loewen C.J. Gaspar M.L. Jesch S.A. Delon C. Ktistakis N.T. Henry S.A. Levine T.P. Science. 2004; 304: 1644-1647Crossref PubMed Scopus (371) Google Scholar), with the exception that choline was present in the current experiment, and the cells were labeled in the I−C+ medium for 20 min prior to the addition of inositol (as opposed to 10-min labeling with 32PinI−C− medium prior to the inositol addition in Loewen et al. (2Loewen C.J. Gaspar M.L. Jesch S.A. Delon C. Ktistakis N.T. Henry S.A. Levine T.P. Science. 2004; 304: 1644-1647Crossref PubMed Scopus (371) Google Scholar)). Assessment of Neutral Lipid Composition by [1-14C]Acetate Labeling—Wild type cells were grown in four different conditions, as described above (i.e. I−C−, I+C−, I−C+, and I+C+). Cultures were grown at 30 °C in the presence of 1 μCi/ml of [1-14C]acetate (specific activity, 57 mCi/mmol) overnight. The next day, cultures were diluted to A600 = 0.1 maintaining label at 1 μCi/ml [1-14C]acetate and allowed to grow until midlogarithmic phase (A600 = 0.5). The cells were then harvested using the same methods described above for 32P labeling experiments. Total lipids were extracted with a mixture of chloroform/methanol (2:1, v/v) (26Folch J. Lees M. Sloane Stanley G.H. J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). The chloroform phase was dried, and the residue was dissolved in chloroform/methanol (1:1, v/v). Steady-state incorporation of label into major neutral lipid classes was determined by thin layer chromatography. Neutral lipids were separated on Whatman Silica Gel 60A HPTLC plates using the solvent system hexane/diethyl ether/formic acid (80:20:2, v/v/v) (27Christie W. Lipid Analysis: Isolation, Separation, Identification, and Structural Analysis of Lipids.in: Christie W. The Oily Press, Bridgwater, UK2003: 105-112Google Scholar). Labeled lipids on the chromatograms were quantified on a STORM 860 PhosphorImager (Amersham Biosciences). Metabolite identity was determined by comparison with the mobility of known standards. Kinetic Analysis of Changes in Neutral Lipid Composition following Inositol Addition—To follow changes in the relative composition of the neutral lipid classes following the addition of inositol, wild type cells were labeled with 1 μCi/ml [1-14C]acetate to steady state, as described above, in I−C+ medium. A 5-ml sample, which served as time 0 control, was taken prior to the addition of inositol, and then inositol was added to a final concentration of 75 μm, and 5 ml of cells were harvested at 2, 5, 15, 30, and 60 min. Neutral lipids were extracted and analyzed as described above for assessment of steady-state neutral lipid composition. Short Time Course Labeling with [1-14C]Acetate—To analyze new synthesis of both neutral lipids and selective phospholipids in a single experiment following inositol addition, 1 μCi/ml [1-14C]acetate was added to cultures of wild type and nte1Δ cells grown to midlogarithmic phase (A600 = 0.5) in I−C+ medium. Samples were taken 10, 20, and 30 min following the addition of label. At 60 min following the addition of label, inositol was added to a final concentration of 75 μm, and samples were collected at 5, 15, 30, and 60 min following the addition of inositol. Lipid extraction and analysis was performed, as described above, for the 32P and 14C steady-state labeling. Phospholipids were separated on Whatman Silica Gel 60A HPTLC plates using the solvent system chloroform/ethyl acetate/acetone/isopropyl alcohol/ethanol/methanol/water/acetic acid (30:6:6:6:16:28:6:2, v/v/v/v/v/v/v/v) (28Weerheim A.M. Kolb A.M. Sturk A. Nieuwland R. Anal. Biochem. 2002; 302: 191-198Crossref PubMed Scopus (121) Google Scholar). The neutral lipid classes were analyzed on HPTLC plates using hexane/diethylether/formic acid (80:20:2, v/v/v) (27Christie W. Lipid Analysis: Isolation, Separation, Identification, and Structural Analysis of Lipids.in: Christie W. The Oily Press, Bridgwater, UK2003: 105-112Google Scholar). Metabolite identity was established based on the mobility of known standards. The amounts of labeled lipids on the chromatograms were quantified on a STORM 860 PhosphorImager (Amersham Biosciences). Short Time Course Labeling in the Presence of Cerulenin—In order to measure the effects of inhibition of de novo fatty acid synthesis on phospholipid synthesis, wild type cells were grown in I−C+ medium at midlogarithmic phase (A600 = 0.5) and were exposed to different concentrations of cerulenin, an inhibitor of de novo fatty acid synthesis (29Nomura S. Horiuchi T. Omura S. Hata T. J. Biochem. (Tokyo). 1972; 71: 783-796Crossref PubMed Scopus (92) Google Scholar). Protocols similar to those described above for short term labeling with either 1 μCi/ml [1-14C]acetate or 100 μCi/ml [32P]orthophosphate were carried out. For experiments involving short term labeling with [1-14C]acetate, wild type cells were grown at 30 °C to A600 = 0.5 in I−C+ or I+C+ medium and treated with concentrations of cerulenin ranging from 0.625 to 10 μg/ml. Label and cerulenin were added simultaneously, and samples were taken at 15, 30, and 45 min following the addition of inositol. Cells were harvested, and phospholipids and neutral lipid classes were extracted and analyzed as described above for 14C steady-state labeling. For short term labeling with [32P]orthophosphate, the procedure described above for short term labeling of phospholipids with 32P of wild type cells was followed. At zero time, both 32P and 10 μg/ml (4.4 × 10−5 m) cerulenin were added simultaneously. Inositol was added to a final concentration of 75 μm to the cultures 20 min after the addition of the label and cerulenin. Samples were collected at the time points indicated. Quantitation of Transcripts by Northern Blotting—Wild type and nte1Δ cells were grown in liquid I−C+ medium to midlogarithmic growth phase, and inositol was added to a final concentration of 75 μm. Cells were harvested by filtration and immediately frozen on dry ice immediately prior to and 5, 15, 30, and 60 min following the addition of inositol. Total RNA from each time point was isolated according to the manufacturer's instructions with the RNeasy minikit (Qiagen, Inc.). 250 ng of total RNA was fractionated on 1.1% glyoxal-agarose gels, transferred to positively charged nylon membrane using Turboblotter (30Burnett W.V. BioTechniques. 1997; 22: 668-671Crossref PubMed Scopus (61) Google Scholar), and probed for INO1, OLE1, KAR2, and ACT1 transcripts using 32P-labeled antisense riboprobes as described (4Jesch S.A. Zhao X. Wells M.T. Henry S.A. J. Biol. Chem. 2005; 280: 9106-9118Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Transcript levels were quantitated by a PhosphorImager. Values were expressed as a ratio of INO1, OLE1, and KAR2 mRNA to ACT1 mRNA levels. To evaluate the expression of the INO1 and OLE1 genes when the de novo synthesis of fatty acids is impaired, wild t
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