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• W2059550531 abstract "We had previously isolated the temperature-sensitive erg26-1 mutant and characterized the sterol defects in erg26-1 cells (Baudry, K., Swain, E., Rahier, A., Germann, M., Batta, A., Rondet, S., Mandala, S., Henry, K., Tint, G. S., Edlind, T., Kurtz, M., and Nickels, J. T., Jr. (2001) J. Biol. Chem. 276, 12702–12711). We have now determined the defects in sphingolipid metabolism inerg26-1 cells, examined their effects on cell growth, and initiated studies designed to elucidate how might changes in sterol levels coordinately regulate sphingolipid metabolism inSaccharomyces cerevisiae. Using [3H]inositol radiolabeling studies, we found that the biosynthetic rate and steady-state levels of specific hydroxylated forms of inositolphosphorylceramides were decreased in erg26-1 cells when compared with wild type cells. [3H]Dihydrosphingosine radiolabeling studies demonstrated that erg26-1 cells had decreased levels of the phytosphingosine-derived ceramides that are the direct precursors of the specific hydroxylated inositol phosphorylceramides found to be lower in these cells. Gene dosage experiments using the sphingolipid long chain sphingoid base (LCB) hydroxylase gene, SUR2, suggest that erg26-1 cells may accumulate LCB, thus placing one point of sterol regulation of sphingolipid synthesis possibly at the level of ceramide metabolism. The results from additional genetic studies using the sphingolipid hydroxylase and copper transporter genes, SCS7 and CCC2, respectively, suggest a second possible point of sterol regulation at the level of complex sphingolipid hydroxylation. In addition, [3H]inositol radiolabeling of sterol biosynthesis inhibitor-treated wild type cells and late sterol pathway mutants showed that additional blocks in sterol biosynthesis have profound effects on sphingolipid metabolism, particularly sphingolipid hydroxylation state. Finally, our genetic studies in erg26-1 cells using the LCB phosphate phosphatase gene, LBP1, suggest that increasing the levels of the LCB sphingoid base phosphate can remediate the temperature-sensitive phenotype of erg26-1cells. We had previously isolated the temperature-sensitive erg26-1 mutant and characterized the sterol defects in erg26-1 cells (Baudry, K., Swain, E., Rahier, A., Germann, M., Batta, A., Rondet, S., Mandala, S., Henry, K., Tint, G. S., Edlind, T., Kurtz, M., and Nickels, J. T., Jr. (2001) J. Biol. Chem. 276, 12702–12711). We have now determined the defects in sphingolipid metabolism inerg26-1 cells, examined their effects on cell growth, and initiated studies designed to elucidate how might changes in sterol levels coordinately regulate sphingolipid metabolism inSaccharomyces cerevisiae. Using [3H]inositol radiolabeling studies, we found that the biosynthetic rate and steady-state levels of specific hydroxylated forms of inositolphosphorylceramides were decreased in erg26-1 cells when compared with wild type cells. [3H]Dihydrosphingosine radiolabeling studies demonstrated that erg26-1 cells had decreased levels of the phytosphingosine-derived ceramides that are the direct precursors of the specific hydroxylated inositol phosphorylceramides found to be lower in these cells. Gene dosage experiments using the sphingolipid long chain sphingoid base (LCB) hydroxylase gene, SUR2, suggest that erg26-1 cells may accumulate LCB, thus placing one point of sterol regulation of sphingolipid synthesis possibly at the level of ceramide metabolism. The results from additional genetic studies using the sphingolipid hydroxylase and copper transporter genes, SCS7 and CCC2, respectively, suggest a second possible point of sterol regulation at the level of complex sphingolipid hydroxylation. In addition, [3H]inositol radiolabeling of sterol biosynthesis inhibitor-treated wild type cells and late sterol pathway mutants showed that additional blocks in sterol biosynthesis have profound effects on sphingolipid metabolism, particularly sphingolipid hydroxylation state. Finally, our genetic studies in erg26-1 cells using the LCB phosphate phosphatase gene, LBP1, suggest that increasing the levels of the LCB sphingoid base phosphate can remediate the temperature-sensitive phenotype of erg26-1cells. sterol response element-binding protein sterol response element inositolphosphorylceramide mannose inositolphosphorylceramide mannose diinositolphosphorylceramide long chain sphingoid base long chain sphingoid base phosphate glycerol-3-phosphate dehydrogenase promoter gas chromatography/mass spectrometry temperature-sensitive Sphingolipids are ubiquitous membrane lipids that are found in all eukaryotic cells. They are structural components of lipid bilayers (1Hannun Y.A. Bell R.M. Science. 1989; 243: 500-507Crossref PubMed Scopus (1111) Google Scholar), have emerged as signaling molecules generated in response to a variety of growth modulators (2Hannun Y.A. Luberto C. Trends Cell Biol. 2000; 10: 73-80Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 3Mandala S.M. Prostaglandins Other Lipid. Mediat. 2001; 64: 143-156Crossref PubMed Scopus (84) Google Scholar, 4Spiegel S. Cuvillier O. Edsall L.C. Kohama T. Menzeleev R. Olah Z. Olivera A. Pirianov G. Thomas D.M., Tu, Z. Van Brocklyn J.R. Wang F. Ann. N. Y. Acad. Sci. 1998; 845: 11-18Crossref PubMed Scopus (190) Google Scholar, 5Mathias S. Pena L.A. Kolesnick R.N. Biochem. J. 1998; 335: 465-480Crossref PubMed Scopus (621) Google Scholar), and have been found to play an important role in regulating vesicular and lipid trafficking events in higher eukaryotes (6McMaster C.R. Biochem. Cell Biol. 2001; 79: 681-692Crossref PubMed Scopus (70) Google Scholar, 7Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8157) Google Scholar, 8Bagnat M. Keranen S. Shevchenko A. Shevchenko A. Simons K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3254-3329Crossref PubMed Scopus (503) Google Scholar). With regards to human pathology, a number of diseases have been attributed to the inappropriate trafficking and/or metabolism of sphingolipids. Human Niemann-Pick disorders are characterized by the intercellular mislocalization and accumulation of sphingolipid and cholesterol in lysosomes (9Ory D.S. Biochim. Biophys. Acta. 2000; 1529: 331-339Crossref PubMed Scopus (125) Google Scholar, 10Carstea E.D. et al.Science. 1997; 277: 228-231Crossref PubMed Scopus (1230) Google Scholar, 11Schuchman E.H. Desnick R.J. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Metabolic Basis of Inherited Diseases. McGraw-Hill, New York1995: 2601-2624Google Scholar), and a number of rather severe sphingolipid storage diseases result from the inability of a diseased cell to properly catabolize sphingolipids (12Pagano R.E. Puri V. Dominguez M. Marks D.L. Traffic. 2000; 11: 807-815Crossref Scopus (94) Google Scholar, 13Puri V. Watanabe R. Dominguez M. Sun X. Wheatley C.L. Marks D.L. Pagano R.E. Nat. Cell Biol. 1999; 1: 386-388Crossref PubMed Scopus (255) Google Scholar). The mechanisms regulating sphingolipid metabolism at the molecular level are not that well understood. However, there is some evidence suggesting that sphingolipid biosynthesis may be regulated coordinately with cholesterol metabolism in higher eukaryotes. Storey et al. (14Storey M.K. Byers D.M. Cook H.W. Ridgway N.D. Biochem. J. 1998; 336: 246-257Crossref Scopus (75) Google Scholar) showed that sphingomyelin and ceramide biosynthesis could be blocked by the addition of the HMG-CoA reductase inhibitor lovastatin. They also demonstrated that a cholesterol auxotrophic Chinese hamster ovary cell line harboring defects in SREBP1 processing and cholesterol biosynthesis had greatly decreased levels of sphingomyelin (14Storey M.K. Byers D.M. Cook H.W. Ridgway N.D. Biochem. J. 1998; 336: 246-257Crossref Scopus (75) Google Scholar). However, others showed that lovastatin treatment of CaCo-2 cells did not result in changes in sphingolipid biosynthesis (15Chen H. Born E. Mathur S.N. Field F.J. J. Lipid Res. 1993; 34: 2159-2167Abstract Full Text PDF PubMed Google Scholar). There is work from several laboratories showing that sterol esterification rapidly occurs in response to membrane depletion of sphingomyelin (16Slotte J.P. Bierman E.L. Biochem. J. 1988; 250: 653-658Crossref PubMed Scopus (274) Google Scholar, 17Slotte J.P. Hedstrom G. Rannstrom S. Ekman S. Biochim. Biophys. Acta. 1989; 985: 90-96Crossref PubMed Scopus (118) Google Scholar, 18Lange Y. Steck T.L. J. Biol. Chem. 1997; 272: 13103-13108Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) and is accompanied by down-regulation of de novosterol biosynthesis brought about by sterol-dependent inhibition of SREBP processing (19Nohturfft A. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 17243-17250Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Recently, Worgall et al. (20Worgall T.S. Johnson R.A. Seo T. Gierens H. Deckelbaum R.J. J. Biol. Chem. 2002; 277: 3878-3885Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) showed that increasing ceramide levels or inhibiting ceramidase activity in Chinese hamster ovary cells decreases SRE-mediated transcription, presumably by reducing the levels of the transcriptionally active forms of SREBP-1 and SREBP-2. Thus, ceramide may mediate sphingolipid-dependent regulation of sterol metabolism in animals. Genetic studies in the yeast Saccharomyces cerevisiae have hinted at the possibility of a similar coordinate regulatory system in lower eukaryotes. Genome expression studies using antifungals targeting sterol biosynthesis have revealed that many lipid metabolic genes are transcriptionally regulated in response to changes in sterol levels (21Dimster-Denk D. Rine J. Phillips J. Scherer S. Cundiff P. DeBord K. Gilliland D. Hickman S. Jarvis A. Tong L. Ashby M. J. Lipid Res. 1999; 40: 850-860Abstract Full Text Full Text PDF PubMed Google Scholar, 22Bammert G.F. Fostel J.M. Antimicrob. Agents Chemother. 2000; 44: 1255-1265Crossref PubMed Scopus (192) Google Scholar). Among those genes regulated were the SUR2 LCB hydroxylase gene required for the production of phytosphingosine and the LCB1 serine palmitoyltransferase gene involved in the first step of sphingolipid biosynthesis (23Haak D. Gable K. Beeler T. Dunn T. J. Biol. Chem. 1997; 272: 29704-29710Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 24Grilley M.M. Stock S.D. Dickson R.C. Lester R.L. Takemoto J.Y. J. Biol. Chem. 1998; 273: 11062-11068Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Vik and Rine (25Vik A. Rine J. Mol. Cell. Biol. 2001; 21: 6395-6405Crossref PubMed Scopus (201) Google Scholar) have defined an SRE that is common to many ERG genes and is found in the promoter sequence of LCB1 (25Vik A. Rine J. Mol. Cell. Biol. 2001; 21: 6395-6405Crossref PubMed Scopus (201) Google Scholar). Several studies examining antifungal resistance in yeast have demonstrated a genetic interaction between sterol and sphingolipid metabolism and cell viability (26Oh C.S. Toke D.A. Mandala S. Martin C.E. J. Biol. Chem. 1997; 272: 17376-17384Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 27Silve S. Leplatois P. Josse A. Dupuy P.H. Lanau C. Kaghad M. Dhers C. Picard C. Rahier A. Taton M., Le Fur G. Caput D. Ferrara P. Loison G. Mol. Cell. Biol. 1996; 16: 2719-2727Crossref PubMed Scopus (66) Google Scholar, 28Ladeveze V. Marcireau C. Delourme D. Karst F. Lipids. 1993; 28: 907-912Crossref PubMed Scopus (33) Google Scholar). S. cerevisiae should be an excellent model in which to study sphingolipid metabolism and regulation. Sphingolipids are essential for yeast cell viability, emphasizing their critical role in growth (29Dickson R.C. Wells G.B. Schmidt A. Lester R.L. Mol. Cell. Biol. 1990; 10: 2176-2181Crossref PubMed Scopus (61) Google Scholar). Almost all of the genes that are required for sphingolipid biosynthesis and metabolism have been cloned, allowing for the precise modulation of sphingolipid levels in this organism (30Dickson R.C. Lester R.L. Biochim. Biophys. Acta. 1999; 1426: 347-357Crossref PubMed Scopus (171) Google Scholar) (Fig. 1). In addition,S. cerevisiae synthesizes only three complex sphingolipids, inositolphosphorylceramide (IPC), mannose inositolphosphorylceramide (MIPC), and mannose diinositolphosphorylceramide (MIP2C), making the interpretation of the physiological roles of sphingolipids much more straightforward (31Lester R.L. Dickson R.C. Adv. Lipid Res. 1993; 26: 253-274PubMed Google Scholar). Recently, we isolated the conditional erg26-1 yeast mutant in a genetic screen designed to obtained sphingolipid metabolic mutants (32Baudry K. Swain E. Rahier A. Germann M. Batta A. Rondet S. Mandala S. Henry K. Tint G.S. Edlind T. Kurtz M. Nickels J.T., Jr. J. Biol. Chem. 2001; 276: 12702-12711Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Our biochemical studies demonstrated that erg26-1cells were defective in 4α-carboxylsterol-C3 dehydrogenase activity, one of the three enzyme activities required for the conversion of 4,4-dimethylzymosterol to zymosterol. The defect in 4α-carboxylsterol-C3 dehydrogenase activity in erg26-1cells caused the abnormal accumulation of specific zymosterol intermediates and had an effect on neutral and phospholipid biosynthesis and metabolism. We now have gone back and examined in detail how the erg26-1 mutation affects sphingolipid biosynthesis and cell viability. The yeast strains used in this study are derived from W303-1A (MATa leu2-3, 112 trp1-1 ura3-1 his3-11, 15 can1-100). The yeast strains were grown in either YEPD (1% yeast extract, 2% Bacto-peptone, 2% glucose) or in synthetic minimal medium containing 0.67% yeast nitrogen base (Difco) supplemented with the appropriate amino acids and adenine. Yeast transformations were performed using the procedure described by Ito et al. (33Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar). For routine propagation of plasmids, Escherichia coli XL1 Blue cells were used and grown in LB medium supplemented with ampicillin (200 μg/ml). Yeast null mutants were generated by the one-step disruption method of Rothstein (34Rothstein R.J. Methods Enzymol. 1983; 101: 20-78Crossref PubMed Scopus (3419) Google Scholar) using individual YIp deletion constructs (35Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) or the KanMXcassette (36Longtine M.S. McKenzie A.r. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4193) Google Scholar). Yeast strains harboring individual deletions were verified by PCR analysis. The LEU2- andURA3-containing 2μ plasmids, pRS425 and pRS426, respectively, were used to construct the various high copy vectors used in this study (35Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). The glycerol-3-phosphate dehydrogenase promoter (GPD) (37Mumberg D. Muller R. Funk M. Gene (Amst.). 1995; 156: 119-122Crossref PubMed Scopus (1603) Google Scholar) was obtained from the vector pJR1133 that was provided by Drs. Jasper Rine and Chris Beh (University of California, Berkeley, CA). The GPD promoter was subcloned into pRS425 and pRS426, and these vectors were used for the overexpression studies. A 2μ vector overexpressing CCC2 was kindly provided by Dr. Valeria Culotta (Johns Hopkins University School of Public Health, Baltimore, MD). CCC2 was excised from this vector and subcloned into pRS426 that contained the GPD promoter. All of the genomic sequences subcloned into the various vectors were obtained by PCR amplification using the high fidelity Pfu polymerase. All of the DNA sequences that were generated by PCR were sequenced and compared with the yeast genome data base. Starting cultures for all of the lipid radiolabeling experiments were grown overnight at 27 °C to exponential phase in synthetic complete media or selective minimal media.A 600 were then taken, and the cultures were diluted to 5 × 106 cells/ml and preincubated at 27 and 37 °C for 3 h before antifungal treatment and/or radiolabeling. Radiolabeled lipids were resolved by TLC and visualized by autoradiography (Kodak XAR5). The percentage value of each lipid species was determined by densitometry using Bio-Rad model GS-670 Imaging Densitometer and Molecular Analyst software, version 1.4.1. For pulse radiolabeling of complex sphingolipids, the cultures were incubated with 5 μCi/ml [3H]myo-inositol for 30 min. For steady-state labeling, the cultures were incubated with 1 μCi/ml [3H]myo-inositol for 5 h. Radiolabeled sphingolipids were extracted with ethanol, water, diethyl ether, pyridine, 4.2 n ammonium hydroxide (15:15:5:1:0.018), treated with methylamine as described (38Clarke N.G. Dawson R.M. Biochem. J. 1981; 195: 301-306Crossref PubMed Scopus (186) Google Scholar), and analyzed by one-dimensional TLC using chloroform, methanol, acetic acid, and water (16:6:4:1.6). When necessary, wild type cultures were preincubated with specified antifungal compounds as described for the analysis of sterols (see below). For pulse radiolabeling of ceramides, cultures were incubated with 5 μCi/ml [3H]dihydrosphingosine for 20 min. For steady-state radiolabeling, the cultures were incubated with 5 μCi/ml [3H]dihydrosphingosine for 30 min, pelleted, and washed in media lacking radiolabel, and chased for an additional 3 h. To help resolve the various ceramides species, wild type cultures were incubated in the absence and presence of 140 μmfumonisin B1. Radiolabeled ceramides were extracted with ethanol, water, diethyl ether, pyridine, 4.2 n ammonium hydroxide (15:15:5:1:0.018) and analyzed by one-dimensional TLC using chloroform, methanol, acetic acid (95:4.5:0.5) (23Haak D. Gable K. Beeler T. Dunn T. J. Biol. Chem. 1997; 272: 29704-29710Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). For pulse labeling of sterols, antifungal-treated cultures were incubated at 30 °C with 1μCi/ml [14C]acetate for 30 min. For steady-state labeling of sterols, the cultures were incubated at 30 °C with 5 μCi/ml [14C] acetate for 4 h. Radiolabeled sterols were extracted using chloroform, methanol (2:1) and analyzed by one-dimensional TLC using petroleum ether, diethyl ether, acetic acid (70:30:1). In all cases, the cultures were preincubated for 1 h in the absence and presence of a specified concentration of antifungal compound (40 ng/ml fenpropimorph, 2 μg/ml fluconazole, 2 μg/ml terbinafine) prior to radiolabeling cells for sterol analysis. For pulse labeling of phospholipids, the cultures were incubated with 50 μCi/ml [32P]orthophosphate for 20 min. For steady-state radiolabeling, the cultures were incubated with 25 μCi/ml [32P]orthophosphate for several generations. Radiolabeled phospholipids were extracted using the spheroplast method of Atkinson et al. (39Atkinson K. Fogel S. Henry S.A. J. Biol. Chem. 1980; 255: 6653-6661Abstract Full Text PDF PubMed Google Scholar) and analyzed by one-dimensional TLC as described previously (38Clarke N.G. Dawson R.M. Biochem. J. 1981; 195: 301-306Crossref PubMed Scopus (186) Google Scholar). To analyze total fatty acids, the cells were cultured at 27 °C in synthetic media to a density of ∼1 × 107 cells/ml. The cells were harvested by centrifugation and resuspended in an equivalent volume of fresh media prewarmed at 27 °C (control cultures) or 37 °C. Incubation of all cultures continued for three additional hours at the indicated temperatures. The cells were harvested again and washed with sterile water to remove residual media. Fatty acid methyl esters were prepared by HCl methanolysis of whole cell lipids (40Browse J. McCourt P.J. Somerville C.R. Anal. Biochem. 1986; 152: 141-145Crossref PubMed Scopus (399) Google Scholar). Whole cells were collected by centrifugation in glass screw-capped test tubes. All of the residual water was removed by aspiration, and the cell pellets were resuspended in 1 ml of 1 m methanolic HCl, 5% 2,2-dimethoxypropane (v/v). Test tubes was purged with nitrogen and incubated at 85 °C for 1 h. Fatty acid methyl esters were extracted from cooled samples by the addition of 1 ml of hexane, ethyl ether (1:1 v/v) and 1 ml of 0.9% NaCl. The samples were vigorously mixed and then centrifuged to advance phase separation. The organic phase was collected and passed over a silica gel mini-column preconditioned with hexane, ethyl ether (1:1 v/v) solvent and then washed with a minimal amount (1–2 ml) of solvent: The column flow-through was dried under nitrogen, and dried fatty acid methyl esters were resuspended in 200 μl of hexane. The samples of fatty acid methyl esters were analyzed by gas-liquid chromatography on a Hewlett-Packard model 6890 gas chromatograph using a 30-m HP-5 column. Instrument conditions were as follows: injection port temperature, 220 °C; detector temperature, 300 °C; initial oven temperature, 120 °C. After a 1-min hold at 120 °C, the oven temperature was increased at 2 °C/min up to a temperature of 150 °C followed by a 4 °C/min increase to a final temperature of 300 °C and held for 5 min. Identification of cellular fatty acid species was by comparison of retention times to those of standard compounds and by GC/MS analysis using data base files of fatty acid methyl ester spectra. GC/MS analysis was performed on a Hewlett-Packard model 6890 gas chromatograph and a Hewlett-Packard model 5973 Mass Selective Detector. The run conditions were identical to those described above for gas-liquid chromatography. We first analyzed the complex sphingolipid compositions of wild type and erg26-1 cells using lipid radiolabeling studies and TLC. In these studies, we examined the rates of biosynthesis and steady-state levels of sphingolipids. Our experiments were carried out at both the permissive and restrictive temperatures for erg26-1 cell growth. The results of these studies are shown in Fig. 2. Using [H3]inositol radiolabeling studies, we found thaterg26-1 cells harbored defects in the rate of biosynthesis of several hydroxylated IPC species (Fig. 2). These defects were detected at both temperatures in erg26-1 cells. Using yeast mutants lacking individual sphingolipid hydroxylase activities (23Haak D. Gable K. Beeler T. Dunn T. J. Biol. Chem. 1997; 272: 29704-29710Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 24Grilley M.M. Stock S.D. Dickson R.C. Lester R.L. Takemoto J.Y. J. Biol. Chem. 1998; 273: 11062-11068Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar,42Fu D. Beeler T.J. Dunn T.M. Yeast. 1995; 11: 283-292Crossref PubMed Scopus (144) Google Scholar), we were able to determine what IPC species were affected by theerg26-1 mutation. Our results revealed thaterg26-1 cells grown at the permissive temperature had an increased rate of biosynthesis of IPC-C (4.2-fold) while exhibiting a decreased rate of biosynthesis of IPC-D (8.5-fold) when compared with wild type cells grown under the exact conditions (Fig. 2,Pulse, 27°, ERG26 versus erg26-1). IPC-B, MIPC, and MIP2C biosyntheses were all unaffected in erg26-1 cells grown at this temperature. Strikingly, we found that IPC biosynthesis was almost completely shut down in temperature-shifted erg26-1 cells. The biosynthetic levels of the three predominant IPC species detected inerg26-1 cells at the permissive temperature, IPC-B, IPC-C, and IPC-D, were all drastically reduced in temperature-shiftederg26-1 cells (Fig. 2, Pulse, 37°,ERG26 versus erg26-1). In contrast, we observed only a slight decrease in MIPC biosynthesis (2.3-fold) and a slight increase in the biosynthesis of MIP2C in temperature-shifted erg26-1 cells (1.4-fold) (Fig. 2,Pulse, 37°, ERG26 versus erg26-1). When we examined steady-state sphingolipid composition, we found thaterg26-1 cells harbored defects in their ability to sustain normal levels of the major hydroxylated IPC species, IPC-B, IPC-C, and IPC-D (Fig. 2, Steady-State). A decrease in the levels of these three IPC species was detected at both temperatures inerg26-1 cells (Fig. 2, Steady-State,27° and 37°). In the case of IPC-B, we did not detect any accumulation of this complex sphingolipid inerg26-1 cells at either temperature. Whereas the steady-state levels of IPC-C were decreased 15- and 7.3-fold, the IPC-D levels were decreased 6.2- and 4-fold, at 27 and 37 °C, respectively. On the other hand, steady-state MIPC levels were only slightly decreased (1.8-fold, 27o ; 2.0-fold,37o ), whereas MIP2C levels were not affected in these cells at either temperature. To ascertain what step(s) in the sphingolipid pathway is regulated by sterol levels in erg26-1 cells, we determined the rates of biosynthesis and metabolism of the hydroxylated ceramides using [H3]dihydrosphingosine radiolabeling and TLC analysis (23Haak D. Gable K. Beeler T. Dunn T. J. Biol. Chem. 1997; 272: 29704-29710Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Using this method, we were able to determine the biosynthetic and metabolic levels of four of the five hydroxylated ceramide species known to be synthesized in yeast cells (Fig. 1). We found that erg26-1 cells were defective in their ability to synthesize and accumulate the phytosphingosine-containing ceramides, ceramide-B and ceramide-C (Fig. 3). Ceramide-B is the precursor for ceramide-C, where ceramide-C is produced through the action of the sphingolipid hydroxylase, Scs7p (Fig. 1) (23Haak D. Gable K. Beeler T. Dunn T. J. Biol. Chem. 1997; 272: 29704-29710Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). The rate of biosynthesis and steady-state levels of ceramide-B and -C were most severely reduced at the restrictive growth temperature (Fig. 3, B and D). The steady-state levels of ceramide-B and -C in temperature-shifted erg26-1cells were only 22 and 28% of that seen in wild type cells (Fig.3 D). The rate of biosynthesis and steady-state levels of the dihydrosphingosine-containing ceramides, ceramide-A and ceramide-B′, in erg26-1 cells were similar to those seen in wild type cells (Fig. 3). We were unable to resolve ceramide-D using this TLC method. Therefore, we cannot say whether the biosynthetic and/or the metabolic level of this ceramide species was affected by the erg26-1 mutation. C26 fatty acids are used exclusively to synthesize complex sphingolipids in yeast (43Daum G. Lees N.D. Bard M. Dickson R. Yeast. 1998; 14: 1471-1510Crossref PubMed Scopus (529) Google Scholar). We reasoned that the defects seen in complex sphingolipid metabolism in erg26-1cells may cause the accumulation of C26 fatty acids, which in turn may have secondary effects on total fatty acid metabolism. Thus, we determined the steady-state levels of various fatty acid species in wild type and erg26-1 cells at the permissive and restrictive temperatures using GC/MS analysis. We found that at either temperature, erg26-1 cells contained dramatically less C10:0 and C12:0 fatty acid species while overaccumulating C26:0 fatty acid (Fig.4 A). erg26-1 cells had 4- and 3-fold reductions in C10:0 and C12:0 fatty acids while accumulating ∼3-fold higher C26:0 fatty acid at both growth temperatures. In addition, we found that erg26-1 cells also accumulated low levels of C20:0, C20:1, and C24:0 fatty acid species at the restrictive temperature (∼0.03–0.10% of total fatty acid) (not shown). In contrast, the levels of the major fatty acid species in yeast cells, C16:0, C16:1, C18:0, and C18:1, were similar inerg26-1 and wild type cells at both temperatures (Fig.4 B). The loss of proper ceramide-B levels inerg26-1 cells could be due either to a decrease in its biosynthesis or an increase in its turnover or both. This may lead to the accumulation of cytotoxic levels of LCB and/or LCBP at high temperatures (44Kim S. Fyrst H. Saba J. Genetics. 2000; 156: 1519-1529Crossref PubMed Google Scholar, 45Jenkins G.M. Richards A. Wahl T. Mao C. Obeid L. Hannun Y.A. J. Biol. Chem. 1997; 272: 32566-32572Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 46Mandala S.M. Thornton R., Tu, Z. Kurtz M.B. Nickels J.T., Jr. Broach J. Menzeleev R. Spiegel S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 150-155Crossref PubMed Scopus (235) Google Scholar, 47Mandala S.M. Thornton R. Galve-Roperh I. Poulton S. Peterson C. Olivera A. Bergstrom J. Kurtz M.B. Spiegel S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7859-7864Crossref PubMed Scopus (174) Google Scholar). To begin to examine whether LCB and/or LCBP may accumulate in erg26-1 cells and contribute to the observed growth defect, we took a genetic approach and first determined how altering the gene dosage of individual hydroxylase genes affectederg26-1 viability. In particular, we were interested in determining how the increased gene dosage of the SUR2 gene required for the biosynthesis of the LCB phytosphingosine affectederg26-1 viability (23Haak D. Gable K. Beeler T. Dunn T. J. Biol. Chem. 1997; 272: 29704-29710Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 24Grilley M.M. Stock S.D. Dickson R.C. Lester R.L. Takemoto J.Y. J. Biol. Chem. 1998; 273: 11062-11068Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Wild type and erg26-1 cells carrying individual high copy plasmids overexpressing SUR2, SCS7, orCCC2 from the constitutive GPD promoter were examined for their ability to grow at the permissive and restrictive growth temperatures. SUR2 and SCS7 encode for sphingolipid hydroxylases (23Haak D. Gable K. Beeler T. Dunn T. J. Biol. Chem. 1997; 272: 29704-29710Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 24Grilley M.M. Stock S.D. Dickson R.C. Lester R.L. Takemoto J.Y. J. Biol. Chem. 1998; 273: 11062-11068Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), whereas CCC2 encodes for a copper transporter that is required for Cu2+ uptake (Fig.1) (42Fu D. Beeler T.J. Dunn T.M. Yeast. 1995; 11: 283-29" @default.
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