Portal de Periódicos da CAPES

S-Adenosyl-L-homocysteine Hydrolase, Key Enzyme of Methylation Metabolism, Regulates Phosphatidylcholine Synthesis and Triacylglycerol Homeostasis in Yeast

2008; Elsevier BV; Volume: 283; Issue: 35 Linguagem: Inglês

10.1074/jbc.m800830200

1083-351X

Nermina Malanović, Ingo E. Streith, Heimo Wolinski, Gerald N. Rechberger, Sepp D. Kohlwein, Oksana Tehlivets,

Cancer-related gene regulation

In eukaryotes, S-adenosyl-l-homocysteine hydrolase (Sah1) offers a single way for degradation of S-adenosyl-l-homocysteine, a product and potent competitive inhibitor of S-adenosyl-l-methionine (AdoMet)-dependent methyltransferases. De novophosphatidylcholine(PC)synthesisrequiresthreeAdoMet-dependent methylation steps. Here we show that down-regulation of SAH1 expression in yeast leads to accumulation of S-adenosyl-l-homocysteine and decreased de novo PC synthesis in vivo. This decrease is accompanied by an increase in triacylglycerol (TG) levels, demonstrating that Sah1-regulated methylation has a major impact on cellular lipid homeostasis. TG accumulation is also observed in cho2 and opi3 mutants defective in methylation of phosphatidylethanolamine to PC, confirming that PC de novo synthesis and TG synthesis are metabolically coupled through the efficiency of the phospholipid methylation reaction. Indeed, because both types of lipids share phosphatidic acid as a precursor, we find in cells with down-regulated Sah1 activity major alterations in the expression of the INO1 gene as well as in the localization of Opi1, a negative regulatory factor of phospholipid synthesis, which binds and is retained in the endoplasmic reticulum membrane by phosphatidic acid in conjunction with VAMP/synaptobrevin-associated protein, Scs2. The addition of homocysteine, by the reversal of the Sah1-catalyzed reaction, also leads to TG accumulation in yeast, providing an attractive model for the role of homocysteine as a risk factor of atherosclerosis in humans. In eukaryotes, S-adenosyl-l-homocysteine hydrolase (Sah1) offers a single way for degradation of S-adenosyl-l-homocysteine, a product and potent competitive inhibitor of S-adenosyl-l-methionine (AdoMet)-dependent methyltransferases. De novophosphatidylcholine(PC)synthesisrequiresthreeAdoMet-dependent methylation steps. Here we show that down-regulation of SAH1 expression in yeast leads to accumulation of S-adenosyl-l-homocysteine and decreased de novo PC synthesis in vivo. This decrease is accompanied by an increase in triacylglycerol (TG) levels, demonstrating that Sah1-regulated methylation has a major impact on cellular lipid homeostasis. TG accumulation is also observed in cho2 and opi3 mutants defective in methylation of phosphatidylethanolamine to PC, confirming that PC de novo synthesis and TG synthesis are metabolically coupled through the efficiency of the phospholipid methylation reaction. Indeed, because both types of lipids share phosphatidic acid as a precursor, we find in cells with down-regulated Sah1 activity major alterations in the expression of the INO1 gene as well as in the localization of Opi1, a negative regulatory factor of phospholipid synthesis, which binds and is retained in the endoplasmic reticulum membrane by phosphatidic acid in conjunction with VAMP/synaptobrevin-associated protein, Scs2. The addition of homocysteine, by the reversal of the Sah1-catalyzed reaction, also leads to TG accumulation in yeast, providing an attractive model for the role of homocysteine as a risk factor of atherosclerosis in humans. S-Adenosyl-l-homocysteine hydrolase (Sah1, 2The abbreviations used are:Sah1S-adenosyl-l-homocysteine hydrolaseAdoMetS-adenosyl-l-methionineHcyhomocysteineAdoHcyS-adenosyl-l-homocysteinePCphosphatidylcholinePEphosphatidylethanol-amineTGtriacylglycerolChocholineINO1inositol-3-phosphate synthaseCho2phosphatidylethanolamine methyltransferaseOpi3phospholipid methyltransferaseMET25O-acetylhomoserine sulfhydrylaseSTR4cystathionine-β-synthaseERendoplasmic reticulumYPDyeast extract/peptone/dextroseSPEsolid-phase extractionHPLChigh performance liquid chromatographyMSmass spectroscopyGFPgreen fluorescent proteinESIelectrospray ionization. EC 3.3.1.1.) is one of the most highly conserved enzymes from bacteria to mammals (1Mushegian A.R. Garey J.R. Martin J. Liu L.X. Genome Res. 1998; 8: 590-598Crossref PubMed Scopus (145) Google Scholar, 2Tehlivets O. Hasslacher M. Kohlwein S.D. FEBS Lett. 2004; 577: 501-506Crossref PubMed Scopus (28) Google Scholar). It plays a key role in the regulation of transmethylation reactions in all eukaryotic organisms by catalyzing the degradation of S-adenosyl-l-homocysteine (AdoHcy), the potent product inhibitor of AdoMet-dependent methyltransferases (see Fig. 1) (3Wolfe M.S. Borchardt R.T. J. Med. Chem. 1991; 34: 1521-1530Crossref PubMed Scopus (222) Google Scholar, 4Lee H. Kim J.H. Chae Y.J. Ogawa H. Lee M.H. Gerton G.L. Biol. Reprod. 1998; 58: 1437-1444Crossref PubMed Scopus (38) Google Scholar, 5Chiang P.K. Gordon R.K. Tal J. Zeng G.C. Doctor B.P. Pardhasaradhi K. McCann P.P. FASEB J. 1996; 10: 471-480Crossref PubMed Scopus (742) Google Scholar). Targets of AdoMet-dependent methyltransferases include a wide spectrum of cellular compounds, such as DNA (5Chiang P.K. Gordon R.K. Tal J. Zeng G.C. Doctor B.P. Pardhasaradhi K. McCann P.P. FASEB J. 1996; 10: 471-480Crossref PubMed Scopus (742) Google Scholar, 6Lindsay H. Adams R.L. Biochem. J. 1996; 320: 473-478Crossref PubMed Scopus (26) Google Scholar), mRNA (5Chiang P.K. Gordon R.K. Tal J. Zeng G.C. Doctor B.P. Pardhasaradhi K. McCann P.P. FASEB J. 1996; 10: 471-480Crossref PubMed Scopus (742) Google Scholar), histones H3 and H4 (7Strahl B.D. Grant P.A. Briggs S.D. Sun Z.W. Bone J.R. Caldwell J.A. Mollah S. Cook R.G. Shabanowitz J. Hunt D.F. Allis C.D. Mol. Cell. Biol. 2002; 22: 1298-1306Crossref PubMed Scopus (431) Google Scholar, 8Davis C.D. Ross S.A. Nutr. Rev. 2007; 65: 88-94Crossref PubMed Scopus (89) Google Scholar), and other proteins as well as smaller metabolites, including lipids, e.g. phosphatidylethanolamine (9Vance D.E. FEBS Lett. 2006; 580: 5430-5435Crossref PubMed Scopus (3) Google Scholar). Thus, by regulating AdoHcy levels in the cell, Sah1 activity has pleiotropic effects on gene expression, signal transduction, and lipid biosynthesis. In addition, the Sah1-catalyzed reaction is reversible, adding to the regulatory complexity of trans-methylation reactions. Whereas in vitro the equilibrium of the Sah1 reaction is in the biosynthetic direction to form AdoHcy from adenosine and homocysteine (10De La Haba G. Cantoni G.L. J. Biol. Chem. 1959; 234: 603-608Abstract Full Text PDF PubMed Google Scholar), quick metabolization of adenosine to ATP and inosine and homocysteine to methionine and cysteine and subsequently, glutathione, favors the catabolic direction of the reaction in vivo (11Hoffman D.R. Marion D.W. Cornatzer W.E. Duerre J.A. J. Biol. Chem. 1980; 255: 10822-10827Abstract Full Text PDF PubMed Google Scholar). Therefore, in vivo accumulation of homocysteine and/or adenosine favors the bio-synthetic reaction, resulting in AdoHcy accumulation under these conditions (12Isa Y. Mishima T. Tsuge H. Hayakawa T. J. Nutr. Sci. Vitaminol. (Tokyo). 2006; 52: 479-482Crossref PubMed Scopus (10) Google Scholar, 13Moffatt B.A. Stevens Y.Y. Allen M.S. Snider J.D. Pereira L.A. Todorova M.I. Summers P.S. Weretilnyk E.A. Martin-McCaffrey L. Wagner C. Plant Physiol. 2002; 128: 812-821Crossref PubMed Scopus (141) Google Scholar). S-adenosyl-l-homocysteine hydrolase S-adenosyl-l-methionine homocysteine S-adenosyl-l-homocysteine phosphatidylcholine phosphatidylethanol-amine triacylglycerol choline inositol-3-phosphate synthase phosphatidylethanolamine methyltransferase phospholipid methyltransferase O-acetylhomoserine sulfhydrylase cystathionine-β-synthase endoplasmic reticulum yeast extract/peptone/dextrose solid-phase extraction high performance liquid chromatography mass spectroscopy green fluorescent protein electrospray ionization. Many pathological disease states have been related to altered Sah1 function. Hypomethylation of DNA and high homocysteine/AdoHcy levels were shown to be associated with the pathology of cardiovascular diseases in mammals (14Zaina S. Lindholm M.W. Lund G. J. Nutr. 2005; 135: 5-8Crossref PubMed Scopus (118) Google Scholar, 15Dayal S. Bottiglieri T. Arning E. Maeda N. Malinow M.R. Sigmund C.D. Heistad D.D. Faraci F.M. Lentz S.R. Circ. Res. 2001; 88: 1203-1209Crossref PubMed Scopus (198) Google Scholar, 16Castro R. Rivera I. Struys E.A. Jansen E.E. Ravasco P. Camilo M.E. Blom H.J. Jakobs C. Tavares de Almeida I. Clin. Chem. 2003; 49: 1292-1296Crossref PubMed Scopus (347) Google Scholar). Mice deficient in methylene tetrahydrofolate reductase, necessary for homocysteine to methionine remethylation, exhibit hyperhomocysteinemia and decreased methylation capacity along with neuropathology and aortic lipid deposition (17Chen Z. Karaplis A.C. Ackerman S.L. Pogribny I.P. Melnyk S. Lussier-Cacan S. Chen M.F. Pai A. John S.W. Smith R.S. Bottiglieri T. Bagley P. Selhub J. Rudnicki M.A. James S.J. Rozen R. Hum. Mol. Genet. 2001; 10: 433-443Crossref PubMed Scopus (506) Google Scholar). A defect in hepatic phosphatidylethanolamine (PE) to phosphatidylcholine (PC) methylation leads to liver steatosis in mice (18Li Z. Agellon L.B. Allen T.M. Umeda M. Jewell L. Mason A. Vance D.E. Cell Metab. 2006; 3: 321-331Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar) and was also shown to be associated with diabetes in mice and rats (19Pan H.J. Agate D.S. King B.L. Wu M.K. Roderick S.L. Leiter E.H. Cohen D.E. FEBS Lett. 2006; 580: 5953-5958Crossref PubMed Scopus (19) Google Scholar, 20Hartz C.S. Nieman K.M. Jacobs R.L. Vance D.E. Schalinske K.L. J. Nutr. 2006; 136: 3005-3009Crossref PubMed Scopus (20) Google Scholar). Complete loss of Sah1 function in mammals is deleterious for growth and development; deletion of the SAH1 locus is embryonic lethal in mice (21Miller M.W. Duhl D.M. Winkes B.M. Arredondo-Vega F. Saxon P.J. Wolff G.L. Epstein C.J. Hershfield M.S. Barsh G.S. EMBO J. 1994; 13: 1806-1816Crossref PubMed Scopus (99) Google Scholar). However, recently patients deficient in Sah1 and exhibiting only 3-20% of mean control Sah1 activity were identified that displayed severe myopathy and mental retardation (22Buist N.R. Glenn B. Vugrek O. Wagner C. Stabler S. Allen R.H. Pogribny I. Schulze A. Zeisel S.H. Baric I. Mudd S.H. J. Inherited Metab. Dis. 2006; 29: 538-545Crossref PubMed Scopus (56) Google Scholar, 23Baric I. Fumic K. Glenn B. Cuk M. Schulze A. Finkelstein J.D. James S.J. Mejaski-Bosnjak V. Pazanin L. Pogribny I.P. Rados M. Sarnavka V. Scukanec-Spoljar M. Allen R.H. Stabler S. Uzelac L. Vugrek O. Wagner C. Zeisel S. Mudd S.H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4234-4239Crossref PubMed Scopus (178) Google Scholar, 24Baric I. Cuk M. Fumic K. Vugrek O. Allen R.H. Glenn B. Maradin M. Pazanin L. Pogribny I. Rados M. Sarnavka V. Schulze A. Stabler S. Wagner C. Zeisel S.H. Mudd S.H. J. Inherited Metab. Dis. 2005; 28: 885-902Crossref PubMed Scopus (64) Google Scholar). Conflicting results exist as to the essential function of Sah1 in yeast (see below). In addition to AdoHcy catabolism, Sah1 plays an important role both in mammals and yeast in homocysteine production, which is required for cysteine and glutathione synthesis (25Finkelstein J.D. J. Nutr. 2006; 136: 1750-1754Crossref PubMed Google Scholar, 26Mendoza-Cozatl D. Loza-Tavera H. Hernandez-Navarro A. Moreno-Sanchez R. FEMS Microbiol. Rev. 2005; 29: 653-671Crossref PubMed Scopus (341) Google Scholar). Whereas in mammalian cells homocysteine is produced exclusively by Sah1, yeast possesses an additional option to synthesize homocysteine through the sulfur assimilation pathway that allows it to utilize sulfate as a source of sulfur (Fig. 1) (27Hansen J. Johannesen P.F. Mol. Gen. Genet. 2000; 263: 535-542Crossref PubMed Scopus (98) Google Scholar, 28Breton A. Surdin-Kerjan Y. J. Bacteriol. 1977; 132: 224-232Crossref PubMed Google Scholar, 29Thomas D. Surdin-Kerjan Y. Microbiol. Mol. Biol. Rev. 1997; 61: 503-532Crossref PubMed Scopus (537) Google Scholar). In yeast as in mammalian cells the major membrane phospholipid, PC, is synthesized either de novo by sequential methylation of PE by AdoMet-dependent methyltransferases (Cho2 and Opi3 in yeast) or via the Kennedy pathway from diacylglycerol (DG) and CDP-activated choline (9Vance D.E. FEBS Lett. 2006; 580: 5430-5435Crossref PubMed Scopus (3) Google Scholar, 30Vance J.E. Vance D.E. Biochem. Cell Biol. 2004; 82: 113-128Crossref PubMed Scopus (267) Google Scholar, 31de Kroon A.I. Biochim. Biophys. Acta. 2007; 1771: 343-352Crossref PubMed Scopus (81) Google Scholar). Although in yeast PC de novo synthesis is the dominating pathway, in mammals only 30% of total hepatic PC is synthesized by PE methylation de novo (9Vance D.E. FEBS Lett. 2006; 580: 5430-5435Crossref PubMed Scopus (3) Google Scholar). Nevertheless, phospholipid methylation in mammals appears to be a major AdoMet-consuming pathway, since hepatic PC de novo synthesis is responsible for about 50% of plasma homocysteine levels (32Stead L.M. Brosnan J.T. Brosnan M.E. Vance D.E. Jacobs R.L. Am. J. Clin. Nutr. 2006; 83: 5-10Crossref PubMed Scopus (207) Google Scholar). Accordingly, the highest Sah1 activity levels were detected in liver (21Miller M.W. Duhl D.M. Winkes B.M. Arredondo-Vega F. Saxon P.J. Wolff G.L. Epstein C.J. Hershfield M.S. Barsh G.S. EMBO J. 1994; 13: 1806-1816Crossref PubMed Scopus (99) Google Scholar). Previously, we have shown that SAH1 transcription in yeast is regulated in response to the lipid precursors, inositol and choline, and is coordinated with genes involved in phospholipid biosynthesis (2Tehlivets O. Hasslacher M. Kohlwein S.D. FEBS Lett. 2004; 577: 501-506Crossref PubMed Scopus (28) Google Scholar). This transcriptional regulation requires two transcriptional activators Ino2/Ino4 as well as the repressor Opi1 and is dependent on the phospholipid metabolite, phosphatidic acid (PA), in the endoplasmic reticulum (33Loewen 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 (369) Google Scholar). Ino2/Ino4 bind as heterodimeric activation complex to UASINO sequences present in the 5′ regions of all phospholipid biosynthetic genes (34Chen M. Hancock L.C. Lopes J.M. Biochim. Biophys. Acta. 2007; 1771: 310-321Crossref PubMed Scopus (66) Google Scholar, 35Bachhawat N. Ouyang Q. Henry S.A. J. Biol. Chem. 1995; 270: 25087-25095Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). In the absence of lipid precursors, Opi1, which may interact with Ino2 in the nucleus to block transcription, is retained in the endoplasmic reticulum by binding to PA (33Loewen 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 (369) Google Scholar). The presence of inositol leads to rapid consumption of PA pools in the ER and subsequent translocation of Opi1 into the nucleus to repress Ino2/Ino4-dependent transcription (33Loewen 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 (369) Google Scholar, 36Gaspar M.L. Aregullin M.A. Jesch S.A. Nunez L.R. Villa-Garcia M. Henry S.A. Biochim. Biophys. Acta. 2007; 1771: 241-254Crossref PubMed Scopus (60) Google Scholar, 37Jesch S.A. Liu P. Zhao X. Wells M.T. Henry S.A. J. Biol. Chem. 2006; 281: 24070-24083Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Defective PE to PC methylation in cho2 or opi3 mutants leads to PA accumulation and, accordingly, to derepression of phospholipid biosynthetic genes, in particular INO1, which encodes inositol 3-phosphate synthase, the most highly regulated gene in this regulatory circuit (38Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acid Res. Mol. Biol. 1998; 61: 133-179Crossref PubMed Google Scholar, 39Carman G.M. Henry S.A. J. Biol. Chem. 2007; 282: 37293-37297Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Depletion of Sah1 activity in yeast results in massive accumulation of lipid droplets and triacylglycerols during logarithmic growth (2Tehlivets O. Hasslacher M. Kohlwein S.D. FEBS Lett. 2004; 577: 501-506Crossref PubMed Scopus (28) Google Scholar). Here we show for the first time that Sah1 activity controls PC synthesis in yeast and that a decreased PC de novo synthesis both in Sah1-depleted cells and in cho2 and opi3 mutants defective in PE to PC methylation is accompanied by an increase in triacylglycerol levels. Reduction of Sah1 activity leads to deregulated INO1 expression, comparable with that observed in cho2 and opi3 mutants. Furthermore, homocysteine supplementation leads to AdoHcy accumulation through the Sah1-catalyzed reaction in vivo and, subsequently, inhibition of PE methylation and TG accumulation. We also show that the sulfur assimilation pathway in yeast is essential for survival in the absence of Sah1. These data demonstrate that Sah1 is not only important for regulating AdoMet-dependent methyl transfer reactions. It is also essential for providing homocysteine as a precursor for cysteine and glutathione synthesis in the absence of sulfur assimilation, which is lacking in mammalian cells. Taken together, Sah1 activity has a major impact on cellular lipid homeostasis, and its deficiency results in dysregulated lipid metabolism, leading to an imbalance of phospholipid and triacylglycerol synthesis, with implications for mammalian lipid-associated disorders. Strains and Culture Conditions—The strains used in this study, except for the AID strain, are derived from the BX back-ground and are listed in Table 1. The tetO7-SAH1 strain, expressing SAH1 under control of the heterologous tetO7 promoter, was obtained from Open Biosystems. Precultures were grown in YPD complete media containing 10 g/liter yeast extract (Difco), 20 g/liter peptone (Difco), and 20 g/liter glucose (Merck) or in minimal SD media containing 6.7 g/liter yeast nitrogen base without amino acids (Difco), 20 g/liter glucose (Merck) and supplemented with amino acids and bases as described (2Tehlivets O. Hasslacher M. Kohlwein S.D. FEBS Lett. 2004; 577: 501-506Crossref PubMed Scopus (28) Google Scholar). Synthetic, inositol- and choline-free media that also lacked threonine (-I/-C/-threo) contained 10% glucose (40Jesch S.A. Zhao X. Wells M.T. Henry S.A. J. Biol. Chem. 2005; 280: 9106-9118Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Where indicated, methionine (20 μg/ml), cysteine (0.1 mm), inositol (80 μm), choline (1 mm), and homocysteine (1 mm) were added. Deuterated homocysteine (d4-Hcy, CDN Isotopes, Quebec, Canada) was added to the -I/-C/-threo medium at 1 mm. For repression of SAH1 expression under control of the tetO7 promoter, doxycycline was added at concentrations ranging from 0 to 50 μg/ml, as indicated. Media were solidified by the addition of 20 g/liter agar.TABLE 1Strains used in this studyStrainGenotypeSourceYNM1MATa his3Δ1 met25Δ0 leu2Δ0 ura3Δ0 lys2Δ0Laboratory strainYNM8MATa his3Δ1 leu2Δ0 ura3Δ0 lys2Δ0This studyYNM9MATa his3-1 leu2-0 pSAH1::kanR-tet07-TATA URA3::CMV-tTAThis studyYNM2MATa his3-1 leu2-0 met25-0 pSAH1::kanR-tet07-TATA URA3::CMV-tTAOpen BiosystemsYNM3MATa his3Δ1 leu2Δ0 met25Δ0 ura3Δ0 cho2::kanMX4EuroscarfYNM4MATa his3Δ1 leu2Δ0 met25Δ0 ura3Δ0 opi3::kanMX4EuroscarfYNM5MATa his3Δ1 met25Δ0 leu2Δ0 ura3Δ0 lys2Δ0 met6::kanMX4 str4::kanMX4This studyYNM6MATα his3Δ1 leu2Δ0 ura3Δ0 lys2Δ0 met6::kanMX4 str4::kanMX4This studyYNM7MATa/α; his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 lys2Δ0/LYS2 MET25/met25Δ0 ura3Δ0/ura3Δ0 sah 1::kanMX4/SAH1EuroscarfYNM10MATα his3Δ1 leu2Δ0 ura3Δ0 lys2Δ0 pUG36[Opil-GFP]This studyYNM11MATα his3Δ1 leu2Δ0 ura3Δ0 lys2Δ0 sah 1::kanMX4 pUG36[Opil-GFP]This studyAIDMATa/α ade1/ade1 ino1-13/ino1-13S.A. Henry Open table in a new tab Cloning of the MET25 Gene and Repair of the met25 Mutation—The MET25 wild type gene was amplified from BY4742 genomic DNA by PCR using 5′-ACTAATTAAGTTAGTCAAGGCGCCA-3′ and 5′-TCATTACGCACACTCATGGTTTTT-3′ primers and Tag DNA polymerase (Fermentas) following standard procedures. The obtained 2.7-kilobase MET25 fragment was transformed into yeast strains YNM1 and YNM2 (both met25) by the method of Gietz (41Gietz R.D. Schiestl R.H. Willems A.R. Woods R.A. Yeast. 1995; 11: 355-360Crossref PubMed Scopus (1704) Google Scholar), replacing the met25 locus with the MET25 wild type gene. Correct integration at the MET25 locus and methionine prototrophy of the resultant transformants was confirmed by colony PCR and growth tests on media plates lacking methionine, respectively. AdoHcy Quantification—AdoHcy was extracted by the method of Gellekink (42Gellekink H. van Oppenraaij-Emmerzaal D. van Rooij A. Struys E.A. den Heijer M. Blom H.J. Clin. Chem. 2005; 51: 1487-1492Crossref PubMed Scopus (101) Google Scholar) with minor modifications. Yeast cells were grown overnight in 5 ml of YPD at 30 °C with shaking, inoculated in 100 ml of YPD medium to A600 = 0.1, and grown for additional 72 h. Cells were washed with sterile water and inoculated to A600 = 0.025 in -I/-C/-threo medium with or without supplements as indicated. Over a period of 25 h samples were collected, stored at -70 °C, or processed immediately. For AdoHcy extraction 100 A600 units of yeast cells were harvested, resuspended in 5 ml of 0.091 m acetic acid, and broken with glass beads in a Mercken-schlager homogenizer (Braun Bio-tech International) under CO2 cooling. The supernatant was separated from glass beads by centrifugation. 15 nmol of d3-AdoMet (CDN Isotopes) were added as internal standard to every sample before solid-phase extraction (SPE) on Bond Elut PBA columns (Varian Inc.) (42Gellekink H. van Oppenraaij-Emmerzaal D. van Rooij A. Struys E.A. den Heijer M. Blom H.J. Clin. Chem. 2005; 51: 1487-1492Crossref PubMed Scopus (101) Google Scholar). AdoHcy was separated by HPLC (Rheos 2000 pump, Flux Instruments, Basel, Switzerland) using an ACE®3 C18 column (100 × 2.1 mm, Advanced Chromatography Technologies, Aberdeen, UK). The gradient started from 96% solvent A (0.1% formic acid in water) and increased to 100% solvent B (acetonitrile, 0.1% formic acid) over 9 min at a flow rate of 100 μl/min. AdoHcy concentration was measured on an LCQ Duo Ion Trap mass spectrometer (Thermo, San Jose, CA) using MS/MS-Scans for m/z 385 (AdoHcy), 389 (d4-AdoHcy) and 402 (d3-AdoMet) and with a TSQ-7000 Triple Quadrupole mass spectrometer performing the multiple reaction monitoring (MRM) scans for m/z 385→136 (AdoHcy), m/z 389→136 (d4-AdoHcy), and m/z 402→136 (d3-AdoMet). Calibration curves of AdoMet and AdoHcy (Sigma Aldrich) in the range of 1 to 50 μm were used for quantification. Lipid Analysis—Yeast lipids were extracted by the Folch method (43Folch J. Lees M. Sloane Stanley G.H. J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). Briefly, 50 A600 units of yeast cells grown under identical conditions as used for AdoHcy extraction (see above) were harvested and broken with glass beads in a Mercken-schlager homogenizer (Braun Biotech International) under CO2 cooling. Lipids were extracted using chloroform/methanol 2:1 (v/v). Samples were applied onto silica gel TLC plates (Merck) with an automated sampler (Camag Automatic TLC Sampler 4). Total lipids were separated using light petroleum/diethyl ether/acetic acid 32:8:0.4 (v/v/v). Triacylglycerols and total phospholipids were visualized by carbonization and quantified by densitometric scanning as described (44Schneiter R. Daum G. Methods Mol. Biol. 2006; 313: 75-84PubMed Google Scholar) using triolein as the standard. Phospholipid composition was analyzed by normal-phase HPLC using a YMC-Pack™ Diol column (250 × 4.6 mm, 5 μm) on an Agilent 1100 HPLC system (Agilent Technologies, Waldbronn, GER) equipped with an evaporative light scattering detector (PL-ELS 1000, Polymer Labs, Amherst, MA). The gradient was based on a previously reported method (45Sas B. Peys E. Helsen M. J. Chromatogr. A. 1999; 864: 179-182Crossref PubMed Scopus (50) Google Scholar) and changed from acetone to acetone/methanol (46.2/53.8 v/v) in the presence of hexane (7 volume %), acetic acid (1.9%), and triethylamine (1.6%) over 32 min at a flow rate of 0.4 ml/min. Quantification of phospholipids was achieved by external standardization using lipid standards from Avanti Polar Lipids Inc. (Alabaster, AL). RNA Isolation and Real Time PCR—An overnight culture grown in minimal medium was inoculated to A600 = 0.05 in -I/-C/-threo synthetic medium with or without doxycycline supplementation and grown for 6 h at 30°C under shaking. RNA was prepared using SV Total RNA Isolation System (Promega). 1 μg of total RNA was converted into cDNA using Superscript™ reverse transcriptase (Invitrogen). Expression of SAH1 (primers 5′-CCGAAGTTACCTGGTCCTCTTTGA-3′ and 5′-ACCGGAAGCGGCAATAGC-3′ and TagMan probe 5′-CGACTCAAGATCATGCCGCCGC-3′) and INO1 genes (primers 5′-GGAATGACGTTTATGCTCCTTTTAA-3′ and 5′-GTCCCAACCAGAGACGACAAA-3′ and TagMan probe 5′-CTGTTGCCCATGGTTAGCCCAAACG-3′) was measured by real time PCR using Universal PCR Master Mix (Applied Biosystems) and normalized to ACT1 (primers 5′-CGCCTTGGACTTCGAACAAG-3′ and 5′-GACCATCTGGAAGTTCGTAGGATT-3′ and TagMan probe 5′-TGCAAACCGCTGCTCAATCTTCTTCAAT-3′). Calculation of relative expression was according to the suppliers' protocol. Opi Test—The test for Opi phenotype was performed as described previously with minor modifications (46Greenberg M.L. Goldwasser P. Henry S.A. Mol. Gen. Genet. 1982; 186: 157-163Crossref PubMed Scopus (57) Google Scholar, 47McGee T.P. Skinner H.B. Bankaitis V.A. J. Bacteriol. 1994; 176: 6861-6868Crossref PubMed Google Scholar). Briefly, strains were grown overnight in minimal medium, and 5-μl aliquots of A600 = 1 dilutions each were spotted onto -I/-C/-threo plates with or without doxycycline supplementation as indicated. The plates were incubated at 30 °C for 24 h and sprayed with a suspension of an inositol auxotrophic tester strain (AID). Secretion of inositol resulting in a growth ring of the tester strain around the spotted cultures was scored after further incubation for 24-48 h at 30 °C. Microscopic Analysis of Opi1-GFP Localization—Wild type and sah1 mutant cells expressing C-terminal GFP-tagged Opi1 from plasmid pUG36 (48Niedenthal R.K. Riles L. Johnston M. Hegemann J.H. Yeast. 1996; 12: 773-786Crossref PubMed Scopus (366) Google Scholar) were grown overnight in minimal SD media lacking uracil. Before microscopy, cells were inoculated into fresh -I/-C/-ura media containing 2 μg/ml methionine and 1 mm cysteine (see above) and sequentially re-inoculated in 3 cycles (8-12 h each) to obtain a logarithmically growing cell population with homogeneous Opi1-GFP expression. GFP fluorescence was analyzed on a Leica TCS4d confocal microscope with 488-nm excitation and 525/50-nm band pass filter detection. Quantitative image analysis was performed using Image J software (rsb.info.nih.gov/ij). Briefly, GFP fluorescence intensity profiles were calculated along a linear axis through the cell, normalized, and plotted (see also Fig. 6C). Intensity maxima (±S.D.), corresponding to the nuclear rim/ER, were correlated to intensity values inside and outside the nucleus, from at least 20 cells/nuclei in each preparation. Sah1 Is Essential in Yeast in the Absence of Sulfur Assimilation—Previous studies have yielded ambiguous results as to an essential function of Sah1 in yeast. To address the reported discrepancies and to generate a conditional system that allowed systematic studies of the impact of Sah1 on lipid metabolism, we made use of a strain expressing SAH1 under control of the doxycycline-regulatable tetO7 promoter (Fig. 2A). As shown in Fig. 2B, expression of SAH1 in strain YNM2 (tet07SAH1 met25), as determined by real time PCR, was elevated compared with wild type during growth without doxycycline supplementation, consistent with previous results showing that the tetO7 promoter is rather strong in the absence of repressor. However, SAH1 expression was strongly dependent on the doxycycline concentration and was completely abolished in the presence of 2 μg/ml doxycycline in the medium. This concentration was also sufficient to fully inhibit growth of strain YNM2 (Fig. 2C). The addition of cysteine or homocysteine only marginally restored growth of this strain in the presence of 2 μg/ml doxycycline; however, it indicated to us that the essential function of Sah1 may be related to methionine or cysteine homeostasis. Indeed, MATa strains of the Euroscarf collection (and strains from the tetO7 promoter replacement collection) contain a mutation in the MET25 gene, encoding O-acetylhomoserine sulfhydrylase, which renders the sulfur assimilation pathway inactive (49Thomas D. Barbey R. Surdin-Kerjan Y. J. Biol. Chem. 1990; 265: 15518-15524Abstract Full Text PDF PubMed Google Scholar). Because in yeast sulfur assimilation is an integral part of homocysteine and methionine homeostasis, we have repaired the met25 deletion in strain YNM2 by replacing the mutated allele with the MET25 wild type gene. Indeed, the addition of doxycycline to the medium did not affect the growth of strain YNM9 (tet07SAH1 MET25), demonstrating that Sah1 is only essential in the absence of Met25. To analyze this further, we have subjected strain YNM7 heterozygous for sah1 and met25 mutations (sah1/SAH1 met25/MET25) to tetrad analysis (Fig. 2D). On methionine-containing plates, the resulting progeny developed either big (MET25 or met25), medium size (sah1 MET25), or very small (sah1 met25) colonies. Supplementation of either homocysteine or cysteine in combination with methionine, but not methionine alone, supported slow growth of sah1 or sah1 met25 deletion strains (Fig. 2D). Taken together, these data demonstrate that (i) Sah1 is not an essential enzyme and is only indispensable for the yeast cell in the absence of sulfur assimilation and (ii) elimination of Sah1 function results in strongly reduced growth that cannot be completely rescued by amino acid supplementation, as a consequence of lacking AdoHcy catabolism. Because sulfur assimilation is a