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Originally published In Press as doi:10.1074/jbc.M308696200 on August 22, 2003

J. Biol. Chem., Vol. 278, Issue 44, 43051-43059, October 31, 2003
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Regulation of S-Adenosylmethionine Levels in Saccharomyces cerevisiae*

Sherwin Y. Chan{ddagger} and Dean R. Appling§

From the Department of Chemistry and Biochemistry, The Institute for Cellular and Molecular Biology and The Biochemical Institute, The University of Texas, Austin, Texas 78712

Received for publication, August 6, 2003 , and in revised form, August 22, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, used to methylate homocysteine in methionine biosynthesis. Methionine can be activated by ATP to give rise to the universal methyl donor, S-adenosylmethionine (AdoMet). Previously, a chimeric MTHFR (Chimera-1) comprised of the yeast Met13p N-terminal catalytic domain and the Arabidopsis thaliana MTHFR (AtMTHFR-1) C-terminal regulatory domain was constructed (Roje, S., Chan, S. Y., Kaplan, F., Raymond, R. K., Horne, D. W., Appling, D. R., and Hanson, A. D. (2002) J. Biol. Chem. 277, 4056–4061). Engineered yeast (SCY4) expressing Chimera-1 accumulated more than 100-fold more AdoMet and 7-fold more methionine than the wild type. Surprisingly, SCY4 showed no appreciable growth defect. The ability of yeast to hyperaccumulate AdoMet was investigated by studying the intracellular compartmentation of AdoMet as well as the mode of hyperaccumulation. Previous studies have established that AdoMet is distributed between the cytosol and the vacuole. A strain expressing Chimera-1 and lacking either vacuoles (vps33 mutant) or vacuolar polyphosphate (vtc1 mutant) was not viable when grown under conditions that favored AdoMet hyperaccumulation. The hyperaccumulation of AdoMet was a robust phenomenon when these cells were grown in medium containing glycine and formate but did not occur when these supplements were replaced by serine. The basis of the nutrient-dependent AdoMet hyperaccumulation effect is discussed in relation to homocysteine biosynthesis and sulfur metabolism.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Folate derivatives, which are present in virtually every known organism and cell type, mediate one-carbon (C1) transfers that play essential roles in several key cellular processes. Folate-dependent one-carbon metabolism is necessary for the syntheses of purines, thymidylate, formylmethionyl-tRNA, glycine, serine, and methionine (1, 2). Furthermore, many methylated molecules such as nucleic acids, proteins, and lipids are formed by transmethylation reactions with S-adenosylmethionine. This latter compound is synthesized indirectly by a folate-dependent pathway (Fig. 1) and is second only to ATP as the most abundantly used cofactor in metabolic reactions. In addition to serving as a methyl group donor, the propylamine group of decarboxylated S-adenosylmethionine can be used in the synthesis of spermidine from putrescine; donation of a second propylamine group results in the formation of spermine.



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  		FIG. 1.
Methyl group metabolism in yeast. The re-methylation, transulfuration, and sulfur assimilation pathways are shown. Genes and enzymes catalyzing individual reactions are: 1, SHM1 and SHM2, serine hydroxymethyltransferase (SHMT; EC 2.1.2.1 [EC] ); 2, MET12 and MET13, MTHFR (EC 1.5.1.20 [EC] ); 3, MET6, methionine synthase (EC 2.1.1.14 [EC] ); 4, SAM1 and SAM2, S-adenosylmethionine synthetase (EC 2.5.1.6 [EC] ); 5, SAH1, S-adenosylhomocysteine hydrolase (EC 3.3.1.1 [EC] ); 6, CYS4, CBS (EC 4.2.1.22 [EC] ); 7, CYS3, cystathionine {gamma}-lyase (EC 4.4.1.1 [EC] ); 8, MET14, adenylylsulfate kinase (EC 2.7.1.25 [EC] ); 9, MET17, O-acetylhomoserine (thiol)-lyase (EC 2.5.1.49 [EC] ). X represents any methyl group acceptor.

 

Methylenetetrahydrofolate reductase (MTHFR)1 participates in methionine metabolism as shown in Fig. 1. Saccharomyces cerevisiae expresses two isozymes of MTHFR encoded by the MET12 and MET13 genes (3). The methyl group of CH3-THF is transferred to homocysteine in the synthesis of methionine. Methionine can be incorporated into proteins, but the bulk of it is activated by ATP to generate S-adenosylmethionine (AdoMet). AdoMet is a high energy sulfonium compound that is utilized in the majority of biological methylation reactions (4). The other product of AdoMet-dependent methylation is S-adenosylhomocysteine (AdoHcy), which is rapidly hydrolyzed by AdoHcy hydrolase, encoded by the yeast SAH1 gene (5). Homocysteine regenerated by this reaction can re-enter the methyl cycle or participate in the transulfuration pathway to synthesize cysteine.

Because homocysteine is the central component in methyl group biogenesis, the synthesis and metabolism of this nonprotein amino acid is tightly controlled. Coordinated regulation of the re-methylation and transulfuration pathways is important in determining the intracellular fate of homocysteine. AdoMet plays an important role in the regulation of these two competing pathways by inhibiting MTHFR (6–8). AdoMet also activates cystathionine {beta}-synthase (CBS) in mammalian cells (9), but this regulation is absent in yeast (10, 11). However, AdoMet is required for full cysteine-mediated transcriptional regulation of sulfur assimilation genes (5). Therefore, regulation by AdoMet would potentially prevent the unnecessary use of activated C1 units and limit the buildup of homocysteine. Elevated levels of homocysteine in human plasma or urine are indicative of a deficiency in the re-methylation or transulfuration pathways and can be detrimental to human health. However, it is not clear if homocysteine is directly pathogenic. In fact, recent studies with S. cerevisiae suggest that AdoHcy may be the causative agent (12).

The MTHFR encoded by MET13 has been shown to be NADPH-dependent and inhibited by AdoMet in vivo (8). This physiologically irreversible reaction commits the methyl group carried by THF to methionine synthesis (13–15). Therefore, the regulation of MTHFR is crucial for C1 metabolism in all organisms because the MTHFR reaction has the potential to trap folates in the methylated form (16–18). An excessive accumulation of CH3-THF may be detrimental to the de novo synthesis of nucleic acids that use non-methylated folates as substrates. A metabolically engineered yeast strain (SCY4) expressing an AdoMet-insensitive, chimeric yeast-plant MTHFR (Chimera-1) is able to accumulate greatly elevated pools of methionine and AdoMet (8). Although the folate pool is enlarged, the distribution of folate derivatives in SCY4 is not shifted toward the CH3-THF derivative (8). In the present study it is demonstrated that the hyperaccumulation of AdoMet is detrimental to yeast if the excess AdoMet cannot be sequestered in the vacuole using polyphosphate as a cationic exchanger. Furthermore, it is shown that the transulfuration pathway plays a vital role in regulating AdoMet biosynthesis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—General chemicals of the highest grade commercially available were obtained from Sigma or Fisher. The high performance liquid chromatography (HPLC) of AdoMet and AdoHcy used 1-heptanesulfonic acid from Sigma and Spherisorb® 5-µm ODS2 columns supplied by Waters Corp. (Milford, MA). Quinacrine dihydrochloride was purchased from Acros Organics (Fisher), and acid-washed glass beads were from Sigma. Oligonucleotide primers were synthesized and purified by Integrated DNA Technologies, Inc. (Coralville, IA).

Strains and Growth Conditions—The S. cerevisiae strains used in this study are described in Table I. Rich media for culturing yeast (YPAD) contained 1% Bacto yeast extract, 2% Bacto peptone, adenine hemisulfate (20 mg liter–1), and 2% glucose. Synthetic minimal medium (YMD) contained 0.7% yeast nitrogen base without amino acids (Difco Bacto®) and 2% glucose supplemented with the following where indicated: 375 mg liter–1 L-serine, 30 mg liter–1 L-leucine, 20 mg liter–1L-histidine-HCl, 20 mg liter–1 L-tryptophan, 20 mg liter–1 uracil, 20 mg liter–1 L-methionine, 30 mg liter–1 L-lysine-HCl, 20 mg liter–1 glycine, 250 mg liter–1 formate, 30 mg liter–1 glutathione, reduced form, 30 mg liter–1 L-cysteine-HCl, and 300 mg liter–1 Geneticin® (G418 sulfate). Cultures were grown at 30 °C in a rotary shaker at 200 rpm or if solid media was required, 2% Bacto agar was added. Pre-sporulation medium contained 0.8% Bacto yeast extract, 0.3% Bacto peptone, 10% glucose, and 2% Bacto agar. Minimal sporulation medium contained 1% potassium acetate and 2% Bacto agar. Escherichia coli XL1-Blue (Stratagene®) was used as a host for propagation of plasmids in 2YT culture medium consisting of 1.6% Bacto tryptone, 1% Bacto yeast extract, and 0.5% NaCl supplemented with 50 µg ml–1 ampicillin.


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  		TABLE I
Yeast strains

 

The vacuole mutants DAY4vac, SCY4vac, and SCY4vac SER1 were constructed by mating DAY4 or SCY4 with yeast strain Y15305 [GenBank] (vps33::kanMX4), obtained from the European S. cerevisiae Archives for Functional Analysis (EUROSCARF) deletion collection (Frankfurt, Germany). The resulting diploids were sporulated by incubation on presporulation medium at 30 °C for 2 days followed by transfer to minimal sporulation medium supplemented with leucine and uracil and incubation for 5–7 days at 24 °C. Tetrads were incubated in 200 µl of sterile H2O, 2 µl of 1 M 2-mercaptoethanol, and 3 µl of 10 mg ml–1 lyticase (Sigma) at 30 °C for 6–8 min and dissected using a Zeiss Tetrad microscope system (Carl Zeiss Inc., Thornwood, NY). Desired haploids were selected based on nutritional requirements and PCR analysis.

The polyphosphate mutants DAY4pop and SCY4pop were constructed by gene replacement. Genomic DNA was isolated by the method described by Hoffman (19) from the EUROSCARF strain Y10212 [GenBank] (vtc1::kanMX4). The VTC1 locus of Y10212 [GenBank] was amplified by PCR using Taq DNA polymerase and primers 5'-CCCAGGATGCGCCTATATCG-3' (VTC1-kanMX4-5') and 5'-AGCGCTTGGTTGTACTACGC-3' (VTC1-kanMX4-3'). The amplified 2.7-kb fragment containing the 1.6-kb kanMX4 module (20) was purified using a QIAquick PCR purification kit (Qiagen) and transformed into the DAY4 and SCY4 strains using lithium acetate (21). Polyphosphate mutants were identified after isolating genomic DNA from Geneticin-resistant cells and performing PCR analysis of the VTC1 locus using primers 5'-TTAGCACGTGTCTCGGAGAG-3' (PHM4-5') and 5'-CTCAGCGTACCATTAGGCCC-3' (PHM4-3').

The cystathionine {beta}-synthase mutants were constructed by amplifying the URA3 cassette out of pJR-URA3 (22) using Taq DNA polymerase and the primers 5'-CTATATCCGGCATATGCAGTCCACACGGCATTACCGTTTCAACAGCTATGACCATG-3' (CYS4-URA3–5'; the sequence homologous to CYS4 is in italics) and 5'-ACAGTGACGTTTACAGATAGGACGACCGGATGAGCGACTGGTAAAACGACGGCCAGT-3' (CYS4-URA3-3'; the sequence homologous to CYS4 is in italics). The PCR product was gel-purified using a QIAquick gel extraction kit (Qiagen) and introduced into the uracil auxotrophs DAY4 and SCY4. Deletion of CYS4 was confirmed by isolating genomic DNA from uracil prototrophs and performing PCR analysis of the CYS4 locus using primers 5'-TGGCACGTGATAGTAGTGGC-3' (CYS4-5') and 5'-CGGTGTCAAGAAATGACGGC-3' (CYS4-3') to establish the DAY4cbs and SCY4cbs strains.

Growth Studies—The growth of {Delta}vps33 yeast strains was studied by first growing a 5-ml culture in rich medium for 18 h. Overnight cultures were washed twice with 5 ml of sterile H2O, resuspended in 3 ml of sterile H2O, and used to inoculate 10 ml of synthetic minimal medium supplemented with glycine, formate, leucine, histidine, tryptophan, and uracil at an OD600 = 0.05. Cells were grown at 30 °C in a rotary shaker, and the turbidity of the cultures at 600 nm was measured approximately every 2 h with a Hewlett Packard 8452A diode array spectrophotometer (Agilent Technologies, Palo Alto, CA). Growth rates were calculated using data points in the linear portion of the exponential growth phase.

Visualization of Vacuoles—Yeast cells to be labeled with quinacrine were grown to mid-log growth phase in YPAD medium supplemented with 50 mM NaH2PO4, pH 7.6. Quinacrine was added to 1-ml aliquots of cells to a final concentration of 200 µM. After incubation at 30 °C for 5 min, cells were pelleted by centrifugation (15,000 x g, 30 s) and resuspended in 30 µl of YPAD with 50 mM NaH2PO4, pH 7.6, and 1% low melting temperature agarose. Observation of cells at x100 magnification employed a Zeiss Axioplan microscope equipped with a Universal Arclamp Power Supply XB075 HBO100, narrow band green fluorescent protein filter set (Axio) with excitation at 480 nm and emission at 520 nm with a bandwidth of 10 nm, dichroic mirror at 505 nm, and standard narrow band for eliminating auto-fluorescence. Data were collected as photographs of random fields using a Roper Scientific camera RTE/CCD-1300-Y/HS (Trenton, NJ). Each field was photographed using filters for both Nomarski optics (175-ms exposure time) and epifluorescence (5-s exposure time) and analyzed with MetaMorph Imaging Series software (Universal Imaging Corp., Downingtown, PA).

Determination of AdoMet and AdoHcy Pools—Yeast cells were grown in YMD medium supplemented with leucine, histidine, tryptophan, uracil, and either serine or glycine and formate. Cells were harvested in mid-log growth phase and washed with distilled water. To measure the levels of AdoMet and AdoHcy, 300-mg samples of wet cells were resuspended in 500 µl of sterile H2O and lysed by vortexing with glass beads for 5 x 30 s. Protein from clarified lysates was precipitated with ethanol (final concentration of 70%) at –20 °C for 15 min and removed by centrifugation (13,000 x g, 20 min, 4 °C). The supernatant was evaporated to dryness in vacuo, and the residue was redissolved in sterile H2O and analyzed by isocratic HPLC (23). A Waters Spherisorb® 5-µm ODS2 column (4.6 x 250 mm) was used with a Beckman Coulter® System Gold 126 Solvent Module and 166 Detector (Fullerton, CA) equipped with a Rheodyne 7725i injector (Cotati, CA). The mobile phase, consisting of 50 mM NaH2PO4, 10 mM 1-heptanesulfonic acid, and 20% (v/v) methanol, pH 4.4, was pumped through the column during the 17-min run. This chromatographic program was run at a flow rate of 0.8 ml min–1 at 24 °C after a 20-µl sample injection, and the eluate was monitored at 260 nm.

Folate Determination—Yeast strains were grown, harvested, and washed as described for AdoMet and AdoHcy determination. Pelleted cells were resuspended in twice their wet weight of extraction buffer (50 mM HEPES, 50 mM CHES, 0.2 M 2-mercaptoethanol, and 2% (w/v) sodium ascorbate, pH 7.85). Glass beads were added at 1.5 times the wet weight of the cell pellet. The samples were heated at 100 °C for 10 min and lysed by vortexing for 4 min. After centrifugation (20,000 x g, 30 min, 4 °C), supernatants were decanted, and their volumes were measured and stored at –70 °C until analysis. Folate derivatives were separated by ion-pair reverse phase HPLC and quantified using the Lactobacillus rhamnosus (ATCC 7469) microbiological assay (24, 25).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Growth of SCY4 in Medium Supplemented with Serine Versus Glycine and Formate—A yeast strain (SCY4) was previously engineered in which the coding sequence of Chimera-1 replaced the chromosomal MET13 gene in the wild type DAY4 strain (8). The growth rates of these two ser1 mutant strains in synthetic minimal medium supplemented with serine, leucine, histidine, tryptophan, and uracil were similar (doubling times, 2.5 ± 0.2 h). When glycine and formate replaced serine in YMD medium, the doubling time of SCY4 (11 ± 0.2 h) was nearly twice as long as that of the wild type strain expressing the native yeast MET13 MTHFR (6.5 ± 0.2 h). Concurrent with the decrease in growth rate, SCY4 accumulates >100-fold more AdoMet than DAY4 in minimal medium supplemented with glycine and formate but not in medium with serine (8).

Intracellular Compartmentation of AdoMet—AdoMet is distributed between the cytosol and the vacuole in yeasts, with up to 80% of cellular AdoMet reported in the vacuoles of wild type cells (26–28). The ability of yeast to hyperaccumulate AdoMet was investigated by studying the role of the vacuole in removing excess AdoMet from the metabolically active pool. VPS33, encoding a protein essential in vacuolar morphogenesis and function (29), was deleted in DAY4 and SCY4 to establish the vacuole mutant strains DAY4vac and SCY4vac. No vacuoles were present in the vps33 mutants, as observed by labeling the cells with quinacrine (Fig. 2). Cells treated with quinacrine were viewed with Nomarski and fluorescence optics, and identical frames of the same cells were photographed. In SCY4 cells (Fig. 2, A and B), the fluorescent bodies within the cells indicate the presence of vacuoles (Fig. 2B). In contrast, SCY4vac (Fig. 2, C and D) has no vacuoles, as shown by the lack of fluorescence in these cells (Fig. 2D).



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  		FIG. 2.
Quinacrine labeling of vacuoles as visualized by epifluorescence. SCY4 cells (VPS33) were viewed with Normarski (panel A) and fluorescence (panel B) optical filters. Panels C and D are photographs of SCY4vac cells ({Delta}vps33).

 

The growth of the vacuole mutants was studied by first growing them in rich medium and then switching them to synthetic minimal medium containing glycine and formate (with the other required nutrients of leucine, histidine, tryptophan, and uracil), where they would hyperaccumulate AdoMet. The turbidity of the liquid cultures was followed over 3 days (Fig. 3). DAY4vac grew regularly, displaying a very short lag phase and reaching stationary phase at 30 h (Fig. 3A). On the other hand, SCY4vac divided only a few times before growth ceased (Fig. 3A). Furthermore, when SCY4vac was transferred from minimal medium containing serine to that containing glycine and formate, there was an immediate arrest in growth (Fig. 3B). DAY4vac again grew normally under these conditions.



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  		FIG. 3.
Growth of vacuole mutants. A, the growth of DAY4vac (MET13 {Delta}vps33) and SCY4vac (met13::CHIMERA-1 {Delta}vps33) was studied directly in synthetic minimal medium supplemented with glycine and formate. B, the growth of the same cells was also examined in YMD minimal medium containing glycine and formate after an initial period of 4 h in minimal medium with serine. The switch in medium is indicated by the arrow, where half of the cells were kept in serine-containing medium and the other half was transferred to medium supplemented with glycine and formate.

 

The intracellular concentration of AdoMet in the vacuole mutants was studied by growing the cells initially in medium containing serine to mid-log growth phase (OD600 = 2) before switching them to medium supplemented with glycine and formate. After only one cell division, SCY4vac accumulated 6-fold higher levels of AdoMet (309 ± 28 nmol g–1 wet weight) compared with DAY4vac (54 ± 4 nmol g–1 wet weight).

Sequestration of AdoMet in Vacuoles—Polyphosphate, which is found almost exclusively in the vacuole (30), has been championed to act as a sink for many positively charged molecules, including basic amino acids, divalent cations, and AdoMet (31). VTC1, which is involved in vacuolar polyphosphate accumulation (32), was deleted in DAY4 and SCY4 by gene transplacement (Fig. 4). Polyphosphate synthesis is blocked in vtc1 mutants (32); growth of these mutants was tested on medium containing glycine and formate (Fig. 5). The wild type strain, DAY4, as well as DAY4pop ("pop," signifying cells defective in polyphosphate synthesis) and SCY4 all exhibited growth on medium that permits AdoMet hyperaccumulation. However, SCY4pop, which does not have the polyanion but has vacuoles, is not viable on medium with glycine and formate (Fig. 5).



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  		FIG. 4.
Deletion of VTC1 in DAY4 and SCY4. PCR primers PHM4-5' and PHM4-3' amplify a 930-bp fragment from the wild type VTC1 gene and a 2080-bp product from the vtc1::kanMX4 disrupted gene through incorporation of the 1.6-kb kanMX4 cassette at the VTC1 locus (see "Experimental Procedures"). MW, 1-kb molecular mass ladder. Lane 1, DAY4 (VTC1); lane 2, DAY4pop ({Delta}vtc1); lane 3, SCY4 (VTC1); lane 4, SCY4pop ({Delta}vtc1).

 


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  		FIG. 5.
Growth of {Delta}vtc1 mutants. DAY4 (MET13 VTC1), DAYpop (MET13 {Delta}vtc1), SCY4 (met13::CHIMERA-1 VTC1), and SCY4pop (met13::CHIMERA-1 {Delta}vtc1) were incubated at 30 °C for 3 days on YMD medium supplemented with glycine, formate, leucine, histidine, tryptophan, and uracil.

 

Intracellular Folate Levels in Yeast Strains DAY4 and SCY4 —The pools of 10-CHO-THF, THF, and CH3-THF were determined by HPLC combined with microbiological assay (24, 25) from cells grown in minimal medium supplemented with serine, leucine, histidine, tryptophan, and uracil (Table II). The most abundant folate derivative in both strains was CH3-THF, constituting >85% of the total folate pool. The other coenzymes (10-CHO-THF and THF) each represented <15% of the total folate in both strains (Table II). It is significant that the folate pool expansion in SCY4 over DAY4 in serine-supplemented medium (16.1 nmol g–1 wet weight) can be almost completely accounted for by the CH3-THF derivative alone (Table II).


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  		TABLE II
Folate levels in DAY4 and SCY4 yeast strains grown with serine in medium

Yeast were grown to mid-log growth phase (OD600 = 2.7-2.9) in minimal medium supplemented with serine, leucine, histidine, tryptophan, and uracil. Folate species were determined by HPLC and microbiological assay of column effluents. Values are reported as means of triplicates.

 

Nutrient-dependent Accumulation of AdoMet—The ability of yeast to hyperaccumulate AdoMet in selective medium was studied by testing the viability of SCY4vac on different media involving combinations of serine, glycine, and formate. Because AdoMet hyperaccumulation restricts the growth of SCY4vac, this represents a simple assay to test for elevated AdoMet content. Of the nine different media examined, two do not permit the growth of SCY4vac, namely media containing glycine and formate (as described above) and formate alone (Table III). Serine or high levels of glycine (100 mg ml–1) can rescue SCY4vac on these two media. Furthermore, a SER1 wild type version of SCY4vac (SCY4vac SER1) was viable and did not hyperaccumulate AdoMet when grown in medium with glycine plus formate (78 ± 2 nmol g–1 wet weight). In medium containing serine, SCY4vac SER1 had 21 ± 1 nmol of AdoMet g–1 wet weight. This suggests that serine may prevent overproduction of AdoMet by regulating the reductive flux of formate through the trifunctional C1-THF synthase ADE3 gene product. Alternatively, serine may deplete homocysteine from the methyl cycle by driving the cystathionine {beta}-synthase reaction toward the synthesis of cystathionine (Fig. 1).


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  		TABLE III
Growth of SCY4vac on different media

SCY4vac (met13::CHIMERA-1 {Delta}vps33) was streaked on nine different media involving combinations of serine, glycine, and formate with leucine, histidine, tryptophan, and uracil. The growth of SCY4vac was scored after incubation for 5 days at 30 °C.

 

Deletion of CYS4 —To assess the role of the transulfuration pathway in alleviating AdoMet hyperaccumulation, the CYS4 gene encoding cystathionine {beta}-synthase was deleted in strains expressing MTHFRs encoded by MET13 and CHIMERA-1 (Fig. 6). Disruption of CYS4 was accomplished by integration of the URA3 deletion cassette, comprising of the S. cerevisiae URA3 marker gene and terminal flanking sequences that are homologous to the 5'- and 3'-untranslated regions of the CYS4 gene. Because the transulfuration pathway represents the only route for cysteine biosynthesis in yeast (33, 34), cys4 mutants are auxotrophic for cysteine, and this growth requirement can be met with either cysteine or glutathione (GSH). Because GSH is more efficiently transported into cells (5), the cys4 mutants employed in the present study were supplemented with GSH. AdoMet levels were examined in the CBS mutants DAY4cbs and SCY4cbs grown with either serine or glycine and formate (Fig. 7). All of the homocysteine in these cells is forced to participate in the methyl cycle because deletion of CBS eliminates the only other possible fate for homocysteine. Therefore, it was surprising that deletion of the CYS4 gene did not cause AdoMet hyperaccumulation. In fact, SCY4cbs accumulated only slightly more AdoMet than DAY4cbs in medium with glycine plus formate (Fig. 7).



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  		FIG. 6.
Deletion of CYS4 in DAY4 and SCY4. PCR primers CYS4-5' and CYS4-3' amplify a 2460-bp fragment from the wild type CYS4 gene. A fragment of 1570 bp is produced from amplification of the cys4::URA3 disrupted gene through incorporation of the 1.2-kb URA3 module at the CYS4 locus (see "Experimental Procedures"). MW, 1-kb molecular mass ladder. Lane 1, DAY4 (CYS4); lane 2, DAY4cbs ({Delta}cys4); lane 3, SCY4 (CYS4); lane 4, SCY4cbs ({Delta}cys4).

 


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  		FIG. 7.
AdoMet levels in {Delta}cys4 mutants. Intracellular levels of AdoMet in yeast strains DAY4cbs (MET13 {Delta}cys4) and SCY4cbs (met13::CHIMERA-1 {Delta}cys4) were determined after cells were grown to mid-log growth phase (OD600 = 2.7–2.9) in synthetic minimal medium supplemented with leucine, histidine, tryptophan, glutathione, and either serine or glycine and formate. Data are expressed as means of three separate determinations ± S.E.

 

Regulation of AdoMet Production by Cysteine—It was recently reported that cysteine is required to repress the transcription of two key enzymes in the sulfur assimilation pathway (5). Adenylylsulfate kinase encoded by MET14 and O-acetylhomoserine (thiol)-lyase encoded by MET17 are both negatively affected by cysteine. Because DAY4cbs and SCY4cbs were provided with glutathione in the media, they have a readily available source of cysteine. The enzymes encoded by MET14 and MET17 are involved in the de novo synthesis of homocysteine (Fig. 1). Therefore, cysteine may in turn limit the levels of homocysteine available to accept the methyl group from CH3-THF and, thus, control the extent of AdoMet accumulation. Following this hypothesis, the amount of AdoMet was quantified in the CBS wild type strains DAY4 and SCY4, grown in medium with GSH (Fig. 8A). DAY4 grown in media with serine, glycine, and formate, or glycine, formate, and GSH produced very low levels of AdoMet. Similarly, SCY4 grown with serine has limited amounts of AdoMet but hyperaccumulates AdoMet in medium supplemented with glycine and formate. The addition of 0.1 mM glutathione to YMD medium containing glycine and formate brought the level of AdoMet in SCY4 down to the level seen in DAY4 (Fig. 8A). Additionally, the growth of SCY4vac on medium containing glycine and formate can be rescued by the addition of glutathione or cysteine (Fig. 9). This is very strong evidence to suggest that serine is used by SCY4 to produce cysteine via transulfuration, which in turn limits the amount of homocysteine available to participate in AdoMet synthesis. Therefore, the repressing effect of serine is 2-fold; serine can deplete the homocysteine pool by condensing with homocysteine to form cystathionine in addition to acting through cysteine to inhibit the biosynthesis of homocysteine.



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  		FIG. 8.
AdoMet and AdoHcy content in DAY4 and SCY4 grown with glutathione. The amount of AdoMet (A) and AdoHcy (B) was determined in DAY4 (MET13) and SCY4 (met13::CHIMERA-1) grown to mid-log growth phase (OD600 = 2.7–2.9) in YMD medium supplemented with leucine, histidine, tryptophan, uracil, and either serine, glycine and formate, or glycine, formate, and GSH. The ratio of AdoMet/AdoHcy was calculated from values obtained from the same sets of experiments (C). Data are reported as means ± S.E., n = 3.

 


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  		FIG. 9.
Rescue of SCY4vac growth by cysteine. SCY4vac (met13::CHIMERA-1 {Delta}vps33) is not viable on synthetic minimal medium supplemented with glycine, formate, leucine, histidine, tryptophan, and uracil (1). However, the addition of serine (2), glutathione (3), or cysteine (4) prevented AdoMet hyperaccumulation and permitted growth of this strain; visible colonies were observed after incubation at 30 °C for 4 days.

 

AdoMet as a Cytotoxic Agent—The cytotoxic agent involved in human diseases related to high levels of homocysteine remains controversial (35). It has been proposed that compounds such as homocysteine itself (36), homocysteine thiolactone (37), and S-adenosylhomocysteine (12, 38) are independent risk factors for vascular disease. AdoHcy is a potent inhibitor of most AdoMet-dependent methyltransferases, and thus, it was of interest to determine the AdoHcy content in DAY4 and SCY4. This was performed in parallel with AdoMet analysis through HPLC separation and detection at 260 nm (see "Experimental Procedures"). The AdoHcy levels in the two strains grown with three different media did not vary greatly (Fig. 8B). However, there was a 3-fold higher level of AdoHcy in SCY4 as compared with DAY4 when grown in medium containing glycine and formate. Along with the increases in AdoMet and AdoHcy there was a concomitant 29-fold increase in the AdoMet/AdoHcy ratio in SCY4 grown with glycine and formate compared with the isogenic wild type strain (Fig. 8C).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Yeast expressing a chimeric enzyme, Chimera-1, which is comprised of the Met13p N-terminal domain and the At-MTHFR-1 C-terminal domain, hyperaccumulates <100-fold more AdoMet than an isogenic wild type strain with no ill effect on growth (8). This was surprising given that the expression of most genes governing sulfur metabolism is severely repressed in response to an increase in intracellular AdoMet (39). The present study demonstrates that most of the excess AdoMet is sequestered in the vacuole and, therefore, excluded from the metabolically active cytosolic pool, as occurs when AdoMet is overproduced in yeast cultured with a high level of methionine in the medium (40). When VPS33, encoding for a non-essential Class C vacuolar sorting protein, was deleted in SCY4, this strain had no vacuoles and was not viable under conditions that allow for the accumulation of AdoMet. That S. cerevisiae possesses two AdoMet synthetases has led to the proposal that each isozyme is responsible for one of the two separate AdoMet pools; the SAM1 enzyme is thought to catalyze the formation of the "labile" pool in the cytosol, whereas the SAM2 enzyme provides the "stable" vacuolar pool (41). To corroborate the proposed model it may prove useful to determine the viability of SCY4vac on medium with glycine and formate if SAM2 is also disrupted.

The vacuolar compartmentation of AdoMet required polyphosphate molecules as deletion of VTC1 prevented growth of SCY4 on medium supplemented with glycine and formate. Polyphosphate is found at concentrations of ~120 mM in S. cerevisiae, which accounts for ~40% of total phosphate (30) and 10–20% of the cellular dry weight (31). Approximately 99% of the polyanion is found in the vacuole (30), where it may act as a counterion to maintain a concentration gradient of AdoMet across the tonoplast. Therefore, yeast are able to tolerate high levels of AdoMet if polyphosphate is present to electrostatically interact with AdoMet in the vacuole lumen. The possibility that increased levels of polyphosphate might be able to rescue SCY4vac on medium with glycine and formate is intriguing. However, an initial screen of a yeast multicopy library in SCY4vac did not reveal any overexpression suppressors of AdoMet hyperaccumulation or permit AdoMet accumulation on medium supplemented with glycine and formate.

The transport of AdoMet in spheroplasts has been documented (42–44); however, information regarding the transport of AdoMet in vacuoles is lacking. Sam3p was discovered to function as a high affinity AdoMet permease that mediates transport of the amino acid across the plasma membrane (44). AdoMet is also transported into yeast cells by a low affinity transport system that appears to be carrier-mediated facilitated diffusion (44). Although the transport of several amino acids across the vacuolar membrane has been described (45), the vacuolar transporter responsible for AdoMet uptake remains to be elucidated. Potential candidates include proteins encoded by AVT2, AVT5, AVT7, and YER053C. Sequence analysis predicts that the three Avt proteins are related to the synaptic vesicular transporters found in Caenorhabditis elegans, rat, and mouse; the protein encoded by YER053C is a member of the mitochondrial carrier family. The latter two proteins represent more promising candidates because they have been shown to localize to the vacuole (45, 46).

Although AdoMet hyperaccumulation was a robust phenomenon in strain SCY4 grown in medium with glycine and formate, it did not occur when these supplements were replaced by serine. This was unpredicted because less CH2-THF is available to MTHFR in cells supplemented with glycine (47). Presumably, excess glycine is used in a side reaction of serine hydroxymethyltransferase to hydrolyze CH+-THF to 5-CHO-THF. Because 5-CHO-THF is able to inhibit the activities of CH2-THF dehydrogenase and CH+-THF cyclohydrolase (48), this would curtail flux through the ADE3 gene product, leaving 10-CHO-THF available for purine biosynthesis. Additionally, 5-CHO-THF together with glycine binds and inhibits serine hydroxymethyltransferase, thereby limiting the amount of CH2-THF synthesized from serine. Therefore, the proposed model (47) predicts that less CH2-THF would be available for AdoMet biosynthesis in glycine-grown cells. This is in contrast to the results of the current study where AdoMet hyperaccumulation occurred in those cells grown with glycine and formate but not serine (Table III). However, 5x concentrations of glycine (1.33 mM) prevented overaccumulation of AdoMet. These data suggest that serine is responsible for controlling AdoMet synthesis for two reasons. First, glycine at 5x concentrations produces serine. Glycine at high concentrations can provide both the two-carbon unit and the CH2-THF (via the glycine cleavage system) required for the synthesis of serine via serine hydroxymethyltransferase (49, 50). Moreover, serine itself was able to repress AdoMet hyperaccumulation (Table III).

Through its involvement in the transulfuration pathway, serine may limit the amount of homocysteine available to the methyl cycle (Fig. 1). Serine can condense with homocysteine in a reaction catalyzed by cystathionine {beta}-synthase. Homocysteine stands at the intersection of two competing pathways; the re-methylation and transulfuration pathways consume roughly equal amounts of homocysteine (9, 51, 52). However, when the CYS4 gene was deleted in SCY4, the strain failed to hyperaccumulate AdoMet in medium with serine as well as in medium with glycine and formate. This was shown to be due to glutathione, which was added to the medium to support the nutritional requirement for cysteine caused by the CYS4 disruption. The hydrolysis of glutathione produces cysteine, and Hansen and Johannesen (5) previously showed that cysteine represses the synthesis of homocysteine. Consequently, SCY4 could not hyperaccumulate AdoMet in medium containing GSH. In the same way, GSH and cysteine attenuated the detrimental effect of AdoMet on growth of SCY4vac. This is supported by Ono et al. (53), who showed that Met17p activity was repressed by ~2-fold when cells were incubated in minimal medium with 0.1 mM glutathione or cysteine for 24 h. It should be noted that the transcription of neither MET14 nor MET17 was found to be repressed in cells exposed to glutathione at concentrations of 2 and 10 mM for 2 h, although no results were shown (5). However, a longer time study may be required to show down-regulation of these genes involved in the de novo biosynthesis of homocysteine. Therefore, the ability of serine to inhibit AdoMet synthesis is multi-faceted. Serine can deplete the homocysteine pool by condensing with homocysteine to form cystathionine and, hence, limit the amount of methionine and AdoMet synthesized. In addition, through its role in the synthesis of cysteine, serine acts to repress the biosynthesis of homocysteine. The ability of serine to regulate sulfur metabolism has also been observed in higher organisms where the embryotoxicity of homocysteine in GD10 rat embryos could be attenuated by serine (38). This suggests that the regulatory role of serine is universal and represents a very elegant backup by which wild type cells can regulate AdoMet production after the "primary" feedback inhibition of MTHFR by AdoMet.

It is also possible that serine may regulate the reductive flux of formate through the ADE3 gene product and in fact be working in concert with the transulfuration pathway to regulate AdoMet production. However, two principle lines of evidence suggest that serine does not regulate Ade3p. First, if SCY4 is grown in medium containing serine, limited amounts of homocysteine are expected to be available to accept the methyl group from CH3-THF. This is a result of the regulation of homocysteine biosynthesis by cysteine. Because Chimera-1 is not feedback inhibited by AdoMet, it can potentially reduce all available CH2-THF. Therefore, the CH3-THF pool would be expected to increase if Chimera-1 is irreversible. However, if serine in fact regulates Ade3p, then serine will control the amount of CH2-THF that is produced from formate and in effect regulate the pool of CH3-THF. In the present study, greater than 85% of the total folate pool expansion in SCY4 versus DAY4 with serine-grown cells can be accounted for by the increase in the CH3-THF pool. Therefore, serine does not appear to regulate Ade3p in vivo. Moreover, the CH2-THF dehydrogenase and 10-CHO-THF synthetase activities of Ade3p are not inhibited by serine at levels of up to 3.6 mM.2 Future work in this area should concentrate on studying the ability of serine to regulate the CH+-THF cyclohydrolase activity of Ade3p. It is also possible the serine may regulate the NAD-dependent CH2-THF dehydrogenase encoded by MTD1.

Homocysteine has received much attention as a possible pathogenic compound. However, it is not toxic to yeast, as evidenced by the observation that {Delta}cys4 mutant yeast can accumulate 10-fold higher levels of homocysteine (28 nmol mg–1 protein) than the wild type without negative effects (12). Similarly, AdoHcy has been suggested to be toxic to cells as it is a potent inhibitor of most AdoMet-dependent methyltransferases. In fact, the Km value for AdoMet is often higher than the Ki value for AdoHcy (4). The current study presents evidence to suggest that high levels of AdoMet are also harmful to yeast. In other organisms an increased level of AdoMet has been reported to have deleterious effects. For instance, in rat PC12 cells, AdoMet induces apoptosis by possibly stimulating protein methylation (54). An increase in AdoMet activity also occurs with aging and may contribute to gradual cell death and neurodegenerative disorders (55). In the present study, the wild type strain, DAY4, grown with glycine and formate had 16 nmol of AdoMet g–1 wet weight, whereas up to 1.7 µmol of AdoMet g–1 wet weight was present in SCY4. In previous work, Christopher et al. (12) report that the growth of CBS mutant yeast is inhibited when the AdoMet/AdoHcy ratio is less than 1.5. The AdoHcy content in SCY4 (61 nmol g–1 wet weight) was more than 3-fold higher compared with DAY4 (17 nmol g–1 wet weight) in medium supplemented with glycine and formate. However, even greater amounts of AdoHcy were found in DAY4 (68 nmol g–1 wet weight) and SCY4 (66 nmol g–1 wet weight) grown with serine. These higher AdoHcy levels were not associated with decreased growth rates. This suggests that the 3-fold increase of AdoHcy in SCY4 grown in medium with glycine and formate is benign. Moreover, the increase of AdoHcy in cells grown with glycine and formate was accompanied by a striking 29-fold increase in the AdoMet/AdoHcy ratio in SCY4 over the wild type. Associated with this change was an increase in doubling time of ~2-fold. Therefore, the greater than 100-fold elevation of AdoMet levels in SCY4 is correlated with a dramatically changed AdoMet/AdoHcy ratio and a decreased growth rate. These results imply that elevated levels of AdoMet are detrimental to yeast, particularly when it cannot be sequestered in the vacuole.

This study describes an in depth characterization of the regulation of AdoMet biosynthesis in S. cerevisiae. The complete genetic and biochemical analysis of the methyl cycle in a single eukaryotic organism has and will continue to provide a greater understanding of the regulation of this pathway and the relationship between methionine biosynthesis and other metabolic processes in the cell. Unlike yeast, however, the mammalian CBS is activated by AdoMet (9). This is an important difference because AdoMet is distributed between the cytosol and mitochondria in rat hepatocytes (28). Therefore, it is possible that mammalian cells possess this added regulatory mechanism to maintain homeostasis of AdoMet levels because mammalian mitochondria may not be able to store an excess of AdoMet, as occurs with yeast vacuoles. This possibility warrants further study.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant DK61428. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Present address: Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Nine Cambridge Center, Cambridge, MA 02142-1479. Back

§ To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, The University of Texas at Austin, 1 University Station A5300, Austin, TX 78712-0165. Tel.: 512-471-5842; Fax: 512-471-5849; E-mail: dappling{at}mail.utexas.edu.

1 The abbreviations used are: MTHFR, methylenetetrahydrofolate reductase; AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine; THF, tetrahydrofolate; CH2-THF, 5,10-methylenetetrahydrofolate; CH3-THF, 5-methyltetrahydrofolate; 5-CHO-THF, 5-formyltetrahydrofolate; 10-CHO-THF, 10-formyltetrahydrofolate; CH+-THF, 5,10-methenyltetrahydrofolate; CBS, cystathionine {beta}-synthase; GSH, glutathione, reduced form; HPLC, high performance liquid chromatography; CHES, 2-(cyclohexylamino)ethanesulfonic acid; kb, kilobase; EUROSCARF, European S. cerevisiae Archives for Functional Analysis. Back

2 S. Pike, personal communication. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Arturo De Lozanne (The University of Texas at Austin) for the generous use of the fluorescence microscope.



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 ABSTRACT
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 RESULTS
 DISCUSSION
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