Saccharomyces cerevisiae Expresses Two Genes Encoding Isozymes of Methylenetetrahydrofolate Reductase

Archives of Biochemistry and Biophysics Vol. 372, No. 2, December 15, pp. 300 –308, 1999 Article ID abbi.1999.1498, available online at http://www.idealibrary.com on

Saccharomyces cerevisiae Expresses Two Genes Encoding Isozymes of Methylenetetrahydrofolate Reductase 1 Rhonda K. Raymond, Evdokia K. Kastanos, 2 and Dean R. Appling 3 Department of Chemistry and Biochemistry, The Institute for Cellular and Molecular Biology, and The Biochemical Institute, University of Texas at Austin, Austin, Texas 78712

Received July 1, 1999, and in revised form September 2, 1999

The identification, expression, and assay of two Saccharomyces cerevisiae genes encoding methylenetetrahydrofolate reductases (MTHFR) is described. MTHFR catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, used to methylate homocysteine in methionine synthesis. The MET12 gene is located on chromosome XVI and encodes a protein of 657 amino acids. The MET13 gene is located on chromosome VII and encodes a protein of 599 amino acids. The deduced amino acid sequences of these two genes are 34% identical to each other and 32–37% identical to the human MTHFR. A phenotype for the single disruption of MET12 was not observed, however, single disruption of MET13 resulted in methionine auxotrophy. Double disruption of both MET12 and MET13 also resulted in methionine auxotrophy. Growth of the methionine auxotrophs was supported by both methionine and S-adenosylmethionine. Transcripts of both MET12 and MET13 were detected in total RNA from wild type cells grown in the presence or absence of methionine. The methionine requirement of the met12 met13 double disruptant was complemented by plasmid-borne MET13, but not MET12 even when a multicopy plasmid was used. Furthermore, overexpression of the human MTHFR in the met12 met13 double disruptant complemented the methionine auxotrophy of this strain. In contrast, overexpression of the Escherichia coli metF gene did not complement the methionine requirement of met12 met13 cells. Assays for MTHFR in crude extracts and expression of the yeast proteins in Escherichia coli verified that both MET12 and MET13 encode functional MTHFR isozymes. © 1999 Academic Press 1

This work was supported by NIH Grant RR09276. Present address: National Naval Medical Center, NCI-Navy Medicine Branch, Bethesda, MD. 3 To whom correspondence and reprint requests should be addressed: Fax: (512) 471-5849. E-mail: [email protected]. 2

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Key Words: Saccharomyces cerevisiae; methylenetetrahydrofolate reductase; isozymes; homocysteine.

Methylenetetrahydrofolate reductase (MTHFR) 4 catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH 2-THF) to 5-methyltetrahydrofolate (CH 3-THF). This reaction commits the methyl group carried by tetrahydrofolate to the synthesis of methionine, via methylation of homocysteine, and ultimately S-adenosylmethionine (Fig. 1). Thus, MTHFR is critical in maintaining low cellular homocysteine levels. MTHFR deficiency is the most common inborn error of folate metabolism in humans, resulting in hyperhomocysteinemia, homocystinuria, and hypomethionemia (1). More recently, elevated homocysteine due to MTHFR deficiency has been implicated as a risk factor for vascular disease (2) and neural tube defects (3). Methylenetetrahydrofolate reductases are flavoproteins that can be reduced by either NADPH, as with the mammalian enzymes, or other endogenous cofactors, as with the bacterial enzymes (4). MTHFR activity has been detected in several organisms, including bacteria, parasites, and mammals (5). The pig liver, human liver, and Escherichia coli enzymes have been purified and characterized (6 – 8). A cDNA encoding the human MTHFR has been isolated (9) and expressed in E. coli (2). Isolation and sequencing of the human MTHFR cDNA led to the discovery of a previously unidentified ORF in Saccharomyces cerevisiae that exhibits significant homology to other methylenetetrahydrofolate reductases. As additional yeast genome se4

Abbreviations used: MTHFR, methylenetetrahydrofolate reductase; CH 2-THF, 5,10-methylenetetrahydrofolate; CH 3-THF, 5-methyltetrahydrofolate; Met12p, the protein encoded by MET12; Met13p, the protein encoded by MET13; SGD, Saccharomyces Genome Database at Stanford University; ORF, open reading frame. 0003-9861/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

YEAST METHYLENETETRAHYDROFOLATE REDUCTASE ISOZYMES

FIG. 1. Metabolic scheme for the synthesis and interconversion of methyl groups. Reaction 1 is catalyzed by serine hydroxymethyltransferase (EC 6.3.4.3). Reaction 2 is catalyzed by MTHFR (EC 1.5.1.20). Reaction 3 is catalyzed by methionine synthase (EC 2.1.1.14). Reaction 4 is catalyzed by S-adenosylhomocysteine hydrolase (EC 3.3.1.1). Reaction 5 is catalyzed by cystathionine b-synthase (EC 4.2.1.22). Reaction 6 is catalyzed by cystathionine g-lyase (EC 4.4.1.1). X represents any methylacceptor substrate.

quence became available, a second putative yeast MTHFR was identified that also exhibits significant homology to other methylenetetrahydrofolate reductases. Here we report the cloning, disruption, expression, and assay of two methylenetetrahydrofolate reductases in yeast. Our results indicate that these two genes, designated MET12 and MET13, encode MTHFR isozymes and that the isozyme encoded by MET13 is responsible for most of the activity in the cell. MATERIALS AND METHODS Materials. Common reagents were high-grade commercial products. DIFCO growth media were purchased from VWR (Morrisville, NC). S-adenosylmethionine was purchased from Sigma (St. Louis, MO). Restriction enzymes were purchased from Life Technologies (Grand Island, NY). Oligonucleotide primers were synthesized and purified by Integrated DNA Technologies (Coralville, IA). Taq polymerase was purchased from PE Applied Biosystems (Foster City, CA). Radiolabeled nucleotide [a- 32P]dCTP was purchased from NEN Life Science Products (Boston, MA). MTHFR assay components bovine serum albumin, FAD, menadione, and dimedone were purchased from Sigma (St. Louis, MO). Unlabeled (6R,S)CH 3-tetrahydrofolate was purchased from Schircks Laboratories (Jona, Switzerland) and (6R,S)[ 14CH 3]-tetrahydrofolate was purchased from Amersham Life Science (Cleveland, OH). Strains and growth conditions. The parental S. cerevisiae strain used in this paper was DAY4 (a ura3-52 trp1 leu2 his4 ser1) (10). An isogenic DAY4 diploid was constructed by transforming DAY4 haploids to uracil prototrophy with a plasmid containing the HO endonuclease (11) and allowing the transformants to mate with each other for 6 h at 25°C. Putative diploids were then streaked onto 5-fluoroorotic acid plates (12) in order to select for loss of the HO plasmid. Positive colonies were screened for the proper nutritional phenotype and then tested against a and a haploid strains to verify that they were diploids and could no longer mate. The resulting isogenic diploid strain, DAY4a/a, was used for disruptions of the MET12 and MET13 genes. Disruption of the two putative yeast MTHFR genes in DAY4a/a followed by sporulation and tetrad dissection (described below), resulted in the three haploid strains

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RRY1(a ura3-52 trp1 leu2 his4 ser1 Dmet13), RRY2 (a ura3-52 trp1 leu2 his4 ser1 Dmet12), and RRY3 (a ura3-52 trp1 leu2 his4 ser1 Dmet13 Dmet12). Rich medium (YPD) contained 1% yeast extract, 2% Bactopeptone, and 2% glucose. Nonfermentable media (YPGE) contained 1% yeast extract, 2% Bactopeptone, 2% glycerol, and 2% ethanol. Synthetic minimal medium (YMD) contained 0.7% yeast nitrogen base without amino acids, 2% glucose, and was supplemented with the following nutrients where indicated (final concentration in mg/liter): L-serine (375), L-leucine (30), L-histidine (20), L-tryptophan (20), L-methionine (20), uracil (20), and S-adenosylmethionine (1 mM). Acetate media (YPA) contained 1% yeast extract, 2% bactopeptone, and 1% potassium acetate. Sporulation media (SPM) contained 1% potassium acetate and 0.02% raffinose. Bacterial culture medium (2YT) contained 1.6% tryptone, 1% yeast extract, and 0.5% NaCl. Escherichia coli XL1-Blue (Stratagene) was used as host for plasmid manipulations, and strain BL21(DE3) (Stratagene) was used as host for MTHFR expression. Gene isolation. The clone pNF1002 was obtained from Dr. Errol C. Friedberg (University of Texas Southwestern Medical School, Dallas, TX) and a 2.7-kbp SstI fragment containing the MET12 gene was subcloned into the SstI site of pBluescript II (KS 1) (Stratagene). Subsequently, a 2.4-kbp HindIII–SstI fragment from the pBluescript clone was subcloned into the HindIII–SstI sites of the yeast expression vector pVT101-U (13). To obtain the MET13 clone, two oligonucleotide primers, 59-TCAGCTGCAGCATCGCCATATGAAGATCACAG-39 (MTR2.5; PstI site is underlined) and 59-TTTAACTTGTGACAGGGAGG-39 (MTR2.3), were used in the polymerase chain reaction (PCR) to amplify the MET13 gene from DAY4 yeast genomic DNA that was isolated as described by Sherman et al. (14). The amplified MET13 gene was digested with the restriction enzymes PstI and HpaI and cloned into pUC19 between the PstI and SmaI sites (Life Technologies). The MET13 fragment from pUC19 was then subcloned into the yeast expression vector pVT101-U (13) between the PstI and SstI sites. Additionally, the two genes MET12 and MET13 were cloned into the plasmid pET16b (Novagen) for expression in E. coli. The MET12 gene was PCR amplified from DAY4 yeast genomic DNA using Pfu DNA polymerase (Stratagene) and the two primers 59-CGTGCACTCGAGTTAAACATGTCCATCAGA-39 (FMET12-1; XhoI site is underlined) and 59-TTAGTACTAGAGCTCAGCGGGTCTAGGTTCG-39 (RMET12-1; Bpu1102I site is underlined). Similarly, the MET13 gene was amplified from DAY4 genomic DNA using the two primers 59-TCAGCTGCAGCATCGCCATATGAAGATCACAG-39 (MTR2.5; NdeI site is underlined) and 59-TCATGATCAAAGAACGCTCAGCGGTGTCTGTG-39 (RMET13-1; Bpu1102I site is underlined). The resulting MET12 and MET13 fragments were digested with the indicated restriction enzymes (underlined in primer sequence) and cloned into the corresponding sites in pET16b. Finally, the human and E. coli methylenetetrahydrofolate reductases were subcloned into pVT103-U (13) for expression in yeast. Clones of the human and E. coli methylenetetrahydrofolate reductases in the plasmid pTrc99A (Pharmacia) were generous gifts of the R. Rozen (McGill University, Quebec) and R. G. Matthews (University of Michigan, Ann Arbor, MI) labs, respectively. The 1970-bp human MTHFR coding sequence was PCR amplified from the pTrc99A clone using Pfu DNA polymerase and the primers 59-GACTCATCTAGAGCCATGGTGAACGAA-39 (hfMTR-4; XbaI site underlined) and 59-TGACTGAAGCTTTCATGGAGCCTCCGT-39 (hrMTR-4; HindIII site underlined). Likewise, the 890-bp E. coli MetF gene was PCR amplified using Pfu DNA polymerase and the primers 59-GCGCGCTCTAGAGGTATGAGC TTTTTT-39 (efMTR-4; XbaI site underlined) and 59-GACGCGAAGCTTTTATAAA CCAGGTCG-39 (erMTR-4; HindIII site underlined). Each amplified fragment was digested with HindIII and XbaI and ligated into the respective sites in pVT103-U. DNA manipulation and E. coli transformations were performed as described by Maniatis et al. (15) and isolation of plasmid DNA was performed according to a modified alkaline lysis method (16). DNA sequence confirmations were

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FIG. 2. Disruption of MET12 and MET13 in yeast. (A) Intact MET12 and MET13 genes. (B) URA3-disrupted MET12 and MET13 genes. Arrows indicate oligonucleotide primers MTR59-5, MTR39-2, MTR2.1, and MTR2.6. (C) PCR of yeast genomic DNA from wild type (DAY4) and URA31 transformants of RRY1 and RRY2: lane 1, DAY4 genomic DNA amplified with primers MTR2.1 and MTR2.6; lanes 2 and 3, RRY1 genomic DNA amplified with primers MTR2.1 and MTR2.6; lane 4, DAY4 genomic DNA amplified with primers MTR59-5 and MTR39-2; lanes 5 and 6, RRY2 genomic DNA amplified with primers MTR59-5 and MTR39-2. Abbreviations: MW, 1-kbp ladder molecular weight marker.

performed by the DNA Facility of the Institute of Cellular and Molecular Biology at the University of Texas at Austin. Gene disruption and strain construction. Disruption of MET12 involved digestion of the plasmid pJR-URA3 (17) with HindIII and SstI to liberate the 1.1-kbp URA3 cassette. The URA3 cassette was then blunt ended with T4 DNA polymerase (Life Technologies) and used to replace a 600-bp NsiI fragment of the MET12 gene in pBluescript (Fig. 2). The disrupted MET12 gene was PCR amplified using the plasmid specific primers 59-AACAGCTATGACCATG-39 (#1201) and 59-GTAAAACGACGGCCAGT-39 (#1211). The crude PCR reaction mixture was used to transform DAY4a/a diploids by the lithium acetate procedure (18). Transformants were selected based on ability to grow without uracil supplementation. PCR using Taq polymerase was performed directly on the positive colonies (19) with primers

59-GGTTACATTGG-AAGAGGGAC-39 (MTR59-5) and 59-TCCGATTGGATTTCTGG-39 (MTR39-2) in order to confirm the MET12 disruption. MET12 disrupted diploids were sporulated by inoculating a single yeast colony into 2 ml YPA media and incubating at 30°C for 2 days. The cells were harvested and washed twice with deionized H 2O and then transferred to 3 ml of SPM media supplemented with 25% of the normal concentration of serine, histidine, leucine, tryptophan, and methionine. The cultures were incubated at 25°C for 3– 4 days and then checked for the presence of tetrads under a light microscope. One hundred microliters of cells were incubated with 2 ml of 1 M 2-mercaptoethanol (ICN Biomedical) and 3 ml of 5 mg/ml Lyticase (Sigma) for 20 min and spores were dissected using a Zeiss Tetrad Microscope System. The resulting spores were replica plated onto selective and rich media. Several spores were tested for mating type by mating with an a and a test strain. The URA3 cassette was evicted using the pHM53 based method described by Roca et al. (17) to yield the Dmet12 disruption strain RRY2. Similarly, disruption of MET13 involved the liberation of the URA3 cassette from pJR-URA3 using SstI and HindIII. The cassette was subsequently blunt ended and used to replace the 480-bp EcoRV fragment of MET13 in pUC19 (Fig. 2). The disrupted MET13 gene was PCR amplified using the primers 59-CAAGTCCAAGTACGGTGACC-39 (MTR2.1) and 59-CAGAGAGCTCCTGTTATCGTTGTCTGTG-39 (MTR2.6). DAY4a/a diploids were disrupted at the MET13 locus as described previously and colony PCR was performed using primers MTR2.1 and MTR2.6 to confirm the MET13 disruption. The URA3 cassette was evicted, and mating type tests were performed to generate the Dmet13 disruptant haploid strain RRY1. Finally, in order to obtain the double MTHFR disruptant, strain RRY2 and a met13::URA3 strain (a methionine auxotroph) were crossed to yield diploids disrupted at both the MET12 and MET13 loci. Diploids were selected by their ability to grow without uracil or methionine supplementation. The diploids were sporulated and dissected as described. Spores were selected based on their ability to grow without uracil supplementation (indicating the met13::URA3 allele) and tested for disruption at the MET12 locus using colony PCR (primers MTR59-5 and MTR39-2). The URA3 cassette was evicted, and mating type tests were performed to obtain the double disruptant haploid strain RRY3 (Dmet12 Dmet13). Northern blot analysis. Total yeast RNA was isolated from a 25 ml culture using the Rneasy Mini kit (Qiagen). Formaldehyde gels were prepared and run according to a protocol from the Gottschling lab website (http://www.fhcrc.org/;gottschling/northerns.html). Briefly, the gel was prepared by dissolving 0.9 g of agarose (Life Technologies) in 85 ml of deionized water, then 5 ml of formaldehyde and 10 ml of 103 running buffer (50 mM sodium acetate, 0.2 M MOPS pH 7.0, and 10 mM EDTA) were added. RNA samples were prepared by mixing 12–24 mg RNA 1:2 with freshly made 1.53 loading buffer (750 ml deionized formamide, 75 ml formaldehyde, 150 ml 103 running buffer, 15 ml 5 mg/ml ethidium bromide, 10 ml deionized water) and incubated at 65°C for 5 min. The samples were then chilled and 2 ml of loading dye (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol) was added. The gel was run at 60 V for 4 to 6 h at room temperature. The RNA was transferred overnight from the gel to a GeneScreen nylon membrane (NEN) using standard capillary transfer with 25 mM sodium phosphate pH 6.5 as transfer buffer. The RNA was then immobilized on the membrane by exposure to UV light (UVP Inc. lamp # 34-0034-01) for 1 min. The membrane was incubated at 43°C for at least 1 h in prehybridization buffer (50% formamide, 0.25 M sodium phosphate, pH 7.2, 0.25 M NaCl, 1 mM EDTA, 100 mg/ml denatured salmon sperm DNA, and 7% SDS) and then overnight in hybridization buffer (prehybridization buffer plus probe) (20). The 32 P-labeled MET12 and MET13 probes were synthesized using the Oligolabelling kit (Pharmacia Biotech). Template for MET12 probe synthesis was obtained by PCR amplifying an 800-bp fragment from the MET12-pVT101U clone using the primers 59-CTCAATCGAACGAATCACC-39 (MTR59-1) and 59-TTTAGATGGCATGGAACC-39

YEAST METHYLENETETRAHYDROFOLATE REDUCTASE ISOZYMES (MTR39-1). Template for the MET13 probe synthesis was obtained by amplifying a 1000 bp fragment from the MET13-pVT101U clone using primers 59-ACTTGCACATCTACACCATG-39 (MTR2.7) and 59CAGAGAGCTCCGCTCAGCGGTGTCTGTG-39 (MTR2.6). After overnight hybridization, the membrane was rinsed briefly at room temperature with 0.13 SSC (3.0 M NaCl, 0.3 M sodium citrate, pH 7.0) containing 0.1% SDS and then washed once at 50°C with 0.13 SSC/0.1% SDS for 30 min. The membrane was then washed once at 50°C with 0.13 SSC/1.0% SDS for 1 h and, finally, once with 0.13 SSC/0.1% SDS for 30 min at 50°C. The membrane was then exposed to a Storage Phosphor Screen (Molecular Dynamics) for 1–3 days and imaged using a PhosphoImager 445 SI (Molecular Dynamics). Expression and assay of MTHFR isozymes. Yeast lysates were prepared by growing yeast cultures in YMD plus appropriate supplements and harvesting the cells during log phase growth. Cells were washed and resuspended in breaking buffer (100 mM phosphate buffer, pH 6.3, 1 mM EDTA, and 10% glycerol). Phenylmethylsufonyl fluoride was added to a final concentration of 1 mM along with an amount of chilled glass beads that was equivalent to about 50% of the total volume of the cell suspension. Cells were lysed by alternately vortexing the sample for 30 s and chilling it on ice for 30 s; this procedure was repeated five times. The lysates were centrifuged at 4°C for 30 min at 13,000g and the supernatant was used for enzyme assay. Clones of MET12 and MET13 in pET16b were transformed into the E. coli host strain BL21(DE3). Cells were grown in 25 ml of 2YT media containing 50 mg/ml ampicillin. Expression was induced with 1 mM isopropyl-b-D-thiogalactopyranoside when the cells had reached an OD 550 of approximately 2.0. Induced cells were grown for an additional 3– 4 h, harvested and washed twice with 100 mM potassium phosphate (pH 6.3). Cells were resuspended in sonication buffer (100 mM potassium phosphate pH 6.3, 0.6% BSA, and 9 mM EDTA) and 1 mM phenylmethylsufonyl fluoride. Cell suspensions were sonicated in 10-s bursts three or four times using a Vibra Cell sonicator (model VC40) at setting 80 –100. Crude sonicates were centrifuged at 13,000g for 10 min at 4°C, and the supernatant was used for enzyme assays. E. coli metF was used as a positive control. Plasmid pCAS-5, containing the E. coli MTHFR gene metF (8), was a generous gift of the R. G. Matthews’ lab (University of Michigan, Ann Arbor, MI). Yeast cell lysates or bacterial cell sonicates were assayed as described by Matthews (5), with a few modifications. Briefly, the final reaction mixture contained 122 mM potassium phosphate buffer (pH 6.3), 37 mM FAD, 0.24% BSA, 3.7 mM EDTA, 305 mM (6R,S)[ 14CH 3]tetrahydrofolate (approximately 1800 dpm/nmol), and 0.6 mM sodium ascorbate. For assays of bacterial sonicates, either a saturated menadione solution in 20% methanol or 20% methanol without menadione was included. It was observed that addition of menadione to reactions containing yeast extracts reproducibly reduced the activity detected in these assays (see Results), so deionized water was used in place of the menadione. Cell extract was added to the reaction mixtures and the reactions were incubated at 37°C for 20 min. The reactions were terminated by addition of a 3 mg/ml solution of dimedone in 1 M sodium acetate buffer (pH 4.5), heated for 10 min at 98°C, and then chilled for 2 min on ice. Subsequently, three times the reaction volume of toluene was added and the samples were vortexed for 10 s each. The samples were centrifuged at 4000g for 10 min and an aliquot of the nonaqueous (top) phase of each sample was added to 10 ml of Bio-Safe NA scintillation cocktail (Research Products International). The samples were counted using a Beckman LS 1801 Liquid Scintillation Counter.

RESULTS

Isolation of two yeast genes encoding MTHFR. Isolation and sequencing of the human MTHFR cDNA (9) led to the discovery of a homologous yeast ORF upstream of the RAD1 gene (21). This ORF is located on

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yeast chromosome XVI between nucleotides 504335 and 506308 and its sequence is available in the Saccharomyces Genome Database at Stanford University (SGD ORF YPL023C). A clone containing the putative MTHFR gene was obtained and the gene was subcloned into a sequencing plasmid and a yeast expression vector. The putative gene was designated MET12. Sequence analysis indicated that Met12p is a 657 amino acid protein with a calculated molecular mass of 73,915 Da. Sequence alignment revealed a 32% identity between Met12p and the human MTHFR protein (Fig. 3). As additional yeast sequence information became available, a second putative yeast MTHFR gene was found downstream of the SCS3 gene (22, 23) on yeast chromosome VII between nucleotides 272524 and 274323 (SGD ORF YGL125W). This DNA sequence was amplified from DAY4 yeast genomic DNA using PCR and then cloned. The second gene was designated MET13. Sequence analysis indicated that Met13p is a 599 amino acid protein with a calculated molecular mass of 68,455 Da. Sequence alignment showed that Met13p is 37% identical to the human MTHFR and 34% identical to Met12p (Fig. 3). Disruption of the MET12 and MET13 genes. In order to minimize the occurrence of secondary mutations, the MET12 and MET13 genes were disrupted in a diploid strain. The disruptions were performed by constructing clones of the MET12 and MET13 genes with several hundred nucleotides of internal gene sequence replaced by the 1.1-kbp URA3 cassette. The disrupted gene constructs were amplified such that the resulting fragments contained at least 100 bp of putative MTHFR gene sequence on either side of the URA3 cassette. DAY4a/a diploids were transformed with either the met12::URA3 or met13::URA3 fragments and selected for growth without uracil supplementation. Putative disrupted diploids were confirmed by PCR directly on the colonies. MET12-disrupted diploids yielded two fragments: one at 1370-bp, corresponding to the disrupted genomic copy, and one at 870-bp, corresponding to the wild-type copy (Fig. 2). Similarly, MET13 disruptants also yielded two fragments at 2.0and 1.4-kbp, again with the largest fragment corresponding to the disrupted copy (Fig. 2). Disrupted diploids were then sporulated and dissected to yield the corresponding haploid spores and these spores were replica plated onto selective and rich media. Finally, the URA3 cassette was evicted and the resulting disruptant strains were designated RRY1 (Dmet13) and RRY2 (Dmet12). In order to disrupt both putative MTHFR genes in the same cell, strain RRY2 (Dmet12), a methionine prototroph, and a met13::URA3 strain, a methionine auxotroph, were crossed. The resulting diploids were selected by their ability to grow without uracil and

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FIG. 3. Alignment of the yeast MET12 and MET13 gene products with methylenetetrahydrofolate reductase from human (9) and E. coli (43). The alignment was produced by the GenomeNet WWW Server of Japan (http://www.genome.ad.jp/) using CLUSTAL W. *, identical residues; . and :, varying degrees of conserved substitutions; 2, gap introduced to facilitate alignment; bold letters, point mutation implicated in the thermolabile human enzyme (2); single underline, putative protease-sensitive site (9); double underline, putative SAM binding sequence (9); the 1 above a residue indicates the residues involved in FAD binding (27).

methionine supplementation. Diploids were sporulated and dissected and the resulting spores were selected based upon their ability to grow without uracil, confirming the presence of the met13::URA3 allele. Identification of spores carrying the disruption at the MET12 locus was done by colony PCR using primers MTR59-5 and MTR39-2. Disruption of MET12 gave a

270-bp product versus the 870-bp product seen for the wild-type copy (data not shown). Positive disruptants underwent eviction of the URA3 cassette in order to generate the strain with disruptions at both the MET12 and MET13 loci. The resulting strain was designated RRY3 (Dmet12 Dmet13). Thus RRY1, RRY2, and RRY3 are isogenic with each other and with the parental DAY4 strain except at the MET12 and/or MET13 loci. Phenotypic studies. Strain RRY2, the met12 disruptant, did not exhibit any new nutritional requirements. However, both of the met13-disrupted strains, RRY1 and RRY3, were found to be methionine auxotrophs (Fig. 4). Growth of the methionine auxotrophs was also supported by S-adenosylmethionine (data not shown). Overexpression of the wild-type MET13 gene from a plasmid rescued the methionine requirement of RRY1 and RRY3. However, overexpression of the MET12 gene failed to rescue the methionine requirement of either strain (data not shown). Thus, a functional MET13 gene is necessary and sufficient for methionine synthesis under these conditions. Furthermore, overexpression of both the human (data not shown) and a putative A. thaliana MTHFR (A. Hanson, personal communication) complemented the methionine requirement of the met13 disruptants. Expression of the E. coli MetF gene failed to rescue the methionine requirement (data not shown). All three disruption strains (RRY1, RRY2, and RRY3) were tested for growth on YPGE plates. YPGE media contains only glycerol and ethanol as carbon sources and therefore does not support the growth of cells lacking mitochondrial respiration. All strains grew well on YPGE, indicating that the disruptions of MET12 and MET13 did not significantly effect respiration in the three strains (data not shown). Finally, there was no significant difference between the growth rates of the three disruption strains (RRY1, RRY2, and RRY3) and the wild-type strain, DAY4, when grown in minimal media containing appropriate supplements plus methionine. Doubling times of the various cultures supplemented with methionine were between 2.2 and 2.6 h (data not shown). Without methionine, strains RRY1 (Dmet13) and RRY3 (Dmet12 Dmet13) showed no growth (Fig. 4). The parental DAY4 strain and strain RRY2 (Dmet12) both grew with similar rates in the presence or absence of methionine. Furthermore, overexpression of MET13 from a plasmid in strains RRY3 (Dmet12 Dmet13) and RRY1 (MET12 Dmet13) rescued these cells to near the wild-type growth rate (data not shown) in minimal media lacking methionine. mRNA analysis. Northern blots were performed on total RNA isolated from various strains grown with or without methionine. Results using a MET13 probe indicated that MET13 was expressed in wild-type

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FIG. 4. Growth requirements of MET12 and MET13 disrupted yeast strains. Plus methionine plates contain serine, histidine, leucine, tryptophan, uracil, methionine, and glucose. Minus methionine plates contain the same nutrients but lack methionine. Strains were grown for 3 days at 30°C.

(DAY4) cells grown in the presence or absence of methionine (Fig. 5B, lanes 2 and 3). There was no detectable MET13 transcript in RRY3 (Dmet12 Dmet13) cells (Fig. 5B, lane 1). Results using a MET12 probe indicated that MET12 was also expressed in wild-type cells grown in the presence or absence of methionine, but at lower levels than MET13 (Fig. 5A, lanes 1 and 2).

FIG. 5. Northern blot analysis of total yeast RNA for MET12 and MET13 transcripts. (A) Analysis of total RNA (24 mg/lane) using a MET12 probe: lane 1, total RNA from DAY4 (MET12 MET13) grown without methionine; lane 2, total RNA from DAY4 grown with methionine; lane 3, total RNA from RRY3 (Dmet12 Dmet13); lane 4, total RNA from RRY2 (Dmet12 MET13); lane 5, total RNA from RRY1 (MET12 Dmet13); lane 6, total RNA from RRY3 (Dmet12 Dmet13) overexpressing MET12 from the multicopy plasmid pVT101-U. (B) Analysis of total RNA (12 mg/lane) using a MET13 probe: lane 1, total RNA from RRY3; lane 2, total RNA from DAY4 grown with methionine; lane 3, total RNA from DAY4 grown without methionine. Blots (A) and (B) were exposed for 1 day.

MET12 transcript was not detected in RRY3 cells (Fig. 5A, lane 3) but was readily detected in RRY3 cells overexpressing MET12 (Fig. 5A, lane 6). Furthermore, MET12 transcript was detected in RRY1 (MET12 Dmet13) cells (Fig. 5A, lane 5) but not in RRY2 (Dmet12 MET13) cells (Fig. 5A, lane 4), thus ruling out the possibility that the MET12 probe was cross-hybridizing with MET13 transcript. MTHFR enzyme activity. Assays of MTHFR activity in the forward direction (reduction of CH 2-THF) are difficult in crude cell extracts due to the rapid, nonspecific oxidation of NADPH by the extracts (5). Thus, MTHFR activity was assayed in the reverse direction (oxidation of CH 3-THF). In this assay, the radioactive substrate, [ 14CH 3]THF, is oxidized by the flavin-dependent enzyme to form the product [ 14CH 2]THF. This product decomposes in solution to yield THF and radiolabeled formaldehyde, which is trapped with dimedone and extracted from the reaction mixture with toluene. Finally, an aliquot of the extraction is quantified by scintillation counting. Traditionally, menadione has been used as the in vitro electron acceptor for the MTHFR assay in the reverse direction. However, we found that addition of menadione to reaction mixtures resulted in a significant decrease in the activity levels detected for the putative yeast enzymes. There was approximately a 50% decrease in activity observed for yeast extracts with added menadione versus extracts without added menadione (data not shown). Similar, although less dramatic, results were seen for Met12p and Met13p assayed in E. coli extracts (Table I). The artificial electron acceptor 2,6-dichlorophenol-indophenol was also ineffective with the yeast enzymes (data not shown).

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Methylenetetrahydrofolate Reductase Activity in E. coli and S. cerevisiae Extracts Strain a E. coli strain BL21(DE3) expressing pET16b vector alone pCAS-5 (E. coli MTHFR clone) pET16b-MET12 pET16b-MET13 S. cerevisiae strains DAY4 (MET12 MET13) RRY1 (MET12 Dmet13) RRY2 (Dmet12 MET13) RRY3 (Dmet12 Dmet13)

Menadione

Specific activity b

2 1 2 1 2 1 2 1

0.82 4.11 26.67 35.72 16.22 11.19 7.43 6.91

2 2 2 2

5.66 0.00 4.81 0.00

E. coli was grown in 25 ml 2YT media containing 50 mg/ml ampicillin. S. cerevisiae strains were grown in 1 L YMD with the appropriate supplements plus methionine (strains RRY1 and RRY3) or minus methionine (strains DAY4 and RRY2). b nmol/mg protein/hour. a

Consequently, for all MTHFR assays performed using yeast extracts, menadione was omitted from the assays. Initial assays performed using yeast extracts gave low and inconsistent results. As mentioned previously, elimination of menadione increased the level of activity detected by this assay. Additionally, assay of an E. coli MTHFR clone (8), both with and without menadione, was included as a positive control for the assay (data not shown). Assay of MTHFR activity was performed on each of the four yeast strains DAY4, RRY1, RRY2, and RRY3. Results from these assays show that strain DAY4 (MET12 MET13) and RRY2 (Dmet12 MET13) exhibit essentially equivalent levels of activity (Table I). Furthermore, no activity was detected in either strain RRY1 (MET12 Dmet13) or RRY3 (Dmet12 Dmet13). These results indicate that the enzyme encoded by MET13 is responsible for all of the activity detected in yeast grown under these conditions. This is corroborated by the methionine requirement of both strains RRY1 and RRY3. Finally, the MET12 and MET13 genes were both cloned into the pET16b E. coli expression plasmid. Assays were performed on crude extracts from E. coli transformed with pET16b vector alone (negative control), pET16b-MET12, pET16b-MET13, or the E. coli metF gene (8) (positive control). Expression of both MET12 and MET13 in E. coli resulted in a 9- to 20-fold increase over the endogenous E. coli MTHFR activity (Table I). These results verify that both the MET12 and MET13 genes encode active MTHFR enzymes. However, unlike the stimulation seen with the E. coli

enzyme, menadione had an inhibitory effect on the activity of the yeast enzymes expressed in E. coli (Table I). DISCUSSION

Here we describe the isolation and characterization of two genes, MET12 (YPL023c) and MET13 (YGL125w), encoding methylenetetrahydrofolate reductases in S. cerevisiae. The identity of these two ORFs as MTHFRs was confirmed by several observations. First, the MET12 and MET13-encoded ORFs are homologous to known MTHFRs from other organisms (Fig. 3). Second, disruption of MET13, alone or in conjunction with MET12, produced a strict methionine growth requirement, confirming the recent report from the EUROFAN Systematic Functional Analysis Program (24). The methionine requirement could be complemented by the plasmid expression of MET13 or human MTHFR, but not by MET12. Additionally, results from MTHFR assays with yeast and E. coli extracts confirmed that both MET12 and MET13 encode active MTHFR isozymes. Finally, both MET12 and MET13 transcripts were detected in total RNA isolated from wild type cells. It should be noted that a yeast met13 mutant was first described in 1975 (25); Tizon et al. have confirmed the identity of the original genetically defined MET13 locus as ORF YGL125w (24). Eukaryotic MTHFRs have been previously purified and characterized from both pig liver and human cadaver liver (6, 7). Enzyme from these two sources are flavo-proteins that utilize NADPH as the endogenous source of electrons for the reduction of CH 2-THF to CH 3-THF. The porcine MTHFR is a homodimer, and each subunit is comprised of a 40-kDa catalytic domain and a 37-kDa regulatory domain separated by a protease-sensitive site (26). There is evidence that the human enzyme possesses a similar domain structure (7). The protease-sensitive site of the human enzyme is predicted to be between residues 351 and 374 in a region that contains the highly charged KRREED sequence that is underlined in Fig. 3 (9). The two yeast MTHFRs each contain a similar motif; however, in Met12p the motif is interrupted by a 24-residue insertion (Fig. 3). The 296 amino acid E. coli MTHFR is homologous to the N-terminal 300 amino acids of the eukaryotic MTHFR enzymes (Fig. 3). This region of homology likely encodes the catalytic function of these proteins. A recent report describes the structure of the E. coli MTHFR in which the FAD binding residues have been elucidated (27). Comparison of the protein sequences in Fig. 3 reveals the high conservation of residues now known to be involved in FAD binding (Fig. 3, 1 above residues). The majority of the 58 additional residues that account for the greater length of Met12p over Met13p, lie within the last 70 residues at the end of the

YEAST METHYLENETETRAHYDROFOLATE REDUCTASE ISOZYMES

N-terminal region. A recently identified ORF in Schizosaccharomyces pombe shares 38% identity with MET12 (28). The S. pombe MTHFR sequence also contains additional residues in this section. With the exception of the MET13 isozyme, these sequences contain the highly conserved alanine residue (bold, Fig. 3) implicated in the thermolabile variant of the human MTHFR that is correlated with increased risk for cardiovascular disease (2). The three eukaryotic proteins (Met12p, Met13p, and human) also share homology beyond the N-terminal 300 residues, including the Cterminal domain predicted to contain the S-adenosylmethionine-dependent regulatory function of the human enzyme (9). The putative SAM binding sequence (9) lies in a region of notable similarity in the three eukaryotic proteins (Fig. 3, double underline). The E. coli sequence does not contain the predicted SAM binding sequence, which is in agreement with the fact that the activity of the bacterial enzyme is not affected by SAM. While these results clearly implicate Met13p in producing 5-methyl-THF for methionine biosynthesis in vivo, the metabolic function of the MET12encoded isozyme is not known. However, transcripts from both genes were detected in wild-type cells (Fig. 5), indicating that both genes are expressed under normal growth conditions. MET12 expression was also observed in several of the whole genome analyses recently reported (29 –32). In cells grown with or without methionine, we observed the MET13 transcript to be considerably more abundant than the MET12 transcript. The protein corresponding to Met13p was previously identified using 2D PAGE analysis of [ 35 S]methionine labeled yeast proteins (33). However, this technique has not detected the expression of Met12p in yeast. We were unable to detect any MTHFR enzyme activity above background in strain RRY1 (MET12 Dmet13) (Table I). Given the low abundance of the MET12 transcript, it is possible that the Met12p activity is simply below the detection limits of the assay. Another possibility for the absence of detectable MTHFR activity in RRY1 is that the exogenous methionine required for the growth of this strain may inhibit the activity of the Met12p isozyme. This would not be unprecedented considering that the activity of eukaryotic reductases are susceptible to inhibition by S-adenosylmethionine (SAM) (7, 34), which is a product of methionine metabolism (Fig. 1). Moreover, it is known that S-adenosylhomocysteine (SAH) can partially counteract the inhibition by SAM of the pig liver enzyme (34). However, neither SAH nor methionine had any significant effect on the MTHFR activity detected in crude yeast extracts. Inhibition of the yeast CH 2 -THF reductases by SAM was not tested. One other explanation is that methionine in the medium represses expression of the MET12

307

gene. However, Northern analysis indicated that the MET12 transcript was expressed at a similar or slightly increased level in cells grown with methionine (Fig. 5A). Elucidation of the metabolic role of MET12 will require genetic approaches, currently underway. An interesting difference between the yeast isozymes and other known MTHFRs is the inability of the yeast enzymes to utilize menadione as an in vitro electron acceptor in the reverse reaction. Both yeast enzymes, expressed in E. coli, exhibited good activity in aerobic reactions where O 2 is presumably reoxidizing the reduced flavin on the enzyme. Whereas menadione stimulated the activity of the E. coli enzyme, a slight reduction in activity was observed for the yeast enzymes (Table I). The artificial electron acceptor 2,6dichlorophenol-indophenol (DCPIP) was also ineffective with the yeast enzymes. There are other examples of flavin-dependent reductases that do not use menadione (35). The inability of the yeast enzymes to use these nonphysiological electron acceptors may be due to subtle differences in the active site and surrounding regions, a likely explanation given the sequence divergence between the yeast enzymes and their mammalian homologues. NADP 1 also had no affect on the activity measured. However, this is not surprising since the equilibrium constant for the reduction of CH 2-THF by NADPH is on the order of 10 4–10 7 (34, 36). Therefore, to drive the reaction in the reverse, as in this assay, requires a stronger oxidant such as menadione. In the forward direction, MTHFRs from different organisms utilize a variety of different electron sources such as NADPH in mammals (6, 7), NADH in P. productus (37) and E. coli (8), and ferrodoxin in C. formicoaceticum (38). This variability makes it difficult to predict what the natural yeast reductant will be, but NADPH is a likely candidate since it is utilized by other known eukaryotic MTHFRs. Experiments with purified enzymes will be needed to resolve these questions. MTHFR activity has been previously reported in yeast extracts. Our results are similar to those obtained by Lor and Cossins (39), whereas Zelikson and Luzzati (40) reported levels of activity 100-fold higher. However, the latter measurements were made using the spectrophotometric assay which, as previously noted, is not reliable for crude extracts. The specific activity in wild-type yeast extracts reported here (Table I) is about sevenfold lower than the activity reported for pig liver extracts (6) and about eightfold higher than reported for human liver extracts (7). Finally, it was reported that a peptide related to ORF YGL125w (MET13) is associated with the yeast mitochondrial ribosome (41). However, it is more likely that this identification was due to a sequencing artifact (42), as there is no other evidence that Met13p is a mitochondrial protein.

308

RAYMOND, KASTANOS, AND APPLING

ACKNOWLEDGMENTS A special thanks to Amanda Suarez for her hours of dedicated help with this project. Also, we thank Dr. Christal Sheppard from Dr. Rowena Matthews lab (University of Michigan, Ann Arbor) for teaching us the MTHFR assay, and Drs. Andrew Hanson (University of Florida, Gainesville) and Larry Poulsen (University of Texas at Austin) for helpful discussions.

REFERENCES 1. Rosenblatt, D. S. (1995) in The Metabolic and Molecular Bases of Inherited Disease (C. R. Scriver, A. L. Beaudet, W. S. Sly, and D. Valle, Eds.), pp. 3111–3128, McGraw-Hill, New York. 2. Frosst, P., Blom, H. J., Milos, R., Goyette, P., Sheppard, C. A., Matthews, R. G., Boers, G. J. H., den Heijer, M., Kluijtmans, L. A. J., van den Heuvel, L. P., and Rozen, R. (1995) Nature Genet. 10, 111–113. 3. Wilcken, D. E. L. (1997) Lancet 350, 603– 604. 4. Matthews, R. G. (1984) in Folates and Pterins (R. L. Blakley and S. J. Benkovic, Eds.), pp. 497–553, Wiley, New York. 5. Matthews, R. G., Jencks, D. A., Frasca, V., and Matthews, K. D. (1986) in Chemistry and Biology of Pteridines 1986 (B. A. Cooper and V. M. Whitehead, Eds.), pp. 697–707, Walter de Gruyter, New York. 6. Daubner, S. C., and Matthews, R. G. (1982) J. Biol. Chem. 257, 140 –l145. 7. Zhou, J., Kang, S.-S., Wong, P. W. K., Fournier, B., and Rozen, R. (1990) Biochem. Med. Metab. Biol. 43, 234 –242. 8. Sheppard, C. A., Trimmer, E. E., and Matthews, R. G. (1999) J. Bacteriol. 181, 718 –725. 9. Goyette, P., Sumner, J. S., Milos, R., Duncan, A. M. V., Rosenblatt, D. S., Matthews, R. G., and Rozen, R. (1994) Nature Genet. 7, 195–200. 10. Barlowe, C. K., and Appling, D. R. (1990) Mol. Cell. Biol. 10, 5679 –5687. 11. Russell, D. W., Jensen, R., Zoller, M. J., Burke, J., Errede, B., Smith, M., and Herskowitz, I. (1986) Mol. Cell Biol. 6, 4281– 4294. 12. Boeke, J. D., Trueheart, J., Natsoulis, G., and Fink, G. R. (1987) Methods Enzymol. 154, 164 –175. 13. Vernet, T., Dignard, D., and Thomas, D. Y. (1987) Gene 52, 225–233. 14. Sherman, F., Fink, G. R., and Hicks, J. B. (1986) Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 15. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 16. Feliciello, I., and Chinali, G. (1993) Anal. Biochem. 212, 394 – 401. 17. Roca, J., Gartenberg, M. R., Oshima, Y., and Wang, J. C. (1992) Nucleic Acids Res. 20, 4671– 4672. 18. Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983) J. Bacteriol. 153, 163–168.

19. Sathe, G. M., O’Brien, S., McLaughlin, M. M., Watson, F., and Livi, G. P. (1991) Nucleic Acids Res. 19, 4775. 20. Amasino, R. M. (1986) Anal. Biochem. 152, 304 –307. 21. Yang, E., and Friedberg, E. C. (1984) Mol. Cell. Biol. 4, 2161– 2169. 22. Tizon, B., Rodriguez-Torres, A. M., Rodriguez-Belmonte, E., Cadahia, J. L., and Cerdan, E. (1996) Yeast 12, 1047–1051. 23. Hosaka, K., Nikawa, J., Kodaki, T., Ishizu, H., and Yamashita, S. (1994) J. Biochem. 116, 1317–1321. 24. Tizon, B., Rodriguez-Torres, A. M., and Cerdan, M. E. (1999) Yeast 15, 145–154. 25. Masselot, M., and de Robichon-Szulmajster, H. (1975) Mol. Gen. Genet. 139, 121–132. 26. Matthews, R. G., Vanoni, M. A., Hainfeld, J. F., and Wall, J. (1984) J. Biol. Chem. 259, 11647–11650. 27. Guenther, B. D., Sheppard, C. A., Tran, P., Rozen, R., Matthews, R. G., and Ludwig, M. L. (1999) Nat. Struct. Biol. 6, 359 –365. 28. Yoshioka, S., Kato, K., Nakai, K., Okayama, H., and Nojima, H. (1997) DNA Res. 4, 363–369. 29. DeRisi, J. L., Iyer, V. R., and Brown, P. O. (1997) Science 278, 680 – 686. 30. Chu, S., DeRisi, J., Eisen, M., Mulholland, J., Botstein, D., Brown, P. O., and Herskowitz, I. (1998) Science 282, 699 –705. 31. Spellman, P. T., Sherlock, G., Zhang, M. Q., Iyer, V. R., Anders, K., Eisen, M. B., Brown, P. O., Botstein, D., and Futcher, B. (1998) Mol. Biol. Cell 9, 3273–3297. 32. Holstege, F. C., Jennings, E. G., Wyrick, J. J., Lee, T. I., Hengartner, C. J., Green, M. R., Golub, T. R., Lander, E. S., and Young, R. A. (1998) Cell 95, 717–728. 33. Shevchenko, A., Jensen, O. N., Podtelejnikov, A. V., Sagliocco, F., Wilm, M., Vorm, O., Shevchenko, A., Boucherie, H., and Mann, M. (1996) Proc. Natl. Acad. Sci. USA 93, 14440 –14445. 34. Kutzbach, C., and Stokstad, E. L. R. (1971) Biochim. Biophys. Acta 250, 459 – 477. 35. Wosilait, W., and Nason, A. (1955) Methods Enzymol. 2, 725– 729. 36. Wohlfarth, G., and Diekert, G. (1991) Arch. Microbiol. 155, 378 – 381. 37. Wohlfarth, G., Geerligs, G., and Diekert, G. (1990) Eur. J. Biochem. 192, 411– 417. 38. Clark, J. E., and Ljungdahl, L. G. (1984) J. Biol. Chem. 259, 10845–10849. 39. Lor, K. L., and Cossins, E. A. (1972) Biochem. J. 130, 773–783. 40. Zelikson, R., and Luzzati, M. (1977) Eur. J. Biochem. 79, 285– 292. 41. Kitakawa, M., Graack, H.-R., Grohmann, L., Goldschmidt-Reisin, S., Herfurth, E., Wittmann-Liebold, B., Nishmura, T., and Isono, K. (1997) Eur. J. Biochem. 245, 449 – 456. 42. Graack, H. R., and Wittmann-Liebold, B. (1998) Biochem. J. 329, 433– 448. 43. Saint-Girons, I., Duchange, N., Zakin, M. M., Park, I., Margarita, D., Ferrara, P., and Cohen, G. N. (1983) Nucleic Acids Res. 11, 6723– 6732.