Vol. 174, No. 10

JOURNAL OF BACTERIOLOGY, May 1992, p. 3339-3347

0021-9193/92/103339-09$02.00/0 Copyright © 1992, American Society for Microbiology

Cloning and Characterization of the CYS3 (CYIl) Gene of Saccharomyces cerevisiae BUN-ICHIRO ONO,`* KAZUMA TANAKA,2 KAZUHIDE NAITO,' CHINATSU HEIKE,1 SUMIO SHINODA,1 SUMIYO YAMAMOTO,3 SHINJI OHMORI,3 TAKEHIRO OSHIMA,4 AND AKIO TOH-B2 Laboratory of Environmental Hygiene Chemistry' and Laboratory of Physiological Chemistry,3 Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700, Department of Botany, Tokyo University, Tokyo 113, 2 and Suntory Bio Phanna Tech Center, Chiyoda-machi, Oura-gun, Gunma 370-05, 4 Japan Received 17 June 1991/Accepted 15 March 1992

homoserine O-acetyltransferase (HATase) (EC 2.3.1.31) and O-acetylhomoserine sulfhydrylase (OAH SHLase) (EC 4.2.99.10). Since OAS SHLase and OAH SHLase are identical (42), we refer to this enzyme as OAS-OAH SHLase. In short, the S. cerevisiae biosynthetic pathways of sulfurcontaining amino acids are partly like the enteric bacterial and plant pathways and partly like the mammalian pathway. So it is of interest to see how they have evolved. The simplest speculation is that enzymes catalyzing the same or similar reactions in different organisms have descended from the same ancestor. However, we reached a contrary conclusion in the course of studying the S. cerevisiae CYS3 gene. Our results indicate that S. cerevisiae -y-CTLase is homologous to Escherichia coli -y-CTSase in in vitro functions and that the two enzymes differ in in vivo functions by means of the availability (or unavailability) of substrates. Here, we describe our work and discuss the evolution of these en-

Saccharomyces cerevisiae biosynthetic pathways of the sulfur-containing amino acids cysteine and methionine are shown in Fig. 1. Cysteine is synthesized via two pathways. One consists of serine O-acetyltransferase (SATase) (EC 2.3.1.30) and O-acetylserine sulfhydrylase (OAS SHLase) (EC 4.2.99.8). This is the autotrophic pathway for sulfur utilization, because inorganic sulfur is converted to organic sulfur. It is similar to the enteric bacterial and plant cysteine biosynthetic pathways (for reviews, see references 9 and 37). In enteric bacteria and plants, cysteine sulfur is converted to homocysteine sulfur via transsulfuration consisting of cystathionine y-synthase (-y-CTSase) (EC 4.2.99.9) and cystathionine P-lyase (1-CTLase) (EC 4.4.1.8), and the resultant homocysteine is used for methionine biosynthesis. The same takes place in S. cerevisiae. However, it should be stressed that while S. cerevisiae uses O-acetylhomoserine (OAH) for cystathionine biosynthesis, enteric bacteria and plants use O-succinylhomoserine (OSH) and O-phosphohomoserine, respectively. The other S. cerevisiae cysteine biosynthetic pathway is reverse transsulfuration, which consists of cystathionine ,B-synthase (P-CTSase) (EC 4.2.1.22) and cystathionine y-lyase (-y-CTLase) (EC 4.4.1.1). In this pathway, homocysteine sulfur is converted to cysteine sulfur. This pathway is present in mammals but in neither enteric bacteria nor plants (for a review, see reference 10). Mammals synthesize homocysteine exclusively by demethylation of methionine and convert homocysteine sulfur to cysteine sulfur. In this respect, reverse transsulfuration is a part of the heterotrophic pathway for sulfur utilization. S. cerevisiae synthesizes homocysteine not only by demethylation of methionine but also by sulfuration of homoserine (via OAH) by means of *

zymes.

(A preliminary report of this work was presented at the 2nd International Congress on Amino Acids and Analogues held in Vienna, Austria, in August 1991.)

MATERIALS AND METHODS Strains, plasmids, and growth conditions. S. cerevisiae and E. coli strains used in this study are listed in Table 1. The cysteine-dependent strains were described previously (28, 29). The metl7 strains were constructed by backcrossing strain no. 13 (22) with strain IS66-4C for five cycles (28). Plasmids pBR322 (2), pUC18 and pUC19 (44), YEp24 (3), and YCp19 (39) were used. They were propagated in E. coli K-12 strain DH1 (11). Standard growth media for S. cerevisiae (26, 35) and E. coli (32) were used. The yeast rich medium (YPD) contained 1% yeast extract, 2% peptone, and 2% glucose. The yeast

Corresponding author. 3339

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A DNA fragment containing the Saccharomyces cerevisiae CYS3 (CYII) gene was cloned. The clone had a single open reading frame of 1,182 bp (394 amino acid residues). By comparison of the deduced amino acid sequence with the N-terminal amino acid sequence of cystathionine 'y-lyase, CYS3 (CYI1) was concluded to be the structural gene for this enzyme. In addition, the deduced sequence showed homology with the following enzymes: rat cystathionine y-lyase (41%), Escherichia coli cystathionine -y-synthase (36%), and cystathionine 13-lyase (25%). The N-terminal half of it was homologous (39%o) with the N-terminal half of S. cerevisiae O-acetylserine and O-acetylhomoserine sulfhydrylase. The cloned CYS3 (CYIJ) gene marginally complemented the E. coli metB mutation (cystathionine 'y-synthase deficiency) and conferred cystathionine 'y-synthase activity as well as cystathionine y-lyase activity to E. coli; cystathionine 'y-synthase activity was detected when O-succinylhomoserine but not O-acetylhomoserine was used as substrate. We therefore conclude that S. cerevisiae cystathionine 'y-lyase and E. coli cystathionine -y-synthase are homologous in both structure and in vitro function and propose that their different in vivo functions are due to the unavailability of O-succinylhomoserine in S. cerevisiae and the scarceness of cystathionine in E. coli.

3340

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ONO ET AL.

I

?

HATase

y-CTSase

a

I~~~cys

cysI

yCTLase VyCLaecs

0 -acetylhomoserine |

cystathionine

SATase

sIAlcys4 aet17

OAH SHLase

-CTLase

-CTSase

X

|methionine|

FIG. 1. Biosynthetic pathways of sulfur-containing amino acids in S. cerevisiae. Dotted lines indicate the bacterial and plant pathways; bacteria and plants use O-succinylhomoserine and O-phosphohomoserine, respectively, in place of O-acetylhomoserine (for reviews, see references 9 and 35). Broken lines indicate the mammalian pathway (for a review, see reference 10). The relevant mutations and enzymes are included; for abbreviations of enzymes, see the text.

synthetic minimal medium (SD) was described by Wickerham (41). The bacterial rich medium (LB) consisted of 0.5% yeast extract, 1% tryptone, and 1% NaCl. The bacterial synthetic minimal medium (MS) contained the following (per

TABLE 1. Strains used in this study Strain

S. cerevisiae X2180-1B R28-3 R115 R115-ura+ KT22-1A KT22-1B KT22-1C KT22-1D OK361-3D IS316-4C OK331-2D

KN5-5A IS66-4C NA22-1OC OK307-7A

0K307-3D NA5-2D NA5-2C

KN5-5A OK320-6A E. coli DH1 LA5651

Genotype or description

MATot mal mel gal2 CUP] MA Ta/MA Tot cyrlicyri MATa/MA Ta leu2/+ lys2/+ trpl/+ ura3lura3 cysl/cysl A gene disruptant derived from R115 A spore clone derived from R115-ura+ Same as above Same as above Same as above MATot his7 lys2 tyri MA Tot adel MATot cysl-3 cys3-1 his3 leu2 trpl MATa cysI cys3(cyil)::URA3 leu2 lys2 trpl ura3 MA Ta wild type MATax metl7 MATa cysl-3 cys3-1 MA Ta cysl-3 cys3-1 MA Ta cys2-1 cys4-1 MA Ta cys2-1 cys4-1 MATa cys3(cyil)::URA3 his3 Ieu2 trpl ura3 MA Ta his3 Ieu2 metl 7 trpl F- hsdRI7 endAlI gyrA96 recA I relA4 supE44 thi-1 F-; his gatA gyrA lacYl leuB6 malBI metBI mgIB55OphoA14 rpsLl14 thrl zeg722::TnlO

Reference or sourcea

YGSC This study This study This study This study

This This This This This This This 28 27 29 29 28 28

study study study study study study study

This study This study

11 NIGJ

' YGSC and NIGJ are the Yeast Genetic Stock Center, University of California, Berkeley, and the National Institute of Genetics of Japan, Mishima, Shizuoka,

Japan, respectively.

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hFomocysteine

liter): 5 g of NH4Cl, 1 g of NH4NO3, 2 g of Na2SO4, 3 g of K2HPO4, 1 g of KH2PO4, 0.1 g of MgSO4. 7H20, and 20 g of glucose. Yeast and bacterial strains were grown at 30 and 37°C, respectively. Yeast genetic procedures. Standard yeast genetic procedures were adopted (35). Genetic markers were scored as described by Ono et al. (26). Genetic distances were indicated by centimorgan units (31). DNA manipulation. Extraction of DNA or RNA from S. cerevisiae cells was carried out as described by Sherman et al. (35). Isolation of DNA from E. coli, digestion of DNA with endonucleases, fractionation of DNA by agarose gel electrophoresis, ligation of DNA fragments, nick translation, and reverse transcription were carried out as described by Sambrook et al. (32). Transfer of DNA or RNA from the agarose gel to a nitrocellulose membrane was achieved as described by Southern (38). For DNA-DNA or RNA-DNA hybridization, a DNA- or RNA-bearing nitrocellulose membrane was incubated for 4 h in 0.2 ml (per cm2 of membrane) of 0.05 M sodium phosphate buffer (pH 7.0) containing 0.9 M NaCl, 0.005 M Na2-EDTA, 0.3% sodium dodecyl sulfate (SDS), and 1 mg of salmon sperm DNA per ml. This was followed by an 18-h incubation after the addition of 0.01 to 0.1 ,ug of 32P-labeled DNA (1 to 10 ,uCi) to the solution. The hybridization temperature was 65°C unless stated otherwise. Hybridization was monitored by exposing an X-ray film to the nitrocellulose membrane sheet for 5 to 10 days at -70°C. The film was developed as suggested by the manufacturer (Eastman Kodak Co., Ltd.). The yeast and bacterial transformation procedures were described by Ito et al. (14) and Sambrook et al. (32), respectively. Construction of genomic and cDNA libraries. Genomic DNA of S. cerevisiae strain X2180-1B was extracted and partially digested with restriction endonuclease Sau3AI.

VOL. 174, 1992

CLONING AND CHARACTERIZATION OF CYS3 (CYII)

program.

Enzyme assays. S. cerevisiae strains were grown in liquid SD medium; with cysteine-dependent strains, 30 ,uM glutathione was supplemented. E. coli strains were grown in liquid LB medium containing 50 ,ug of ampicillin per ml. Cells at mid- or late logarithmic phase were harvested and suspended in 20 mM potassium phosphate buffer, pH 7.5, containing 50 ,uM pyridoxal 5'-phosphate and 100 ,uM Na2EDTA. The cells were homogenized, and the cell homogenate was used for enzyme assays after centrifugation and dialysis (28, 29). -y-CTLase and SATase were assayed as described previously (28, 29). y-CTSase was assayed by determining its fy-elimination activity with OSH or OAH as substrate; the reaction conditions were adopted from Kaplan and Guggenheim (15). The reaction mixture contained (in the final

concentrations) 100 mM potassium pyrophosphate (pH 8.2), 0.4 mM pyridoxal 5'-phosphate, and 14 mM OSH or OAH; OSH and OAH were synthesized as described by Nagai and Flavin (21). The reaction was started by the addition of the cell homogenate. oa-Ketobutyrate (from OSH) or pyruvate (from OAH) produced by the enzymatic reaction was measured by the method described by Nakata (23). In this procedure, 2-ethyl (or 2-methyl)-2-hydroxy-6,7-dimethoxyquinoline produced by the reaction of a-ketobutyrate (or pyruvate) and 1,2-diamino-4,5-dimethoxybenzene was measured by either UV absorption at 362 nm or fluorescence emission (excitation at 365 and emission at 476 nm). Activity units were defined as the micromoles of the substrate consumed or the product formed per minute. Protein was measured by the method of Lowry et al. (17) with bovine serum albumin as a standard. The specific activity of the enzyme was defined as units per milligram of protein. SATase was assayed at 30°C, and the other enzymes were assayed at 37°C. Chemicals. Restriction enzymes, linkers, and primers were purchased from Takara Shuzo Co., Ltd., and/or Nippon Gene Co., Ltd. Kits for DNA ligation, reverse transcription, and DNA sequencing were purchased from Takara Shuzo Co., Ltd. Universal probe was supplied by Wakunaga Pharmaceuticals. The biotin detection kit was purchased from Bethesda Research Laboratories. Radioactive chemicals were purchased from Daiichi-kagaku Yakuhin Co., Ltd. Yeast extract, peptone, and tryptone were products of Difco Laboratories. Cycloheximide, cAMP, acetyl coenzyme A, and pyridoxal 5'-phosphate were purchased from Sigma. Other chemicals used were analytical grade. Nucleotide sequence accession number. The nucleotide sequence obtained in this study was deposited in the EMBL DNA data base and assigned accession number 1-2254. RESULTS Cloning of a cycloheximide-inducible gene. The clones of the genomic library (see Materials and Methods) were grown on LB medium containing 50 ,g of ampicillin per ml and then spotted to a nitrocellulose membrane in duplicate. After cell lysis, a set of membranes was probed with test cDNA and the other set was probed with control cDNA (see Materials and Methods). Of 104 clones tested, only one had a markedly stronger hybridization signal with test cDNA than with control cDNA. The plasmid recovered from this colony was designated 69-2-1. The restriction profile of plasmid 69-2-1 is shown in Fig. 2. It contained a 4-kbp insert; according to the sequence analysis, the inserted fragment contained a single open reading frame (ORF) in the indicated orientation (described later). Genomic DNA of strain X2180-1B was digested with several endonucleases, separated by electrophoresis, transferred to nitrocellulose membrane, and probed with plasmid 69-2-1 at 55°C (low stringency) and 65°C (high stringency). The results are shown in Fig. 3. It should be stressed that the hybridization patterns between the two temperatures were indistinguishable. BamHI and HindIll, which did not cut the inserted fragment, gave rise to single bands. EcoRI also gave rise to a single band, although it cleaved the cloned fragment once, indicating that the right and left halves of the cloned fragment are partitioned into two EcoRI fragments of similar sizes in the genome. PstI, which cuts the inserted fragment twice, gave rise to three bands. The most intense band (1.5 kbp) corresponded to the central PstI-PstI subfragment of the insert. From these results, we conclude that the inserted

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Fragments longer than 2 kbp were ligated into the BamHI site of plasmid pBR322 and transformed into E. coli K-12 strain DH1. A total of 104 independent Ampr Tets clones were obtained and used as a genomic library. Since we initially intended to clone genes that would be induced by cyclic AMP (cAMP) in the absence of protein synthesis, we constructed two cDNA libraries. Strain R28-3 (cyrllcyrl; adenylate cyclase deficiency [19]) was grown to logarithmic phase in SD medium containing 0.5 mM cAMP, harvested, washed twice with water, transferred to medium lacking cAMP, and incubated for 4.5 h to deplete cellular cAMP. The culture (400 ml) was divided into halves. A total of 0.5 mM cAMP and 50 ,ug of cycloheximide per ml was added to one (test) but not to the other (control). Both cultures were incubated for 30 min; cells did not grow during this period. Cells were harvested from each culture, and poly(A) RNA was isolated. By using the obtained poly(A) RNAs, 32P-labeled cDNAs (test cDNA and control cDNA) were prepared by the reverse transcription reaction method. Separation and detection of chromosome DNAs. Chromosome DNAs were separated by transverse alternating-field electrophoresis (TAFE) with a Geneline apparatus (Beckman Instruments). Strain 0K361-3D was grown to stationary phase. Cells were embedded in agarose, digested with Zymolyase, and treated with SDS as described by Ono and Ishino-Arao (25). The sample gel was inserted into the separation gel (1.5% agarose). Electrophoresis was achieved by using a two-stage method: 4-s intervals for 30 min at 170 mA and 60-s intervals for 18 h at 150 mA. The gel was photographed after staining with ethidium bromide. Chromosome DNAs were then transferred to a nitrocellulose membrane (38), and hybridization with a suitable probe was carried out at 42°C in 50% formamide-5x Denhardt's solution-Sx SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% SDS-0.1 mg of denatured salmon DNA per ml. Hybridization was monitored by the universal probe method (41). Determination of nucleotide sequence. DNA fragments to be sequenced were inserted into plasmids pUC18 and pUC19. After the preparation of single-stranded DNA of each recombinant plasmid, the nucleotide sequence of the insert was determined by the dideoxy chain-termination method with a suitable primer (33). Chain extension was carried out in the presence of [35S]dATP. 35S-labeled DNA was fractionated by polyacrylamide gel electrophoresis, and the polyacrylamide gel was dried and subjected to autoradiography by exposure to an X-ray film for 16 to 24 h at -70°C. Homology search. The amino acid sequence homology search was done by using the Swiss-Prot data base. Data analyses were achieved by means of the GENENTYX

3341

3342

(1.5) SmaI

(0.9) PstI

(1.1)

(1.2) Pvu II

EcoRV

(1.6) AvaI

(1.7) XhoI

(1.8) Bgll

(1.4)

(2.1 ) KpnI

(2.3) Sal I

(2.4) (2.5)

(2.9) NdeI

PstI

AvaI

EcoRI

(4.4)

Kpn I

(7.5) PstI

(6. 3) Nde I-r-" Pvu I (6.1 )

FIG. 2. Restriction map of plasmid 69-2-1. The circular portion and the linear portion represent pBR322 DNA and the inserted fragment, respectively. The BamHI site of pBR322 was diminished by cloning. The following endonucleases did not cut the inserted fragment: BamHI, Clal, HindIII, HpaI, NcoI, PvuI, ScaI, and XbaI. The open box and the arrow represents an ORF and the direction of transcription of the ORF, respectively (see the text). S and L represent two EcoRI fragments of 2.5 and 5.8 kbp produced by treatment with EcoRI (see the text).

fragment represents a unique sequence of the genome. Plasmid 69-2-1 hybridized to the fastest-moving band of TAFE, indicating that the insert localizes on chromosome I

(Fig. 4).

According to the present cloning protocol, the cloned gene should be induced in the presence of either cAMP or cycloheximide or both. To clarify this point further, we performed the following experiment. Poly(A) RNA was extracted from strain R28-3 (cyrllcyri) grown with or without cycloheximide. The same amounts of poly(A) RNA from the two cultures were fractionated by gel electrophoresis, transferred to a nitrocellulose membrane, and then probed

m H

uH

ItD~~~C

z ~~_

~

6~-

4. 4i

cu

with fragments S and L (Fig. 2). As shown in Fig. 5, a band corresponding to 1.3 kb was detected by both probes. The result indicates that the EcoRI site is in the transcribed region. Although cycloheximide caused an increase in the intensity of the band (Fig. 5), cAMP did not (data not

(1)

(2)

.9

C

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

I1

2.3

2.0

(kp)

65 OC

-

I

55 OC

FIG. 3. Hybridization of genomic DNA with a subfragment of plasmid 69-2-1. Genomic DNA (about 1 mg) of strain X2180-1B was digested with the indicated endonucleases, separated by electrophoresis, and transferred to a nitrocellulose membrane. The membrane was hybridized with the 32P-labeled S fragment (see the legend to Fig. 2) (10 ,ug, 3 pCi) at the indicated temperatures.

FIG. 4. Southern hybridization of chromosomal DNAs with plasmid 69-2-1. Chromosomal DNAs (from about 105 cells) were separated by TAFE and stained with ethidium bromide (lane 1). The DNAs were then transferred to nitrocellulose, hybridized with plasmid 69-2-1, and visualized by the universal probe method (lane 2) (see Materials and Methods). I, chromosome I.

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~ -23

J. BACTERIOL.

ONO ET AL.

VOL. 174, 1992

CLONING AND CHARACTERIZATION OF CYS3 (CYII)

+C

-c

ITGCAGATGTGTCTGGAAGGGTCACACCCGGAATTGCAGGTCTACTACTCGAAGA -841

C

-

3343

Pst' -840

-720

TTGCCGCGATCCGTGACTACAAGCTACACCGAGCGTACCAGCGACAGAAGTACGAGCm CATGCATCAACACAGAAACAATCGCTACCAGGACATTCATTCACCAGGACTTCCACAAGA

-721

AGGTCACCGACCTGCGAGCCAGGCTGCTGAACAGAACCACGCAGACCTGGTACfI^A EcoRV

-600

ACAAGGAGCGCCGCGATATGGATATAGTCATCCCAGATGTCAATTACCACGTCCCCATCA -601

AACTTGATAACAAGACGCTGAGCTGTATCACGGCTACGCCAGCGCAGCALA&

TGCTA

PvuII

1 .3

kbp

-480

TCCCGGCGAGCCCGTGGCAGAGGACCTCGCTTGCGAAAGCATCGAGTACCGCTACAGAGC -I81I CAACCCGGTGGACAAACTCGAAGTCATTGTGGACCGAATGAGGCTCAATAACGAGATTAG CGACCTCGAAGGCCTGCGCAAATAMCCACTCCTTCCCGGTGCTCCTGAGTTGAACCC -361

-360

GCTTAGAGACTCCGAAATCAACGACGACTTCCACCAGTGGGCCCAGTGACCGCCACACTG

Smal

GACCCCATACCACTTCTTTTTGTTATTCTTAAATATGTTGTAACGCTATGTAATTCCArC -241 -240 CTTCATTACTAATAATTAGCCATTCACGTGATCTCAGCCAGTTGTGGCGCCACACTTTTT

TTTCCATAAAAATCGGAAAAGAAAAGAAAAAAATAMCAGTTATTTAAAGCATA

-121

XhoI -120

AGATGCCAGGTAGATGGAACTTGTGCCGTGCCAGATTGAATTTTGAAAGTACAATTGAGG CCTATACACATAGACA M GCACCTTATACATATACACACAAGACAAAACCAAAAAAAAT ..... (TATA box)

-1

.

S

L

ATGACTCTACAAGAATCTGATAAATTTGCTACCAAGGCCATTCATGCCGGTGAACATGTG

121

* (Initiation codon) GACGTTCACGGTTCCGTGATCGAACCCA M CTTTGTCCACCACTTTCAAACAATCTTCT CCAGCTAACGCTATCGGTACTTACGAATACTCCAG^. CAAAATCCTAACAGAGAGAAC Bglll

241 361

481 601

shown). Therefore, we concluded that plasmid 69-2-1 contained a cycloheximide-inducible gene and designated it CYII. Although it was not the gene that we were primarily interested in, we analyzed it because of its inducibility by cycloheximide. Nucleotide sequence of CYI71. We sequenced a span of 2,254 bp covering most of the cloned DNA and found an ORF of 1,182 bp corresponding to 394 amino acid residues (molecular weight = 42,697) (Fig. 6). We therefore conclude that this ORF is the structural part of the CYIJ gene. Since the size of its mRNA was about 1.3 kb (Fig. 4), the noncoding region of the mRNA, including the poly(A) tail, must be about 100 bp. Therefore, we assume that TATACATATA starting at -36, or part of it, is the TATA box for this gene. We assume that TAA-(28 bases)-TAGT-(45 bases)-lTT' starting at 1224 and ending at 1303 is a transcription termination signal, because it resembles TAG--TA(T)GT--TTT (45). The partial N-terminal amino acid sequence (18 residues) of S. cerevisiae y-CTLase was determined (24) and was found to be identical to the N-terminal end of the deduced sequence except for the absence of terminal methionine. Therefore, it is concluded that CYIJ is the structural gene for -y-CTLase. According to the deduced amino acid sequence, -y-CTLase has three methionine residues (including the one for initiation) and no cysteine residue. Moreover, the gene product of CYII (i.e., ly-CTLase) had homology to several enzymes involved in biosynthesis of sulfur-containing amino acids (Fig. 7); the incidence of identical amino acids was 41% with rat y-CTLase and 36 and 25% with E. coli -y-CTSase and ,B-CTLase, respectively. The N-terminal half of S. cerevisiae y-CTLase (amino acids 48 to 222) had homology (39%) to the N-terminal half of S. cerevisiae OAS-OAH SHLase. It must be stressed that the three segments VDNTF (amino acids 178 to 182), SATKY (amino acids 201 to 205), and GHSD (amino acids 208 to 211) are common to the four enzymes mentioned above. It has been claimed that the second segment is part of an active site because pyridoxal 5'phosphate binds to K (lysine) of this element (18). It is likely that the other segments are involved in common functions such as interaction with the sulfur atom. Disruption of cellular CYIU. It is known that cys3 mutations confer deficiency of y-CTLase (29). Therefore, it is likely

721 841 961

1081

120

TTGGAAAGAGCAGTTGCCGCTTTAGAGAAGGCTCAATACGGGTTGGCTTTCTCCTCTGGT TCTGCCACCACCGCCACAATCTTGCAATCGCTTCCTCAGGGCTCCCATGCGGTCTCTATC GGTGATGTGTACGGT1iCCACACAGATACTTCACCAAAGTCGCCAACGCTCACGGTGTG Kpn I GAAACCTCCTTCACTAACGATTTGTTGAACGATCTACCTCAATTGATAAAGGAAAACACC AAATTGGTCTGGATCGAAACCCCAACCAACCCAACTTTGAAGGTCACCGACATCCAAAAG GTGGCAGACCTTATCAAGAAGCACGCTGCCGGCCAAGACGTGATCTTGGTTfiICGAAAC Sall

240

ACCTTCTTGTCCCCATATATCTCCAATCCATTGAACTTCGGTGCAGACATCGTTGTCCAC TCCGCTACAAAGTACATCAACGGTCACTCAGACGTTGTGCTCGGTGTCCTGGCCACTAAT AACAAGCCATTGTACGAGCGTCIGfi TTCTTACAAAACGCCATTGGTGCTATCCCATCT PstI

600

CCTTTCGATGCTTGGTTGACCCACAGAGGTTTGAAGACTMTGCATCTACGTGTCAGACAA GCTGCCCTCAGCGCCAACAAAATCGCTGAAIICTTGGCAGCAGACAAGGAAAACGTTGTC Eco RI GCAGTCAACTACCCAGGTTTGAAGACACACCCTAACTACGACGTAGTGTTAAAGCAACAC CGTGATGCCCTTGGTGGTGGTATGATCTCCTTCAGAATCAAGGGTGGTGCTGAAGCTGCT TCCAAGTTCGCCTCCTCCACAAGACTGTTCACATTGGCCGAATCCCTTGGTGGTATCGAA TCTCTATTGGAAGTGCCCGCTGTGATGACCCACGGTGGTATCCCAAAGGAGGCCAGAGAG

360 480

720 840 960

1080

GGCTCTGGTGTTTTTGACGACTTGGTTAGAATCTCTGTCGGTATTGAAGACACTGACGAT CTTTTGGAAGACATCAAGCAAGCCTTGAAACAAGCCACCACCTAATCGCCAGTGCCACGT *

1201

1200 (TerminatIon codon)

CTCTGCCTTCGACCGGACCTTTTTAAGTACGATAAATATCCTTTTATAAATATATAGTCT

AAAATATCCATTAATACTGTGCTCAATCAATCGTGTTAGATGATTTAGTTTTTTCCAAAT 1321

1320

CGTTATTATAGTGCAGAAGTAGTATACATAAAGGCATA

FIG. 6. Nucleotide sequence of the noncoding strand of the inserted DNA fragment. Restriction sites are underlined. , indicated nucleotide sequence signals (see the text); *, possible signals for transcription termination (see the text).

that CYS3 is the structural gene for -y-CTLase; i.e., CYIJ is identical to CYS3. To test this possibility, we carried out a gene disruption experiment. The 1.2-kbp HindlIl fragment containing URA3 was cut out from plasmid YEp24, and its two ends were altered to the BglII and BamHI sites by using the appropriate linkers. The fragment was then integrated into the BglII site of plasmid 69-2-1 (Fig. 2). The plasmid constructed in this way was transformed to a diploid strain, R115, following digestion with EcoRI and PvuII. Strain R115 (a SATase-deficient diploid) was used because it would avoid possible lethality caused by gene disruption and also facilitate identification of the disruptants; deficiency of both SATase and y-CTLase results in cysteine dependence (29). After transformation, uracil-independent clones were selected. A representative clone was sporulated, and the asci were dissected. We found that uracil dependence and cysteine dependence segregated in 2:2 ratios, respectively; all uracil-independent segregants were cysteine dependent, and all uracil-dependent segregants were cysteine independent. While all spore clones were SATase deficient, only cysteinedependent ones were additionally y-CTLase deficient (Table 2). Therefore, it is undeniable that one of the two CYS3 homologs in diploid strain R115 has been disrupted; that R115 is homozygous for cys1 is also indicated. This conten-

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FIG. 5. Northern hybridization with subfragments of plasmid 69-2-1. Poly(A) RNA (15 pLg) from strain R28-3 treated with (+C) or without (-C) 50 ,ug of cycloheximide per ml was fractionated by agarose gel electrophoresis and then transferred to a nitrocellulose membrane. The membrane was hybridized with the 32P-labeled S and L fragments (see the legend to Fig. 2); for each experiment, 10 p.g of DNA (5 ,Ci) was used for hybridization.

I

3344

ONO ET AL.

J. BACTERIOL.

H11

MTL ----- QESDKFATKAIHAGEHVDVH-GSVIEPISLSTTFKQSSPANAIGTYEY

[21

131

::R----------:Q::I:VRS:LND:EQY:C:VP::H::S:YNFTGFNEPR-AHD: :ADKKLDTQLVNAGRSKKYTLGAVNSVIQ-RASSLVFDSVEAK:HATRNR:N:ELF:

[41

:PSHFDTVQLHAGQENPGDNAHRSRAVPI-YATTSYVFENSKHGSQLFGLEVPG:V:

TABLE 2. Enzymatic activities of the gene disruptants Growth

on

(?)*CCGAAHL:A:::::D::GQSS:-FV:

[5]

[1I

51 SRSQNPNRENLERAVAALENAQYGLAFSSGSATTA-TILQS-LPQ-GSHAVSI

[2] [31

::RG::T:DVVQ::L:E::GGAGAVLTNT:MSAIH-LVTTVF:KP-:DLL:AP G:RGTLTHFS:QQ:HCE::GGAGCVL:PC:A:AV:NS::AF-IE:-:D:VLMT

t41

: :F:: :TSNV: :ERI::: :GGAAA: :V:: :Q:AQT-LAI :G-:AHT:DNI: :T

:::G::T:NC::K:::::DG:KHC:T:A::L:A:T-::THL-:KA-:DEVICM

[51

101

I11

GDVYGGTHRYFTKVANAHGVETSFTN-DLL-ND-LPQLIKENTKLVWIETPTN H:C:::SY:L:DSL:KRGCYRVL:VD-QGD-EQA:RAALA:KP:::LV:S:S: NTA:EPSQDFCS:ILSKL::T::WFD-P:IGA:-IVKHLQP:::I:FL:S:SG SYL::::YNQ:KISFKRF:I:AR:VEG:NP-EE-FEKVFD:R::A:YL::IG: DE:::::N:::RR:RSEF:LKI::VD-------------CSK:::--L:AAIT

[21

131 141 151

151

121 [31

141 151

201

I11 121 131 141 151

SATKYINGHSDVVLGVLATNNKPLYERLQFLQNAIGAIPSPFDAWLTHRG :C:::L:::::::A::VIAKDPDVVTE:AWA:N::VTGGA::SY:LL:: A::::LV::::ANI:TAVC:ARC-W:Q:RENAYLN:QNVDADT:YI:S:: ::::: G::GTTIG: IIVDSG:FPWKDYPEKFPQFSQPAEGYHGTIYNEA :::: :::::::M::LVSV:SDD:N:: :R::: :SL: :V:::: :CY:CC:: *****

5***

251

LKTLHLRVRQAALSANK I AEFLAADKENVVAVNYPGLKTHPNYDVVLKQH

(11

:R: :VP:MEL:QRN:QA:VKY:QTQ-PL:KKLYH:S:PENQGHEIAAR:Q

121 131 141

YGN: AYI :HVRTELLRDLGPLNNPFASFLLLQGVET: SLRAERHGENALK

[51

::HCRSGW:NTFQDGMAV:R::ESN-PR:EK:I::::PS::QHELAKRSA

:R::GV:L::HHE:SL:V::W:-:EHPQ:AR::H:A:PGSKGHEFWKRDF

301

RDALGGGNISFRIKGGAEAASKFASSTRLFTLAESLGGIESLLEVPAVMT

[11 [21

K-GF:-A:L::ELD:DEQTLRR:LGGLS::::::::::V:::ISHA:T:: TGSS:LFSFVLKK:LNN:ELANYLDNFS::SN:Y:W::Y:::ILANQPEH

131 141 151

LAKIWLEQSPYVSWVSYPGL::HSHHENAKKY: SNGF::VL:FGVKDLPNA

:AC--P::V::Y:::TLQH:QV:LKNIK::A:::::::Y:::A:L::I:: 351

(21

HOG I PKEAREGSGVFDDLVR I SVG I EDTDDLLED I KQALKQATT :A:NAP:::AAA:ISET:L:::T::::GE::IA:LENGFRA:NKG

131 141

DKETDPFKLS:AQ:V:N:KLA:NLANVG:AKTLV:APYFTTHKQ (46 residues)

[51

:ASV:EKD:ATL:IS:T:IIL:L:::L::EK:::LG::::A:HP

[11

IAA:RPQGEIDFS--GT:I:LHI:L::V:::IA:LDAGFARIV-

FIG. 7. Comparison of amino acid sequences. The amino acid sequence deduced from the nucleotide sequence of the CYS3 (CYII) gene (this study) ([1]) was compared with the amino acid sequences deduced from the nucleotide sequences of E. coli metB (-y-CTSase) (6) ([2]) and metC (,3-CTLase) (1) ([3]), S. cerevisiae MET17 (OAS-OAH SHLase) (5, 32) ([4]), and rat y-CTLase (7) ([5]). Amino acids, starting from the N termini, were aligned for the best match and were numbered according to amino acids deduced from S. cerevisiae CYS3 (CYII) (y-CTLase). gap; :, an identical amino acid; *, highly conserved amino acid (see the text).

+ + + +

y-CTLase

373 ND ND ND ND ND

4.62 7.62 11.58 1.75 0.52 8.61

-CYS is medium lacking cysteine. +, growth; -, no growth. "ND, not detectable.

CYIJ is in fact identical to CYS3. Hereafter, we refer to the gene as CYS3 or CYS3 (CYII). Effects of the cloned CYS3 (CYII) gene on E. coli. In order to examine whether CYS3 (CYII) would function in E. coli, we transformed strain LA5651 (metB) with plasmid pC5. The DNA-treated cells formed colonies on agar medium lacking methionine after 4 to 5 days of incubation, while the control cells did not. The colonies obtained, however, did not grow in liquid medium lacking methionine. They grew somewhat in liquid LB medium containing 50 ,ug of ampicillin per ml; plasmid pC5 was recovered from the cells, and the same event was repeated if the extracted plasmid was transformed to strain LA5651. When the cells grown in medium containing ampicillin were spread on agar LB medium, 10 to 20% of the resulting colonies did not grow at all on medium lacking methionine, while others showed marginal growth. The results suggest that plasmid pC5 is unstable in E. coli and that it supports only marginal growth without methionine partly because of its instability. The cells grown in liquid LB medium containing 50 ,.Lg of ampicillin per ml were examined for their -y-CTSase and Py-CTLase activities (Table 4). While the host strain, LA5651 (metB), had about one-tenth the y-CTSase activity of strain DH1 (met'), the transformants had -y-CTSase activity comparable to or even higher than that of strain DH1. It should be emphasized that y-CTSase activity was detected when OSH, but not OAH, was used as substrate. The observed enzyme activity contradicted results of no growth or poor growth of cells without methionine. However, this may be taken as an indication that the in vitro assay conditions are different from in vivo conditions for -y-CTSase. In addition, the transformants had higher levels of y-CTLase than the original strain (LA5651) (Table 4). In any case, these re-

-,

tion

was supported by the facts that the cysteine-dependent spore clones complemented cys2 cys4 strains but not cys]

cys3 strains and that the induced cysteine dependence showed linkage (7 centimorgans) to adel. Furthermore, the cysteine-dependent spore clones, like the authentic cysi cys3 strains, gave rise to cysteine-independent clones when transformed with plasmid pC5; pC5 was a shuttle plasmid constructed by inserting the HindIII-HindIII (ARSJ-TRPJCEN4) fragment (from plasmid YCp19) into the HindIll site of plasmid 69-2-1 (i.e., pBR322-CYIJ). The transformants all recovered their y-CTLase activity (Table 3). All of these results and the results of TAFE hybridization indicate that

TABLE 3. Functional complementation of cys3 mutations with the cloned CYIJ gene Strain

IS66-4C OK331-2D OK331-2D-1c OK331-2D-2' KN5-SA KN5-5A-sd

KN5-5A-1d

Growth on -CySa

+

+ + + +

-CYS is medium lacking cysteine. not detectable. Transformants of OK331-2D. " Transformants of KN5-5A.

"ND,

Sp act" (mU/mg of protein) SATase

-y-CTLase

373 ND ND ND ND ND ND

4.62 0.82 9.23 10.33 0.70 8.45 12.91

Downloaded from http://jb.asm.org/ on March 12, 2017 by guest

PTLKVTDIQKVADLIKKHAAGQDVILVVDNTF-LSPYISNPLNFGADIVVH :L:R:V::A:ICH:----:REVGAVS::::::-:::ALQ:::AL:::L:L: I:ME:H:VPAIVAAV--RSVVP:A:INI:::W-AAGVLFKA:D::I:VSIQ :KYN:P:FE:IVAIAH::----GIPV::::::GAGG:FCQ:IKY:::::T: ---------------------:TKLVW:::: :-1:A:FQR: :AL::: :CNC

[11

IS66-4C KT22-1A KT22-1B KT22-1C KT22-1D R115

Sp act" (mU/mg of protein) SATase

VOL. 174, 1992

CLONING AND CHARACTERIZATION OF CYS3 (CYII)

TABLE 4. Enzymatic activities of E. coli strain LA5651 (metB) transformed with plasmid pC5 Sp acta (mU/mg of protein)

y-CTSase

Strain OSH

DH1 LA5651 LA5651-tl LA5651-t2 LA5651-t3

0.27 0.02 0.50 0.45 0.42

± ± ± ± ±

OAH

0.03 0.00 0.10 0.12 0.11

ND ND ND ND ND

y-CTLase 6.7 12.1 19.9 17.4 15.5

± ± ± ± ±

1.3 2.2 3.2 1.1 1.0

a Numbers represent the averages and standard deviations of three independent experiments. For y-CTSase, OSH and OAH were used as substrates; NT and ND, not tested and not detectable (