YEAST

VOL.

7:953-961 (1991)

Molecular Cloning of the y-Glutamylcysteine Synthetase Gene of Saccharomyces cerevisiae YASUYUKI OHTAKE AND SEIZOU YABUUCHI Central Research Laboratories, Asahi Breweries Ltd., Omori-kita 2-13-1,Ota-ku, Tokyo 143, Japan

Received 8 March 1991 ;accepted 1 July 1991

The 4.4 kb SphI DNA fragment ( G S H I )that complementsthe y-glutamylcysteine synthetase-deficientmutation (gshl) of Saccharomyces cerevisiae YHl was cloned into vector plasmid YEp24. Gene disruption of the cloned fragment confirmed that this segment was the same gene as gshl. Mutant strain YHI with this plasmid not only restored y-glutamylcysteine synthetase (GSH-I) activity but the glutathione content and the growth rate. DNA sequence analysis of the SphI fragment showed that the G S H l structural gene contained 2034 bp and predicted a polypeptide of 678 amino acids. The deduced amino acid sequence had about a 45% homology to that of rat kidney GSH-I, but a very low homology (about 26%) to that of Escherichia coli GSH-I. Northern analysis showed that GSHI had been transcribed into an approximately 2.7 kb mRNA fragment. Southern analysis showed that GSHI mapped at chromosome X. KEY WORDS - Yeast; Saccharomyces

cerevisiae; glutathione; glutamylcysteinesynthetase.

Saccharomyces cerevisiae has a relatively large amount of GSH but its GSH-I activity is not high. Glutathione (GSH) is found widely in animals, Kistler et al. (1986) isolated GSH-deficient mutants plants and microorganisms. Published reviews of S . cerevisiae. Genetic analysis revealed that these (Meister and Anderson, 1983; Larsson et al., 1983; mutations belonged to a single complementation Sies, 1985; Douglas, 1987) have reported that GSH group and were recessive. Further characterization has various functions in cell protection against showed that they were GSH-I-deficient mutants xenobiotic toxicity, oxidative stress, protein and (Kistler et al., 1990). We previously reported that DNA synthesis, and amino acid transport. the GSH-I reaction may be the rate-limiting step in GSH is synthesized by two enzymatic reactions, the GSH synthesis of S. cerevisiae (Ohtake et al., that of y-glutamylcysteine synthetase (GSH-I) and 1988, 1989). We also isolated the GSH-I- and GSHthat of glutathione synthetase (GSH-11). GSH-I, the 11-deficient mutants of S. cerevisiae and reported first-step enzyme in GSH synthesis, catalyzes the on some of their properties (Ohtake et al., 1990). formation of y-glutamylcysteine (y-GC) from L- Each mutant was recessive and had a single nuclear glutamic acid and L-cysteine (Cys) in the presence of mutation in GSH biosynthesis. The respective ATP. GSH-11, the second-step enzyme, catalyzes mutations were named gshl and gsh2. The growth the formation of GSH from the y-GC and glycine in of both mutants in minimal medium was less than the presence of ATP. The genes for the GSH-I and that of the parent strain but reached the latter value GSH-I1 of Escherichia coli were cloned (Murata and when GSH was added to the medium. Moreover, Kimura, 1982; Murata et al., 1983) and sequenced the mutants were more sensitive to certain chemical (Watanabe et al., 1986; Gushima et al., 1984). In agents such as thiol-reactive and metal-chelating addition, the cDNA for a large subunit of the GSH- agents. We here describe the cloning of the y-glutaI of rat kidney was cloned and sequenced (Yan and Meister, 1990). Based on the genetic information, mylcysteine synthetase gene ( G S H I ) of S. the structures of GSH-I (Huang et al., 1988) and cerevisiae using the GSH-I-deficient mutant and GSH-I1 (Kato et al., 1987; Yamaguchi et al., 1990) report its nucleotide and deduced amino acid sequences. were analyzed. INTRODUCTION

0749-503>(!9 1/09095349 $05.00 0 1991 by John Wiley & Sons Ltd

954

Y. OHTAKE AND S. YABUUCHI

MATERIALS AND METHODS

0 r

A

Bacteria, yeast strains and plasmids

PLASMID

The strains of Saccharomyces cerevisiae used are shown in Table 1. The gene library of S. cerevisiae based on YEp24 was provided by Dr Kikuchi (Toho University, Japan). YEp24 was used as the vector plasmid for S. cerevisiae. E. coli strain DH1 (F- hsdRl7(R,,mg) recAI endA1 gyrA96 thi-1 supE44 h - ) was used for plasmid propagation. E. coli strain MV1184 (ara A(1ac-pro) strA thi-1 (@80 IacZA M15) A(str-recA)306::Tn10(tetT),F' traD36proAB IacPZA M15) and plasmids pUC 1 18 and p U C l l 9 were used for the DNA sequencing.

pYOGlOOl

0

2 ,

6

4

8

10

12 13.6(kb)

m

20.

m c

7-

ac

vi

m x m x

-

1

6

m" .

VECTOR GSH YEp24

(+I

YEp24

(*I

YEp24

(t)

YEp24

I+)

.

31

a

aa

6

f;

pYOG1011

.......

Table 1. S . cerevisiae strains used and gene disruption analysis Strain

Genotype

YNN27 AH22 YHI YHTl2l

M ATa trpl ura3 gal2 M ATa his4 leu2 M ATa trpl ura3 gal2 gshl MATa his4 leu2 GSH1::LEUZ

YHl

MATa trpl ura3 gal2 gshl

YHT121

MATa his4 leu2 GSHl::LEU2

YNN27

MATa trpl ura3 gal2

YHT121

MATa his4 leu2 GSH1::LEUZ

YHl

MATa trpl ura3 gal2 gshl

AH22

MATa his4 leu2

GSH*

+ +-

+

+

*The glutathione (GSH)-producing ability of the strains is expressed as + - .

Yeast transformation and other recombinant techniques

S. cerevisiae was transformed by the method of Ito et al. (1983). Other recombinant DNA procedures were carried out according to Maniatis et al. (1982). Screening Strain YHl was transformed with the gene library and plated on SD minimal plates (2% glucose, 0.67% Yeast Nitrogen Base w/o amino acid and 2%

Table 2. y-Glutamylcysteine synthetase (GSH-I) activities and the glutathione (GSH), y-glutamylcysteine (y-GC) and L-cysteine (Cys) contents of S . cerevisiae transfonnants GSH-I

Strain/plasmid YHI/pYOGIOll Y H 1/Y Ep24 YNN27

Contents (% w/w)t

Activity (units)*

GSH

y-GC

Cys

0.02 ND

1.038 ND

0.031

0.01

0.604

0.027 ND 0.050

0.059

0.003

*One unit is defined as one pmol of y-GC produced per h per mg protein. tGSH, y-GC and Cys contents are expressed as percentages of the dry cell weight. ND. not detected.

agar) containing 20 pg/ml of L-tryptophan. The plates were incubated at 30°C for 5 days, then used as the master plates for replica plating on SD minimal plates containing 20 pg/ml of L-tryptophan. The replicated plates were incubated at 30°C for 2

MOLECULAR CLONING OF THE y-GLUTAMYLCYSTEINE SYNTHETASE GENE

955

then resuspended in 25ml of l ~ s o r b i t o l .After the addition of Zymolyase 100 000 (Seikagaku Kougyo Co. Ltd., Tokyo) to a final concentration of o 1.00 100 pg/ml, the cell suspension was incubated at 0 0.5030°C with gentle shaking. An equal volume of a two0 fold concentration of YM medium (0.1% yeast 5 extract, 0.2% Bacto-peptone, 1% succinic acid, 3 0.100.002% adenine, 0.67% Yeast Nitrogen Base w/o 2 C!3 0.052 amino acid and 2% glucose) containing 0 . 8 ~ MgSO, was added to the prepared spheroplast suspension. After further incubation at 30°C for 30 0.0li ' ' ' ' ' ' 10 20 30 4 0 50 60 min, cycloheximide was added at a final concenIncubation Period (hr) tration of 50 pg/ml. The suspension was then incuFigure 2. Growth curves of the transformants. The curves bated at 30°C for 2 min and immediately poured are YHl/pYOGlOOl (0),YHl/YEp24 ( 0 ) and YNN27/ onto crushed ice prepared from 50ml of frozen pYOGl001 (U). 1 M sorbitol containing 50 pg/ml of cycloheximide. Spheroplasts were collected by centrifugation for 10 min at 3000rpm and 4"C, washed twice with I M sorbitol containing 50 pg/ml cycloheximide, then resuspended in 20 ml of 10 mM EDTA-3Na, 10mM-NaC1 and 1% SDS. The suspension was emulsified by shaking it with an equal volume of --4----4--+ phenol saturated with water at 60°C, after which it was centrifuged to obtain the aqueous phase. The 4 - t - c + aqueous phase was treated twice at room tempera+ + cture with phenol saturated with water followed by treatment with a mixture of phenol/chloroform --+500 bp (1 : 1). The final aqueous solution was mixed with a I--+ one-tenth volume of 5M-LiCl. The total nucleic Figure 3. Strategy for the sequencing of the G S H l gene of S. acids in the solution was precipitated by adding cerevisiue. The length and direction in the sequencing are shown three volumes of ethanol, then leaving the mixture by arrows. Abbreviations of the restriction sites are given in the at - 20°C for 15 h. The precipitate was dissolved in legend to Figure 1. 0.5 ml of 10 mM-Tris-HC1, pH 7.5, containing 0.5 M-LiCI and 0.5% SDS. Lastly, the poly(A) days, after which colonies that had restored GSH RNA was isolated by the mRNA affinity paper contents were screened by the coloring method of method (Wreschner and Henzberg, 1984). Agarose Apontoweil and Berends (1975). These colonies, gel electrophoresis of the poly(A) RNA was done which developed a violet-red color, were picked with 1.2% agarose in 0.2 M-sodium phosphate from the master plates and their GSH contents buffer (pH 6.8) containing 30% (v/v) formaldehyde measured. (Lehrach et al., 1977). The separated poly(A) RNA was transferred to a nylon membrane (Hybond-N, DNA sequencing Amersham International plc, U.K.) and subjected DNA sequences were determined by the dideoxy to northern hybridization (Meinkoth and Wahl, chain-termination method (Messing, 1983; Vieira 1984). The probe was the EcoRV-BarnHI fragment of G S H l labeled by nick translation with [aand Messing, 1987). 32P]dCTP (Amersham International plc, U.K.) using the procedure of Rigby et al. (1977). Northern blot analysis Strain YNN27 was grown to the early logarith- Mapping ofGSH 1 mic phase at 30°C with shaking in 500 ml of YPD Contour-clamped homogeneous electric field gel medium (1 YOBacto-peptone, 1 % yeast extract and 2% glucose). The cells were harvested by centrifuga- electrophoresis (CHEF) (Chu et al., 1986) of the tion, washed with an equal volume of sterile water, chromosomal standards of S. cerevisiue YNN295

5.00-----l

v

;L

'

--

-- ---

956

Y. OHTAKE AND S. YABUUCHI

+74 GCATGCAGCGGACGAAATACCGAATATTGAATCTCATATTGACTTCCTTCTTCTTCTTGCCCTGACGCTTTGTC +174 CTCTGCATTATGAAGCAGTCCAACGGCAGGATTGGACCCTGAAGTCTGATCAGGTCGCGGGCTCTCGGACGGTACGTTGACCAGAGATTGATAGGAAGGG +274 TGGAATACCTGAGGGGCCTCCGCTATGTCTGAATTCTGTGCATTCGAACCGACCCCTGGAGAAGCGTGCGTTATCGGGTTCTTACTTTTACAGTGATCTT +374 GCTCATCACGGAACTGTAACCAATCGTAGCTGTTCACAACTATCTTCTGGTGAGGATTTACGGTATGATCATGCTGGTAAGCTTCGTTACTCATGCTTTG +474 GTTGGCTTCTTGAAAGTGTCTAGTTCACTTTATGCCCGGTTATAAACATAAACTTTCACAGGGTTCTCTTTGATCACCTCTTTGTTTAATCTTATGAATC +574 CAAGGGATTTTATCGGTCAAAGGGGAAATCAATGCGAAAGACAGTAATGATGAGAGAAAAACTCTCCGTAACCACCAAGTTTGGTTCAGCGCGACGAGAT +674 TTTTATCGATTATCGAGAAAAATACCTGTATATCTACATTTCTATGTCAGTGATATATACTTCTTAGATAAGTTATGCCACCAGTGCATACGCTTACGCA 1 +774 CACACACGTATTCTTGTGCACACGCCTGTTACTTCTTGCAGACATCAGACATACTATTGTAATTCAAAAAAAAAAAGCGAATCTTCCCATGCCTGTTGCT +874 GCTCTTGAATGGCGACAGCCTATTGCCCCAGTGTTCCCTCAACAACCTTGGTA~CG~TAGCGTATCCTGTACCATACTAATTCTCTTCTGC 2 +974 CCAACGACGGCTGCCATTAGTCAGCATGGCGCGCACGTGACTACAACTGTGGCTGGAAAGAACGCCTTTTCGTCCTCCCCGGTTTTTCAGTGAGCCGACTCTACT +lo74 A~TGCTTTTTCATTTTTCACTCAGAAAAACCTG~TTGCCAAATTGGCCATGCTCTGTGCCTCCCTTGACAAAGGACATCTTCCCTGTT~C +1174 GGCGGCTTACCAAAAGTTGAAGCTTGTTCTTGCCTCTTATGAGTGGAG~CGAT~TTGAATCGTTGTGCTGGAGTAGTTGGATCTTTCCACGTGG 1 +1274 TCTCGAGTCACTTGTAGAAGCTGAAAATTGAGCAGATTTAG~GGGCTACATTGTAGGGTGGTTTAGAGTATCGAAAA~CA~GAAGAATAAAGAACG +1373 ATGGGACTCTTAGCTTTGGGCACGCCTTTGCAGTGGTTTGAGTCTAGGACGTACAATGAACACATAAGGGATGAAGGTATCGAGCAGTTGTTGTATATT

*

M

G

L

L

A

L

G

T

P

L

Q

W

F

E

S

R

T

Y

N

E

2

H

'

I

R

R

D

N

D

P

L

F

W

G

D

1

+1472 TTCCAAGCTGCTGGTAAAAGAGACAATGACCCTCTTTTTTGGGGAGACGAGCTTGAGTACATGGTTGTAGATTTTGATGATAAGGAGAGAAATTCTATG E L E Y M V V D F D D K E R N S M L D V C H D K I L T E L N M E . D 34 +1571 CTCGACGTTTGCCATGACAAGATACTCACTGAGCTTAATATGGAGGATTCGTCCCTTTGTGAGGCTAACGATGTGAGTTTTCACCCTGAGTATGGCCGG L D V C H D K I L T E L N M E D S S L C E A N D V S F H P E Y G R 67 +1670 TATATGTTAGAGGCAACACCAGCTTCTCCATATTTGAATTACGTGGGTAGTTACGTTGAGGTTAACATGCAAAAAAGACGTGCCATTGCAGAATATAAG Y M L E A T P A S P Y L N Y V G S Y V E V N M Q K R R A I A E Y K 100 +1769 CTATCTGAATATGCGAGACAAGATAGTAAAAATAACTTGCATGTGGGCTCCAGGTCTGTCCCTTTGACGCTGACTGTCTTCCCGAGGATGGGATGCCCC L S E Y A R Q D S K N N L H V G S R S V P L T L T V F P R M G C P 133 +1868 GACTTTATTAACATTAAGGATCCGTGGAATCATAAAAATGCCGCTTCCAGGTCTCTGTTTTTACCCGATGAAGTCATTAAGAACGCAGACATGTCAGGTTTCCT D F I N I K D P W N H K N A A S R S L F L P D E V I N R H V R F P 166 +1967 AACTTGACAGCATCCATCAGGACCAGGCGTGGTGAAAAAGTTTGCATGAATGTTCCCATGTATAAAGATATAGCTACTCCAGAAACGGATGACTCCATC N L T A S I R T R R G E K V C M N V P M Y K D I A T P E T D D S I 199 +2066 TACGATCGAGATTGGTTTTTACCAGAAGACAAAGAGGCGAAACTGGCTTCCAAACCGGGTTTCATTTATATGGATTCCATGGGTTTTGGCATGGGCTGT Y D R D W F L P E D K E A K L A S K P G F I Y M D S M G F G M G C 232 +2165 TCGTGCTTACAAGTGACCTTTCAGGCACCCAATATCAACAAGGCACGTTACCTGTACGATGCATTAGTGAATTTTGCACCTATAATGCTAGCCTTCTCT S C L Q V T F Q A P N I N K A R Y L Y D A L V N F A P I M L A F S 265 +2264 GCCGCTGCGCCTGCTTTTAAAGGTTGGCTAGCCGACCAAGATGTTCGTTGGAATGTGATATCTGGTGCGGTGGACGACCGTACTCCGAAGGAAAGAGGT A A A P A F K G W L A D Q D V R W N V I S G A V D D R T P K E R G 298 GTTGCGCCATTACTACCCAAATACAACAAGAACGGAACGGATTTGGAGGCATTGCCAAAGACGTACAAGAACGGATAAAGTCCTTGAAATACCA~GTCAAGATATAGT +2363 V A P L L P K Y N K N G F G G I A K D V Q D K V L E I P K S R Y S 331 TCGGTTGATCTTTTCTTGGGTGGGTCGAAATTTTTCAATAGGACTTATAACGACACAAATGTACCTATTAATGAAAAAGTATTAGGACGACTACTAGAG +2462 S V D L F L G G S K F F N R T Y N D T N V P I N E K V L G R L L E 364

Figure 4a

957

MOLECULAR CLONING OF THE V-GLUTAMYLCYSTEINESYNTHETASE GENE

+2561

AATGATAAGGCGCCACTGGACTATGATCTTGCTAAACATTTTGCGCATCTCTACAT~GAGATCCAGTATCTACATTCG~GAACTGTTG~TCAGGAC N D K A P L D Y D L A K H F A H L Y I R D P V S T F E E L L N Q D 397 t2660 A A C A A A A C G T C T T C A A A T C A C T T T G A A A A C A T C C A A A G T A C A A G C A A C C C C G G A C A A A A A G

N 430

K

T

S

S

N

H

F

E

N

I

Q

S

T

N

W

Q

T

L

R

F

K

P

P

T

Q

Q

A

T

P

D

K

K

t2759

GATTCTCCTGGTTGGAGAGTGGAATTCAGACCATTTGAAGTGCAACTATTAGATTTTGAGAACGCTGCGTATTCCGTGCTCATATACTTGATTGTCGAT D S P G W R V E F R P F E V Q L L D F E N A A V L I Y L I V D S I 463 t2858 AGCATTTTGACCTTTTCCGATAATATTAACGCATATATTCATATGTCCAAAGTATGGGAAAATATGAAGATAGCCCATCACAGAGATGCTATCCTATTT S I L T F S D N I N A Y I H M S K V W E N M K I A H H R D A I L F 496 t2957 GAAAAATTTCATTGGAAAAAATCATTTCGCAACGACACCGATGTGGAAACTGAAGATTATTCTATAAGCGAGATTTTCCATAATCCAGAGAATGGTATA

E

K

F

H

W

K

K

S

F

R

N

D

T

D

V

E

T

E

D

Y

S

I

S

E

I

F

H

N

P

E

N

G

I

529 t3056 TTTCCTCAATTTGTTACGCCAATCCTATGCCAAAAAGGGTTTGTAACCAAAGATTGGAAAGAATTAAAGCATTCTTCCAAACACGAGAGACTATACTAT

F 562

P

Q

F

V

T

P

I

L

C

Q

K

G

F

V

T

K

D

W

K

E

L

K

H

S

S

K

H

E

R

L

Y

Y

.-.__

r 7 1 GCI

TATTTAAAGCTAATTTCTGATAGAGCAAGCGGTGAATTGCCAACAACAGCAAAATTCTTTAGAAATTTTGTACTACAACATCCAGATTACAAACATGAT Y L K L I S D R A S G E L P T T A K F F R N F V L Q H P D Y K H D 595 t3254 TCAAAAATTTCAAAGTCGATCAATTATGATTTGCTTTCTACGTGTGATAGACTTACCCATTTAGACGATTCA~AGGTGAATTGACATCCTTTTTAGGA S K I S K S I N Y D L L S T C D R L T H L D D S K G E L T S F L G 628 +3353 GCTGAAATTGCAGAATATGTAAAAAAAAATAAGCCTTCAATAGAAAGCAAATGTTAAACTCCTTTTACTTCGGTTGTGAAAGAAAGTTGACATTATCGA A E I A E Y V K K N K P S I E S K C *

661

678 +3453 TTTGGGTGACACGGTGATTGAAAAAGCAACGACCAGTATTATACCTCTTTTTTTTATTATTCAGTTTATATTTTTGCAAGTGATCTTAAGCATTTCTACA +3553 CAAACTTATGCCAACGTGACCATTTATTATTTTATATAGCAAAAAAAAATGAGGGGCCTTGCAGAACAATTGTTGCGAGTTTCTAATAACAAGCACGTAG

t3653 AATATTGGCCATTTAATTTTTCTCTTCAATTTATAGAATGGTTGTGTTAGTGACAAAAAGAATATTCTTCCCCGCCAGGACTCGAACCT~AATCTC +3* GTTCGTAGCCAGACGCCGTGACCATTGGGCCACGAGGAACAAGAATATAAAGATCTCTGAGGGCAAGGTATGCCTATGTCGCAATAAAATGTTTGTTCCT

+3853 GCGCAAAAGTAAAGTTCTATTAATATACAACTACACAGTTATCGGTTCACACTATTCGATAGTTGTAAAAACCATTTTGATAAAGATATAACAAGGCGTT

+3953 TATTAAGGACATTTTTGCTACAAGTCGTGAAGTATTGATTGTAGGCGATCGTTGGTAACTTTCTCCATATCGGAATATTCAATATTGAACTCACCCCTCC +4053 CTTGCGATAAGCTCCTTAGCTTATTGGTGTAGGTGGTAATTTCCCTTAGTGGCACTTTCGCTTTTATAATTTTTTTTACATTAGAACTGGTCTCTTGAGT +4153 GTTTTTTTTATCTTTGGTTGCGTGTAGGGTTGTAACAGCATCAGAGGGAATATATATCCTATTGCTGTTCTCTGGAGAATTACAAGTGGATGCGCCAGAA +4253 TTTGA~ACGCTTGATTCGTCTTCAATAGACAGAATTTGAGCCTTTCTTGCTCCTGTTAGATCTTGTAACACCGTACCAACATCAGATTGAGGGATTGTTA t4353 GATCAAGATTCATGATGGGTTCCAGAAGGTTCTATTTTTCAGGTTTTAAGTCATTGAGAGCTTTAAAAATTAAGTTTCTAGTAATTTTTAAGATTTCTTG +4410 TGGAGTTTCAATATCAGGTGGGACTGACCAATCGCTAT.TGATTTTGATGGAGCATGC

Figure 4b Figure 4. The GSHl sequence. The DNA sequence of GSHl and the deduced amino acids sequence are shown. The length of the DNA and amino acid sequences are 4410 bases and 678 amino acids. The respective TATA and CAAT boxes are indicated by underlining at 1068, 1131, 1216 and 1255 and at 837,976, 1010 and 1123. Tandem repeats are indicated by arrows at 579 and 1225 ( I ) and at 828 and 1272 (2). The inverted repeat is indicated by the arrows at 3636 and 3650. The polyadenylated sequence is indicated by underlining at 3736. This sequence is found in the DDBJ, EMBL and GenBank Data Libraries under accession number D90220.

was done with a CHEF-DRTMIIsystem (Bio-rad Laboratories, California). The switch time was 60 s for 15 h followed by 90 s for 8 h at 200 V and 4°C. The gel was I .O% agarose in 0-5 x TBE (0.09 M-Tris, 0.09 M-borate, 2 mM-EDTA). The chromosomal standards that separated were transferred to a nylon membrane and subjected to Southern hybridization

(Meikoth and Wahl, 1984).The probe was the same as that used in the northern hybridization. M&mmment Of the G S H , Y-GC and CYS contents and the GSH-IactivitY The GSH, y-GC and Cys contents were measured using high performance liquid chromatography

958

Y. OHTAKE AND S. YABUUCHI

1

2

1

2

Chromosome XII

3,500

1,700

-

-

-

IV xv,vIIXVI XIIIII-

x1vxXI

v VIEIX

-

IEVI 1 -

Figure 5. Northern hybridization analysis of G S H I . A 1Opg sample of the poIy(A) RNA fraction of S. cerevisiue YNN27 was electrophoresed on an acrylamide gel. Lane I shows the 28s (1.7 kb) and 40s (3.5 kb) tRNAs of E. coli as the molecular weight'markers. Lane 2 shows the fragment hybridized with the '*P-labeled EcoRV-EumHI fragment of G S H I ; the position of the fragment is shown by the arrowhead.

(Ohtake et al., 1990; Richie and Lang, 1987). GSH-I activity was measured by the method of Jackson (1 969). RESULTS Molecular cloning of the complementingfragment of gshl Strain YHI was transformed with the gene library based on YEp24. About 5000 transformants were screened by complementation o f g s h l using the method of Apontoweil and Berends (1975). One transformant-restoring GSH was found. A plasmid, named pYOG 1001, was recovered from the transformant, and its restriction map determined (Figure IA). The inserted fragment was about 13.6 kb. The GSH-I activity and the GSH and y-GC contents of the transformant containing the pYOGIOOI were

Figure 6 . Chromosomal mapping of G S H I . The chromosome standards of S. rerevisiue YNN295 (Bio-cad Laboratories, California) (lane 1) were hybridized with the '*P-labeled EcoRVEumHI fragment of GSHI after blotting on a nylon membrane (lane 2). The position of the fragment is shown by the arrowhead.

restored (Table 2). Figure 1 B shows the subcloning strategy. The clone of the 4.4kb SphI fragment complemented gshl. The plasmid containing the SphI fragment was named pYOG1011. ~~~~d~~~~~~~~~ Subcloning showed that the SphI fragment contained the complementing region of gshl. To determine whether the complementing region was identical to the gshl gene, we performed gene disruption (Figure IC). The SphI fragment that contained the complementing region was cloned into the SphI site of p U C l l 9 to construct plasmid pYOG401 I . Plasmid pYOG40 1 1 was digested with EcoRV and XhoI, then ligated with the HpaI-SaA fragment containing LEU2 to construct plasmid pYOG121 I , which was then linearized by digestion with SphI. S. cerevisiae AH22 was transformed with this linearized fragment. The transformant YHT121 showing LEU2' was selected (the gene structure of strain YHT121 was confirmed by

959

MOLECULAR CLONING OF THE 7-GLUTAMYLCYSTEINE SYNTHETASE GENE 70

-S.

80

cerevisiae GSH-I Rat Kidney GSH-I

S. cerevisiae GSH-I KI Rat Kidney

GSH-I

--

-S.

cerevisiae GSHRat Kidney GSH-

S . cerevisiae GSH-I Rat Kidney

GSH-I

S. cerevisiae GSH-

Rat Kidney

GSH-

S. cerevisiae GSH-I S Eat Kidney GSH-

FEt#44-B?$I~BB-$$

481 490 . 500 510 520 530 S. cerevisiae GSH-I S FENIQSTNWQ K S WRVEFR V Eat Kidney GSH-I D t F E , I Q S , N W Q ~ ~ ~ $ P 4 ~ S ~ I ~ ~ " E F R ~ V ~ F E 56

S. cerevisiae GSH-I Rat Kidney

GSH-I

S. cerevisiae G S H - ! 4 ~ Q ~ ~ ~ ~ I & & ~ K6 H E

-

Rat Kidney

GSH-I I- I

SYLE

721 S. cerevisiae GSH-I RLTH-

Eat Kidney

RC---

___

DSK

GSH-I ~QIANE&~&&#&h~E'''

Figure 7. Homologous region in the deduced amino acid sequences of the GSH-Is of S. cerevisiae and rat kidney. The comparison of the GSH-Is of S. cerevisiae, rat kidney and E. coli was made with the program, DNASIS (Hitachi Software Engineering Co. Ltd, Tokyo). Identities are displayed in the open boxes. The respective identities between the GSH-I of S. cerevisiae and rat kidney and between the GSH-I of S. cerevisiae and E. coli are about 45% and 26%.

Southern analysis of the D N A 4 a t a not shown), and its GSH-I activity and GSH content measured. Gene disruption caused the loss of GSH-I activity and the GSH content of the transformant, YHT121. When the transformant, YHT121, was mated with strains YHI and YNN27, the resulting diploid, YHl/YHT121, did not restore GSH-I activity and the GSH content, but the diploid YNN27/YHT121 did, evidence that the cloned fragment was identical to the gshl gene. The cloned gene therefore was named G S H I . Growth curve

Growth curves of the transformants of strain YHl with pYOG1001 and YEp24 and those of strain YNN27 with pYOGl001 are shown in Figure 2. The growth rate of strain YH1 with pYOG1001 was approximately the same as that of strain YNN27 with pYOG1001, but that of strain YHI with YEp24 was not restored.

Nucleotide sequence of GSH 1

Based on the sequencing strategy shown in Figure 3, the 4-4kb SphI fragment was sequenced (Figure 4). The SphI fragment consisted of 4410 bases. The possible open reading frame had the ATG initiation codon at 1275 and the TAA termination codon at 3309 from the upstream SphI site and consisted of 2034 bases corresponding to 678 amino acids. Transcriptional signals, TATA and CAAT boxes, respectively were present at 1068, 1 131,12 16,1255 and 1261,and at 837,976,1010 and 1123. Two sets of tandem repeats were observed at 579 and 1125, and at 828 and 1147. The inverted repeat and the polyadenylated region of the eukaryote, AATAAA, were present respectively at 3636 and 3736. Northern analysis of GSH 1

To determine the length of mRNA of G S H I , we performed northern blot analysis (Figure 5). The

960 '2P-labeled BamHI-EcoRV probe of G S H l was hybridized with the 2.7 kb poly(A) RNA fragment, which indicates that G S H l was transcribed into the 2.7 kb mRNA. This length could cover the deduced GSHI open reading frame. Chromosomal mapping of GSH 1 Southern analysis of S. cerevisiae's chromosomal DNA fragments was done to map G S H l on the chromosome (Figure 6). The chromosomal DNA fragments were separated using a CHEF system. The 32P-labeledBamHI-EcoRV probe of G S H l was hybridized with chromosome X, evidence that GSHI is located in chromosome X. DISCUSSION We cloned the G S H l gene from S. cerevisiae using the screening method of Apontoweil and Berends (1975). Because the gshl mutant did not have y-GC and GSH, its colonies did not develop the violet-red color in the nitroprusside assay, an effective method for selecting colonies that restore GSH. Elsewhere we reported that the growth rates of GSH biosynthesis-deficient mutants were much slower than the growth rate of the parent strain YNN27, but were restored to the rate of the parent strain when GSH was added to the medium (Ohtake et al., 1990). In the study reported here, strain YHI containing the complementing plasmid pYOG1011 restored not only GSH biosynthesis but also growth to the rate of the parent strain. These results suggest that GSH participates in cell proliferation. To determine the functions of GSH, the effects of the growth of GSH biosynthesis-deficient mutants must be clarified. Three functions of GSH may affect the growth rate of S. cerevisiae: one is as the coenzyme of methylglyoxal (MG) metabolism. MG inhibits the growth of S. cerevisiae at a millimolar concentration (Ohtake et al., 1990). MG, which is present in a bypass of the glycolytic pathway, in the aminoacetone cycle, or both, is degraded by a glyoxalase system that consists of glyoxalase I, glyoxalase I1 and GSH. The second function is in the decomposition of hydrogen peroxide (H202).H 2 0 2is formed during aerobic growth and metabolized by GSH peroxidase in the presence of GSH and superoxide dismutase. The third function is in the detoxification of such chemical agents as heavy metals, thiolreactive agents and metal-chelating agents. There were five TATA boxes, four CAAT boxes and two sets of tandem repeats at the 5' flanking region of GSNI. These sequences are believed to

Y. OHTAKE AND S. YABUUCHI

affect the regulation of the transcription of G S H l . There were an inverted repeat at 3636 from the upstream SphI and AATAAA sequence, a polyadenylated region of the eukaryote, at 3736. The transcription of G S H l is thought to terminate at these sequences. Elucidation of the regulation mechanism of GSHI expression should reveal some of the functions of GSH. Northern analysis showed that GSHI was transcribed into the mRNA of about 2.7 kb. This supports the deduced transcription and termination signals. The y-glutamylcysteine synthetase genes of E. coli (Watanabe et a1.,1986) and rat kidney (Yan and Meister, 1990) have also been sequenced, and their deduced amino acid sequences compared (Yan and Meister, 1990). Both sequences showed low overall similarity. We compared the deduced amino acid sequence of S. cerevisiae GSHI with these sequences (Figure 7): the GSH-I of S. cerevisiae showed high overall similarity to that of the rat kidney (about 45%), but low overall similarity to that of E. coli (about 26%). These results suggest that the amino acid sequence of GSH-I is very conservative in the eukaryote. ACKNOWLEDGEMENTS We thank Professor H. Shimatake and Dr T. Kikuchi, Toho University (Japan), for providing the experimental installations and for their valuable discussions of our work. We also thank Dr Y. Kikuchi, Toho University, for providing the yeast gene library. REFERENCES Apontoweil, P. and Berends, W. (1975). Isolation and initial characterization of glutathione-deficient mutants of Escherichia coli KI2. Biochirn. Biophys. Acta 399,10-22. Chu. G.,Vollrath, D. and Davis, R. (1986). Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science 232, 1582-1 585. Douglas, K. T. (1987). Mechanisms of action of glutathione-dependent enzymes. In Meister, A. (Ed.), Advances in Enzymology, vol. 59. John Wiley & Sons Inc., New York, pp. 103-167. Gushima, H., Yasuda, S., Soeda, E., Yokota, M., Kondo, M. and Kimura, A. (1984). Complete nucleotide sequence of the E. coli glutathione synthetase gsh-11. Nucleic Acids Res. 12,9299-9307. Huang, C . , Moore, W. R. and Meister, A. (1988). On the active site thiol of y-glutamylcysteine synthetase: Relationships to catalysis, inhibition, and regulation. Proc. Natl. Acad. Sci. U S A 8 5 , 2 4 6 2 4 6 8 .

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