Gene, 121 (1992) 167-171 0 1992 Elsevier Science Publishers

GENE

B.V. All rights reserved.

167

0378-l 119/92/$05.00

06715

The

phenotype

of a dihydrofolate

reductase

of Succharomyces

mutant

cerevisiae (Gene replacement; mitochondria)

Tun Huang”,

dfrl; DHFR;

B.J. Barclay”,

“ Department of Genetics, Pharmacology,

Received

disruption

by J.A. Gorman:

ofNew

21 January

Edmonton,

yeast; folinic acid; thymidylate;

R.C. von Borstel”

T.I. Kalmanb,

University qf‘iilberia.

State University

mutation;

Alberta, Canada.

York at Buffalo, Buffalo, MY, USA.

1992; Revised/Accepted:

Tel. (403)492-l

fog; DIR; respiration;

methotrexate;

and P.J. Hastings”

108: and ’ Departments

of Medicinal

Chemistry and Biochemical

Tel. (716)636-2850

22 June/23

June 1992; Received

at publishers:

3 July 1992

SUMMARY

We have constructed a dihydrofolate reductase mutant (dfl) of Saccharomyces cerevisiae. The mutant has auxotrophic growth requirements for the Cl metabolites dTMP, adenine, histidine and methionine, similar to those of wild-type (wt) strains grown in the presence of methotrexate (MTX). However, unlike wt strains treated with MTX, the growth requirements of the dfrl mutant are not satisfied by exogenous 5-formyltetrahydrofolic acid (FA; folinic acid) in complex (YEPD) medium. This result is surprising, as yeast cells treated with MTX are expected to be phenocopies of dfrl mutants. The inability of the mutants to metabolize FA suggests that the DFRI gene product may have a role in folate metabolism in addition to its well-characterized function in the reduction of dihydrofolate. From dfrl strains, we have isolated secondary mutants whose growth can be supported by FA in YEPD medium. This FA-utilizing phenotype is attributable to recessive mutations which we have designated fou. In addition to their inability to metabolize FA, the dfrl strains are unable to grow on medium containing the non-fermentable carbon source glycerol, suggesting that the DFRI gene product is also required for mitochondrial function. In order to overcome this lack of respiratory activity in the dfrl mutants, we isolated strains containing a dominant mutation, DIR, which allows growth on glycerol in the presence of antifolate drugs. When crossed into dfrl strains, the DIR mutation conferred respiratory competence. These strains should be useful in a variety of studies on the genetics and biochemistry of folate metabolism in this simple eukaryote.

INTRODUCTION

The reaction catalyzed by dihydrofolate reductase (DHFR; EC 1.5.1.3) is an essential step in the biosynthesis of tetrahydrofolate. Selective inhibition of DHFR by antifolates limits the supply of intracellular tetrahydrofolate derivatives and has profound biochemical and genetic effects in yeast (Barclay et al., 1982). Yeast cells treated with

Correspondence

of Alberta,

to: Dr. P.J. Hastings,

Edmonton,

Alberta

Department

of Genetics,

T6G 2E9, Canada.

University

Tel. (403)492-5376;

(Fax) (403)492-1903.

the DHFR inhibitor MTX are auxotrophic for thymidylate (Laskowski and Lehmann-Brauns, 1973; Little and Haynes, 1979). Thymidylate starvation in yeast causes rapid loss of cell viability (thymine-less death) and a variety of genetic alterations including DNA strand breaks and greatly enhanced levels of mitotic recombination and gene conversion (Barclay and Little, 1978; Little and Haynes, 1979; Kunz et al., 1980; 1986; Barclay et al., 1982). In the

thymidine

thetic medium; ces; TMPI,

Abbreviations: methionine; tase; DIR,

Cl, one-carbon DFRI,

5’-monophosphate;

FOU, gene(s) required

metabolites

or dTMP/adenine/histidine/

gene encoding DHFR; DHFR, gene(s) permitting DHFR-independent

dihydrofolate respiration;

reducdTMP,

FA, folinic acid (5-formyltetrahydrofolate);

for FA utilization;

mt, mitochondrial;

gene encoding

yeast extract/peptone/dextrose extract/peptone/glycerol

MC, Mortimer’s

MTX, methotrexate;

thymidylate

synthase;

syn-

wt, wild type; YEPD,

(see Table II, footnote

(see Table II, footnote

complete

S., Saccharomv-

a).

a); YEPG,

yeast

168 mitochondrial genome, thymidylate starvation induces a variety of point mutations, formation of cytoplasmic petites and gradual loss of mitochondrial DNA (Barclay and Lit-

restriction fragment containing the URA3 gene from plasmid pLG669-Z (Guarente and Ptashne, 1981) into the unique Hind111 site within the coding region of the DFRl gene (Barclay et al., 1988). The dfrl::URAS disruption mutation was shown to be defective in the expression of DHFR activity, as, unlike vectors carrying the DFRI gene, plasmids with the disruption construct were unable to confer resistance to the DHFR inhibitor trimethoprim in

tle, 1978). These authors have proposed that the genetic consequences of thymidylate starvation involve aberrations in DNA replication and repair, but the detailed mechanisms responsible for these genetic effects are still not understood. In addition to thymidylate, yeast cells treated with MTX require adenine, histidine and methionine (Little and

Escherichia coli (Barclay et al., 1988). A BamHI-Sal1 fragment containing dfrl ::URA3 was used to replace the endogenous DFRl gene by transformation in strain YHl

Haynes, 1979). In both yeast synthetic (SD) and complex (YEPD) media, these folate-dependent metabolic end products can be replaced with exogenous FA to reverse the growth inhibition of yeast cells treated with anti-folate drugs

(Table I). The dTMP-permeable strain YH 1 was employed as the parental host for gene replacement. Since the growth requirements of yeast cells treated with MTX could be satisfied by exogenous dTMP, adenine, histidine and methionine (Table II), we included these Cl metabolites in the selection medium. In a yeast transformation following the method of Ito et al. (1983) several hundred transformants prototrophic for uracil were obtained. All Ura+ transformants were concomitantly auxotrophic for dTMP, adenine, histidine and methionine, as expected (Table III). Tetrad analysis indicated that dfrl and URA3 cosegregated in spore clones. Replacement of the dfrl : : URA3 mutation at the DFR 1 locus was also confirmed by Southern blot analysis (data not shown). DHFR activity was not detected in a dfrl strain (YH5) which was used in further studies (data not shown). Isolation of the yeast DHFR-deficient mutant in the presence of the Cl metabolites suggests that the DFRl gene

(Little and Haynes, 1979). In spite of attempts at isolating a variety of fol mutants, Saccharomyces cerevisiae strains deficit in DHFR activity have not been described to date. This has raised the possibility that a null mutation in the DFRI gene might be lethal. However, we have shown that this is not the case by constructing a viable dfrl mutant by gene replacement. We describe here the pleiotropic phenotype of the mutant.

EXPERIMENTAL

AND

DISCUSSION

(a) Construction of a yeast DFR mutant by gene replacement To construct a DHFR-deficient mutant by gene replacement (Rothstein, 1983), we first inserted in vitro a Hind111 TABLE

I

Yeast strains used in this study Genotype

Source

HC4-2B

MATa

Jitrgen Heinisch

UTL-‘IA

MATa urajl-52 IeuZ-3,112 trpl

Jilrgen

YHl

MATa ura3-52 leu2-3,112 trpl tup

This study

YH2

MATa ura3-52 leu2-3,112 trpl tup DIR

This study

YH5

MATa

This study

YH7 YH20-4B

MATa ura3-52 leu2-3,112 trpl tup @rl::URA3 fou MATa ura3-52 leu2-2,112 trpl tup

This study

YH22-1B

MATa

ura3-52 leu2-3,112 trpl tup dfrl::URA3

This study

YH23-IA

MATa

urajl-52 leu2-3,112 trpl tup dfrl::URA3

Haploid

Diploid

strain”

ura3-52 IeuZ-3,112 trpl

ura3-52 IeuZ-3,112 trpl tup dfrl::URA3

This study

fou

This study Source

Parents”

strain”

Heinisch

YH20

HC4-2B

1

This study

YH22

YH20-4B

x YH5

This study

YH23

YH20-4B

x YH7

This study

YH36

YH22-1B

x YH2

This study

YH55 YH57

YH22-1B

x YH5

YH23-1A YH23-1A

x YH5 x YH7

This study This study This study

YH77 a Yeast genetic methods described

have been described

by Little and Haynes

previously

(1979). The DIR mutant

100 pg MTX/S mg sulfanilamidei200

pg dTMP

x YH

(Savage

and Hastings,

YH2 was isolated

(all per ml) at 30°C.

1981). The dTMP-permeable

from YHl

strain YHI was isolated

in glycerol complex medium

supplemented

from UTL:7A

with 1.5 mg KH,PO,,

as

169 TABLE

plain why an E. coli fol mutant can be isolated in spite of the apparent need for formylation of the initiator tRNA

II

Growth

requirements

Growth

medium”

of strain YHl

in the presence

of MTX YHl(DFRZ)b

YEPD

during protein synthesis (Lengyel and Soil, 1969). Studies are in progress to determine whether this alternate pathway exists in yeast.

YEPD + MTX YEPD + MTX + dTMP

(b) Growth response of the dfrl mutant to FA

MC

The Cl metabolites that rescue cells from growth inhibition by MTX can be replaced with FA in both synthetic and complex media (Table IV). We therefore expected that FA should also satisfy the auxotrophic requirement of our dfrZ strains, as MTX-treated cells were assumed to be phenocopies of the dfrl mutant. Surprisingly, we found that FA even at very high concentrations (up to 500 pg/ml) did not support the growth of the dfrl mutant in complex medium, although there was some slow growth in synthetic medium under these conditions. It is interesting to note that a similar mutant phenotype is also found in mammalian DHFR-deficient cells (Urlaub and Chasin, 1980). One explanation for the difference in growth response to exogenous FA between DHFR-deficient mutants and DHFR inhibitor-treated wt cells might be that the DHFR enzyme has another enzymatic activity necessary for metabolizing FA, or that it is part of a multienzyme complex. It is conceivable that this enzymatic activity is absent in DHFRdeficient mutants but present in strains treated with antifolate drugs. The finding that exogenous FA supported the growth of dfrl mutants poorly in synthetic medium, but not in complex medium, suggests that differences in cellular physiology may also affect the metabolism or uptake of this folate derivative in yeast. Conversely, growth of dfrl cells in complex medium may result in the intracellular accumulation of toxic metabolic products (such as a metabolic derivative of dihydrofolate). The auxotrophic requirements for Cl metabolites of the

MC + MTX MC+MTX+Cl MC + MTX - dTMP MC + MTX - adenine MC + MTX - histidine MC + MTX - methionine a Yeast growth media have been described ings, 1981; Sherman metabolic

end products:

- dTMP,

- adenine,

omitted medium

dTMP,

- histidine

from the Cl metabolites

periments,

previously

et al., 1984). Cl represents adenine,

histidine

and - methionine in the growth

or 2 pg/ml in MC medium.

and methionine.

The

For routine ex-

of 25 pg/ml in YEPD

In all MTX-containing

was added. In YEPD

and Hast-

indicate that this was

medium.

MTX was added to a final concentration

sulfanilamide,lml

(Savage

the four folate-dependent

100 pg dTMP/ml

media 5 mg

was added when

indicated. h Growth

is indicated

by ( + ) and no growth

by ( - ). For YHl

see

Table I. TABLE

III

Growth

requirements

Growth

medium”

of the dfrl mutant

YH5

YH5(dfZ::URA3)b

YHI(DFRl)”

YEPD

_

+

YEPD + dTMP MC - uracil

+ _

+ _

MC - uracil + Cl

+

_

MC+Cl MC - dTMP

+

MC - adenine

+ _ _

MC - histidine

_

+ +

MC - methionine

_

+

a See Table II, footnote

+

TABLE

a.

’ See Table I and Table II. footnote

b.

Growth

IV response

of dfrl and MTX-treated

DFRZ strains

to exogenous

FA

is not essential for cell viability as long as these nutritional supplements are present in the growth medium. In addition, tetrad analysis showed that the dTMP requirement segregated 2:2, as expected for a single allele. This result indicates that unlike E. coli DHFR-deficient mutants (Howell et al., 1988; Ahrweiler and Frieden, 1988), yeast dfrl strains do not require a second mutation at the TMPl locus for viability. A recent study by Hamm-Alvarez et al. (1990) has shown that E. coli mutants deficient in DHFR activity still contain considerable amounts of intracellular tetrahydrofolates. This observation suggests that another biosynthetic pathway exists in E. coli for the production of tetrahydrofolate in addition to synthesis via DHFR. This finding may ex-

Growth

medium”

Strainsb YH5 (dfrZ;tURA3)b

YHl

YEPD

_

t

YEPD + FA

_

Y EPD + MTX YEPD + MTX + FA

_ _

f _

MC

_

+ +

MC+FA MC + MTX

+I_

+ _

MCtMTX+FA

+I-

+

a FA was supplemented at the final concentration experiment. See also Table II, footnote a. b See Table III, footnote

b; + / - indicates

of 500pg/ml

poor growth.

(DFRI)

in this

170 dfrl mutants

isolated in this study are similar to those of other yeast mutants (tmp3, foil, fi12) defective in tetrahydrofolate biosynthesis (Luzzati, 1975; Little and Haynes, 1979). However, our dfrl strains differ from these yeast

genome and ultimately causes its degradation (Barclay and Little, 1978; Newlon et al., 1979). We considered that a deficiency in the biosynthesis of thymidylate might be responsible for the respiratory deficiency of the dfrl cells.

mutants in two important respects. Firstly, dfrl mutants fail to grow in complex medium supplemented with FA. Second, they are all petite (see section c).

However, the isolation of stable respiratory-proficient tmpl mutants suggests that there is no connection between auxotrophy for dTMP and an obligate respiratory deficiency in yeast cells, as long as sufficient dTMP is present in the

During the analysis of the growth response of the dfrl mutants to FA, we isolated subclones which grew on FA in YEPD medium. We designated the gene fou for the mutation responsible for this folinic acid utilization phenotype. We then crossed a FA-utilizing dfrl mutant YH7 with a haploid strain to generate the heterozygous (DFRI/dfrl FOU/_fbu) diploid strain YH23. Tetrad analysis of strain YH23 and studies on the growth response of the homoallelic dfrl diploids YH57 (FOU/fou) and YH77 (fozqfou) to exogenous FA in YEPD indicate that this mutation responsible for folate utilization is recessive and is not linked to the DFRI locus (Table V). (c) Respiratory competence of dfrl mutants In addition to the defect in FA metabolism, we also found that all dfrl:: URA3 isolates failed to grow on nonfermentable carbon sources. We first considered the possibility that the respiratory deficiency of the dfrl transformants might be a consequence of our selective method. To test this, a respiratory-competent diploid (YH22) heterozygous for the dfrl disruption was constructed. All dfrl mutant spores in tetrads of this diploid did not grow on nonfermentable medium, even in the presence of concentrations of dTMP up to 200 pg/ml. Furthermore, from a heterozygous dfrrl fou diploid (YH23), we were unable to isolate grande dfrl segregants in the presence of high concentrations of FA and/or dTMP. Moreover, all homozygous dfrl mutant strains (YH55, YH57, YH77) were found to be deficient in sporulation, characteristic of yeast petite diploids. The above observations indicate that the DFRl gene product is essential for respiratory activity in yeast. Previous studies have shown that thymidylate starvation in yeast mutants deficient in thymidylate synthase activity (tmpl and cdc21) is highly mutagenic for the mitochondrial TABLE

V

Utilization Growth

of exogenous

medium”

YEPD + FA Y EPD + MTX + FA

FA by a secondary

dfrrl isolate

Strains’ YH5

YH7

YH57

YH77

FOU

.fou

FO U/f&

f&/fOU

_

+ +

_

’ See Table IV. ’ See Table I and Table II, footnote

b.

+ _

+

growth media. As we failed to isolate respiratory-competent segregants by using higher concentrations of dTMP (up to 200 pg/ml), the respiratory deficiency of the yeast dfrl mutant could not be explained solely by mutagenic effects of thymidylate stress on the mt genome. We considered it more likely that the respiratory deficiency of dfrl mutants was the result of blockage in the initiation step of mt protein synthesis. Smith and Marcker (1968) have demonstrated that in vivo initiation of yeast mt protein synthesis requires formylmethionyl-tRNAf”“‘. The formyl group is donated by lo-formyltetrahydrofolate. Therefore, depletion of cellular tetrahydrofolate pools caused by deficiency in DHFR activity would block mt protein synthesis. Studies by Wintersberger and Hirsch (1973a,b) have suggested that MTX induces petite mutations through inhibition of mt protein synthesis. However. Shannon and Rabinowitz (1988) reported that deletion of the yeast MIS1 locus encoding the mt Cl-tetrahydrofolate synthase, a trifunctional polypeptide responsible for the interconversion of various specific Cl derivatives of tetrahydrofolate including the production of lo-formyltetrahydrofolate, has no effect on growth on a nonfermentable carbon source such as glycerol. It is not known whether the initiation of mt protein synthesis can occur in the absence of formylation of methionyl-tRNA”‘“’ or if the activity of cytoplasmic C 1-tetrahydrofolate synthase encoded by the ADE3 gene can provide sufficient intracellular levels of activated one-carbon units for yeast mt protein synthesis. Isolation of viable mammalian mutants deficient in DHFR activity (Urlaub and Chasin, 1980) raises similar questions. Studies on the enzymatic defects in folate-requiring strains have suggested that two parallel sets of enzymes involved in folate coenzyme-mediated Cl metabolism are differentially distributed between yeast mitochondria and cytoplasm (Zelikson and Luzzati, 1976; 1977; Appling, 1991). It has been demonstrated that two genes @DE3 and MZSI) are responsible for the Cl-tetrahydrofolate synthases of yeast cytoplasm and mitochondria, respectively (Staben and Rabinowitz, 1986; Shannon and Rabinowitz, 1988; Barlowe and Appling, 1990). Interestingly, a misl mutant is respiratory-competent, suggesting that a functional MIS1 gene is completely dispensable for mt activity. In the study by Zelikson and Luzzati (1977) the tmp3 mutant was shown to be petite and deficient in the mt enzymes serine transhydroxymethylase, dihydrofolate redfrl mutant

171 ductase and thymidylate synthase, but not in their cytoplasmic counterparts. The mutant requires dTMP, adenine, histidine and methionine for growth. However, it is not clear why a mutant strain apparently deficient only in mt enzymes would have an auxotrophic phenotype, as yeast rho” strains have no auxotrophic requirements. Our results indicate that the DHFR activity encoded by the DFRI gene is required both for the biosynthesis of dTMP, adenine, histidine and methionine and for mt function in yeast. In a further attempt to obtain a respiratory-competent dfrl strain, we isolated mutants which were able to grow on glycerol in the presence of antifolate drugs and the Cl metabolites. We supposed that these mutants bypassed the inhibitory effect of MTX on mt respiratory function. A heterozygous diploid YH36 made from one such mutant strain (YH2) was also able to grow on glycerol in the presence of antifolates and the Cl metabolites. From the diploid YH36 made by a cross of this mutant with a dfrl strain, respiratory-competent dfvl segregants were readily isolated as spore segregants on either YEPD medium containing 100~8 dTMP/ml or YEPG medium containing 200~8 dTMP/ml. These results indicate that a dominant mutation is responsible for the respiratory competence of both yeast DFRl strains treated with antifolate drugs and the dfrl mutants. We have designated the mutation responsible for this dihydrofolate reductase-independent respiration as

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The phenotype of a dihydrofolate reductase mutant of Saccharomyces cerevisiae.

We have constructed a dihydrofolate reductase mutant (dfr1) of Saccharomyces cerevisiae. The mutant has auxotrophic growth requirements for the C1 met...
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