Redox regulation of AMP synthesis in yeast: a role of the Bas1p and Bas2p transcription factors

Molecular Microbiology (2000) 36(6), 1460±1469

Redox regulation of AMP synthesis in yeast: a role of theBas1p and Bas2p transcription factors

BenoõÃt Pinson,1,2 Odd S. Gabrielsen2 and

Bertrand Daignan-Fornier1*1Institut de Biochimie et GeÂneÂtique Cellulaires, CNRS

UPR9026, 1, rue Camille Saint-SaeÈns, F-33077 Bordeaux

Cedex, France.2Department of Biochemistry, University of Oslo, PO Box

1041 Blindern, N-0316 Oslo, Norway.

Summary

Expression of yeast AMP synthesis genes (ADE

genes) was severely affected when cells were grown

under oxidative stress conditions. To get an insight

into the molecular mechanisms of this new transcrip-

tional regulation, the role of the Bas1p and Bas2p

transcription factors, known to activate expression of

the ADE genes, was investigated. In vitro, DNA-

binding of Bas1p was sensitive to oxidation. How-

ever, this sensitivity could not account for the

regulation of the ADE genes because we showed,

using a BAS1-VP16 chimera, that Bas1p DNA-binding

activity was not sensitive to oxidation in vivo.

Consistently, a triple cysteine mutant of Bas1p (fully

resistant to oxidation in vitro) was unable to restore

transcription of the ADE genes under oxidative

conditions. We then investigated the possibility that

Bas2p could be the oxidative stress responsive

factor. Interestingly, transcription of the PHO5 gene,

which is dependent on Bas2p but not on Bas1p, was

found to be severely impaired by oxidative stress.

Nevertheless, a Bas2p cysteine-free mutant was not

sufficient to confer resistance to oxidative stress.

Finally, we found that a Bas1p±Bas2p fusion protein

restored ADE gene expression under oxidative con-

ditions, thus suggesting that redox sensitivity of ADE

gene expression could be due to an impairment of

Bas1p/Bas2p interaction. This hypothesis was further

substantiated in a two hybrid experiment showing

that Bas1p/Bas2p interaction is affected by oxidative

stress.

Introduction

AMP synthesis from 5 0-phosphoribosyl-1-pyrophosphate

is a conserved biosynthesis pathway, involving 12 success-

ive enzymatic steps. In yeast, the enzymes of this

pathway are encoded by the ADE genes. In the presence

of exogenous adenine in the medium, expression of these

genes is downregulated at the transcriptional level

(Daignan-Fornier and Fink, 1992; Denis et al., 1998).

Two transcription factors, Bas1p and Bas2p (Pho2p),

activate expression of these genes (Daignan-Fornier and

Fink, 1992; Denis et al., 1998) and are required for proper

regulation by adenine (Zhang et al., 1997). Adenine

regulation requires several purine salvage enzymes

(Guetsova et al., 1997) and probably affects the interac-

tion between Bas1p and Bas2p (Zhang et al., 1997) rather

than their ability to bind DNA or their nuclear localization

(Pinson et al., 1998).

Bas1p and Bas2p also activate expression of three

genes in the histidine biosynthesis pathway (Arndt et al.,

1987; Springer et al., 1996; Denis et al., 1998). In a gcn4

background, mutations that abolish the BAS1 or BAS2

function lead to an histidine auxotrophy (Arndt et al.,

1987) and therefore their function can be easily scored in

vivo. Moreover, bas1 and bas2 mutants are bradytrophic

for adenine, probably because expression of some ADE

genes is not sufficient to sustain AMP supply (Arndt et al.,

1987).

Bas2p, first identified as Pho2p, is a homeodomain

protein that, together with Pho4p, activates expression of

several phosphate utilization genes in response to

inorganic phosphate availability (Vogel et al., 1989).

Bas1p is not required for phosphate utilization pathway

regulation. Bas1p contains a Myb-like DNA-binding

domain located in the N-terminal part of the protein

(Tice-Baldwin et al., 1989). This DNA-binding domain is

composed of three imperfect tandem repeats containing

highly conserved tryptophan residues, which are essential

for binding to DNA (Pinson et al., 1998). Interestingly, the

Bas1p DNA-binding domain contains a cysteine residue

that is conserved among most of the Myb family

members. This residue was shown to be critical for

redox regulation of c-Myb DNA binding in vitro ( Myrset

et al., 1993).

Indeed, several mammalian transcription factors,

including c-Fos, c-Jun, NF-kB, p53 and c-Myb, are able

to bind in vitro to DNA under reducing conditions, whereas

Q 2000 Blackwell Science Ltd

Accepted 12 April, 2000. *For correspondence. E-mail B. [email protected]; Tel. (133) 5 5699 9055; Fax (133) 55699 9059.

the addition of an oxidizing agent, such as diamide,

abolishes their interaction with DNA (Abate et al., 1990;

Toledano and Leonard, 1991; Xanthoudakis et al., 1992;

Hainaut and Milner, 1993; Hupp et al., 1993; Myrset et al.,

1993). Specific cysteine residues in the DNA-binding

domain of these proteins have been shown to be required

for this redox response (Abate et al., 1990; Toledano and

Leonard, 1991; Xanthoudakis et al., 1992; Myrset et al.,

1993; Rainwater et al., 1995). This behaviour has been

observed in a growing number of transcription factors,

involved in various biological processes, suggesting that it

reflects a general response to oxidative stress, thereby

allowing downregulation of multiple genes. Nevertheless,

because most of these studies have been carried out

mainly in vitro, the physiological relevance of this redox

response remains to be clarified. We therefore wished to

determine whether a similar redox regulation could exist for

the yeast transcription factor Bas1p and its target genes.

In this paper, we show that several yeast purine

biosynthesis genes are subjected to redox regulation in

vivo. We also document the effect of oxidative stress on

the function of Bas1p and Bas2p. Our results imply that, in

vivo, the oxidative sensitivity of ADE genes can not be

explained only by DNA-binding impairment of Bas1p and

Bas2p, but could instead be due to a defective interaction

between these transcription factors.

Results

Expression of AMP biosynthesis genes is impaired by

oxidative stress

We first examined whether oxidative conditions could

affect the expression of AMP biosynthesis genes in vivo.

The ADE1, ADE5,7 and ADE17 genes were previously

shown to be tightly regulated by Bas1p (Daignan-Fornier

and Fink, 1992; Rolfes et al., 1997; Denis et al., 1998),

which binds directly on their promoters (Rolfes et al.,

1997; Pinson et al., 1998). Expression of ADE1, ADE5,7

and ADE17 transcripts decreased after a 15 min treat-

ment with the oxidizing agent diamide, and were hardly

detectable after 30 min (Fig. 1A). Similar results were

obtained after treatment with sodium selenite, another

oxidizing agent (Fig. 1B). Treatment with these oxidizing

agents had no effect on the amount of messenger of HIS5

or ADE16 (Fig. 1A and B), two genes in the histidine and

purine biosynthesis pathways, respectively, which are

unaffected by mutations in the BAS1 gene (Denis et al.,

1998; see also Fig. 1A,B lanes `Bas1p-'). Finally, expres-

sion of control transcripts such as rRNA or ACT1 was not

significantly affected by the mutation in BAS1 or by the

oxidizing agent (Fig. 1A). To confirm this result by a

different approach, we monitored Bas1p-regulated genes

in a trx1 trx2 double-mutant strain. In this mutant, the

production of thioredoxin is deficient (Muller, 1991) and

high levels of oxidized glutathione are observed (Muller,

1996), leading to a decreased reducing power. Expres-

sion of the same set of genes in this strain was assayed

by Northern blot in the absence of external oxidizing

agent. As expected, the ADE1 and ADE17 transcripts

were far less abundant in the thioredoxin mutant than in

the isogenic wild-type strain, while expression of HIS5,

ADE16 and ACT1 was unaffected (Fig. 1C), thus con-

firming the diamide and sodium selenite results.

Bas1p binding to DNA in vitro is affected by oxidation in a

cysteine-dependent manner

The molecular mechanism leading to the redox regulation

of ADE genes was then investigated. By analogy with the

situation described for c-Myb ( Myrset et al., 1993), we

first tested whether this regulation could be due to a

defective DNA binding of either the Myb-like transcription

factor Bas1p or the homeodomain protein Bas2p. DNA

binding of purified Bas1p and Bas2p proteins was tested

using the electrophoretic mobility shift assay (EMSA) in

the presence of increasing concentrations of the oxidizing

agent diamide. The results presented in Fig. 2A show that

Fig. 1. The effect of oxidation on transcription of Bas1p targetgenes.A, B. The effect of diamide (A) or sodium selenite (B) on ADE1,ADE5,7, ADE16, ADE17 and HIS5 expression. The bas1 strainL3080 was transformed with plasmids B836 (Bas1p±) or P79(Bas1p1) and grown in SD 1 casamino acid medium to an OD600

of 0.5. Then, diamide (3 mM) or sodium selenite (1 mM) was addedto the medium and total RNA were extracted at indicated times.C. The effect of mutations in the thioredoxin genes on ADE1, ADE16,ADE17 and HIS5 transcription. Strains EMY60 (TRX1 TRX2), orEMY63 (trx1trx2) were grown in SD 1 casamino acid medium,containing 0.15 mM of adenine, to an OD600 of 0.5.In A, B and C, equal loading on each lane was monitored by ethidiumbromide staining of the gel showing the large (L. rRNA) and small (S.rRNA) rRNAs and by an ACT1-specific probe hybridization. Eachhybridization was carried out independently and assembled for thefigure.

Redox regulation of AMP synthesis genes in Saccharomyces cerevisiae 1461

Q 2000 Blackwell Science Ltd, Molecular Microbiology, 36, 1460±1469

binding of Bas1p to DNA was totally abolished in the

presence of 5 mM diamide whereas Bas2p binding was

unaffected at concentrations of diamide up to 50 mM. A

narrower range of diamide concentrations allowed us to

show that 1.5 mM was indeed sufficient to impair DNA

binding of GST±HA±Bas1p to DNA (Fig. 2B, panel `wild

type'), although in some experiments intermediary binding

was observed at this concentration (data not shown). The

redox sensitivity of Bas1p was likely to occur through

cysteine residues, which are common molecular targets

for oxidative regulation. Bas1p contains three cysteine

residues, all of which are located in the DNA-binding

domain, one in each Myb-like repeat. Replacement of

these three cysteine residues of Bas1p by hydrophobic

residues (Ala or Val) led to fully functional proteins for

their ability to restore histidine prototrophy in a gcn4

background (Fig. 2C). The corresponding purified mutant

protein was assayed for in vitro binding to DNA by EMSA

and was found to be fully resistant to high concentrations

of diamide (Fig. 2B, panel C82AC153VC206V). A Wes-

tern blot of both the wild type and C82AC153VC206V

purified GST±HA±Bas1p (Fig. 2D) showed that similar

amounts of proteins were used in this experiment.

Altogether, these results demonstrate that, in vitro, the

Myb-like protein Bas1p is sensitive to oxidation in a

cysteine-dependent manner.

Expression of BAS1-regulated genes is still affected by

oxidative stress in the BAS1 triple cys mutant

We then wondered whether the Bas1p triple mutant,

which is totally insensitive to diamide in vitro, would make

ADE17 expression insensitive to diamide in vivo. Expres-

sion of ADE17 was monitored by Northern blot in a bas1-

deleted strain transformed with a centromeric plasmid

carrying either the wild-type BAS1 gene or the triple

cysteine mutant of BAS1. The results (Fig. 3A) clearly

show that the amount of ADE17 transcript was still

downregulated after treatment with diamide, even for the

BAS1 triple mutant which is insensitive to diamide in vitro.

Similarly, the triple BAS1 mutant did not rescue expres-

sion of ADE1 or ADE17 in the trx1trx2 mutant strain

(Fig. 3B). Therefore, whereas the triple cysteine mutant is

sufficient to confer to Bas1p a total resistance to diamide

in vitro, the Bas1p activated genes are still affected by

oxidation in vivo.

Fig. 2. The relative role of the cysteine residues in the sensitivity of Bas1p to oxidation by diamide.A. In vitro sensitivity to diamide of Bas1p and Bas2p. Purified Bas1p and Bas2p (1 mg each) were incubated for 15 min at room temperaturewith increasing concentrations of diamide. Samples were then incubated for 15 min at room temperature with 100 fmol of an ADE5,7promoter-radiolabelled probe and were finally separated on 4% non-denaturing gel electrophoresis. Solid and open arrows indicate Bas1p andBas2p complexed to DNA respectively. Lanes C1 and C2: probe-alone control.B. In vitro sensitivity to diamide of the wild-type (lower panel) and C82AC153VC206V mutant (upper panel) of Bas1p. Purified GST±HA±Bas1p wild-type or cysteine triple mutant (1 mg of each) were incubated with diamide and submitted to EMSA as in Fig. 2A. For theC82AC153VC206V Bas1p mutant, only the part of the gel containing the GST±HA±Bas1p/DNA complexes is shown. Solid arrows indicateBas1p complexed to DNA.C. In vivo effect of the mutations in the three cysteine residues of Bas1p on histidine requirement. L3080 cells were transformed with eitherthe B836 empty plasmid (control), or with a plasmid containing the wild-type BAS1 gene (WT) or the cysteine free BAS1 gene (TM). Serialdilutions of the cells (104, 103, 102 and 101 cells) were spotted on SC medium lacking uracil and supplemented (left panel) or not (right panel)with histidine. Cells were grown for 36 h at 288C.D. Western blot analysis of purified wild-type and mutant GST±HA±Bas1p. Four microgrammes of each purified protein were subjected to 12.5%SDS±PAGE and electroblotted on PVDF membrane. The blot was incubated with anti-HA antibodies (0.5 mg ml21) followed by horseradishperoxidase labelled IgG (1 : 2500) as secondary antibodies and, finally, luminescent substrate before exposure to film. The GST-HA protein(25 kDa) in the control lane is much smaller than the GST±HA±Bas1p fusion (115 kDa) and therefore does not appear on the figure. Lane 1,GST-HA control. Lanes 2 and 3 correspond to wild-type GST±HA±Bas1p and C82AC153VC206V GST±HA±Bas1p mutant respectively.

1462 B. Pinson, O. S. Gabrielsen and B. Daignan-Fornier


Bas1p DNA-binding activity is sensitive to oxidation in

vitro but not in vivo

In an attempt to explain the in vivo/in vitro discrepancy

observed for the Bas1p cys triple mutant, we then tested

whether the redox sensitivity of binding of Bas1p to DNA

in vitro would operate in vivo. Bas1p in the absence of

Bas2p is unable to activate transcription of the ADE genes

(Daignan-Fornier and Fink, 1992). Therefore, to be able to

assay the effect of oxidative stress on BAS1 alone, we

constructed a chimera fusing the 1±705 N-terminal

residues of Bas1p to the transcription activation domain

of VP16. Such constructs, carrying either the wild-type

BAS1 sequence or the triple cysteine mutant described

above, were able to complement the growth defect of a

bas1 bas2 double mutant in the absence of adenine

(Fig. 4A). The expression of ADE genes was assayed in a

bas1bas2 strain, carrying the VP16 fusion, before and

after treatment with diamide. The results (Fig. 4B) show

that the VP16 activation of ADE1 and ADE17 was totally

insensitive to diamide even when the fusion protein

carries the three cysteine residues conferring sensitivity

to oxidation in vitro. To ensure that the oxidative

insensitivity of the Bas1p±VP16 protein was not due to

the lack of the last 106 amino acids of Bas1p in the

chimera, we tested the effect of diamide treatment on an

Fig. 3. The effect of oxidation on the function of either a wild typeor cysteine-less Bas1p.A.The Effect of diamide on ADE17 expression. Strain L3080 wastransformed with plasmids B836 (No), P79 (WT: BAS1 wild type) orP1163 (TM: C82AC153VC206V BAS1 mutant). Transformants weregrown in SD 1 casamino acid medium to an OD600 of 0.5. Thendiamide (3 mM) was added to the medium and total RNA wereextracted and treated as in Fig. 1A.B. The effect of mutations in the thioredoxin genes on ADE1,ADE16, ADE17 and HIS5 transcription. Strains EMY60 (TRX1TRX2) or EMY63 (trx1 trx2) were transformed with plasmids P79(WT) or P1163 (TM) and were grown in SD 1 casamino acidmedium, containing 0.15 mM of adenine, to an OD600 of 0.5. TotalRNA were extracted and treated as in Fig. 1C.

Fig. 4. Target gene activation by Bas1p±VP16 fusion protein isinsensitive to oxidation.A. In vivo effect of the Bas1p±VP16 chimera on adeninerequirement. L3080 and L4233 cells were transformed with eitherthe B836 control plasmid (No), or with a plasmid containing thewild-type BAS1 gene fused (WT±VP16) or not (WT) to thetransactivation domain of VP16, or the BAS1 cysteine free mutantgene fused (TM±VP16) or not (TM) to VP16. Serial dilutions of thecells (104, 103, 102 and 101 cells) were spotted on SC mediumlacking uracil and supplemented (left panel) or not (right panel) withadenine. Cells were grown for 36 h at 288C.B. The Bas1p±VP16 fusion is insensitive to oxidation. Strain L4233was transformed with plasmids B184 (WT±VP16) or B186 (TM±VP16). Transformants were grown in SD 1 casamino acid mediumto an OD600 of 0.5. Then diamide (3 mM) was added to themedium and total RNA were extracted and treated as in Fig. 1A.C. The Bas1p C-terminal region is not responsible for the oxidationsensitivity of the protein. Strain L3080 was transformed with eitherthe control plasmid (Bas1p±) or a plasmid harbouring wild type(WT) or truncated (expressing amino acids 1±664) BAS1 gene.Transformants were grown in SD 1 casamino acid medium to anOD600 of 0.5. Then diamide (3 mM) was added to the medium andtotal RNA were extracted and treated as in Fig. 1A.



active truncated Bas1p protein containing only the 1±664

amino acids (B. Pinson, T. L. Konsgrud, L. Johansen, B.

Daignan-Fornier and O. S. Gabrielsen, submitted). The

results in Fig. 4C clearly show that the truncated version

of Bas1p activates ADE1 and ADE17 genes as well as the

wild-type version (see lanes 2 and 5 in absence of

diamide), and still in an oxidative dependent manner.

Thus, from these results we conclude that the redox

sensitivity of Bas1p DNA binding through cysteine

residues observed in vitro is not responsible for the

redox regulation of ADE genes in vivo. These results

suggest that the latter regulation may not be explained

only by impairment of Bas1p DNA binding function, but

imply that other function(s) of Bas1p or other factor(s)

required for activation of the ADE genes still confer this

sensitivity to oxidation.

PHO5, a BAS2-regulated gene, is affected by redox

regulation independently of BAS1

As mentioned previously, Bas2p is strictly required for

activation of all known Bas1p-regulated genes, so it is a

potential candidate for the redox regulation of ADE genes.

We found that Bas2p binding to DNA was insensitive to

diamide in vitro (Fig. 2A), but we could not rule out that

another function of Bas2p could be sensitive to oxidative

stress. Expression of the PHO5 gene, which is activated

by Bas2p (Pho2p) and Pho4p (Vogel et al., 1989) but

does not require Bas1p (see Fig. 5A), was assayed by

Northern blot under oxidation conditions. As shown in

Fig. 5B, the amount of PHO5 transcript in the trx1 trx2

double mutant is strongly decreased. Similarly, the

expression of the PHO5 gene was found to be severely

affected by oxidizing agents such as diamide or sodium

selenite (Fig. 5C and D). These results show that

expression of PHO5 is affected by the redox state and

suggest that Bas2p is a possible redox sensor.

Expression of PHO5 and purine biosynthesis genes is still

affected by oxidative stress in a BAS2 cysteine triple

mutant.

To evaluate the role of Bas2p in redox regulation, the

three cysteine residues of Bas2p (positions 200, 282, 369)

were changed to Ala, Ala and Ser, respectively, as

described in Experimental procedures. This triple

cysteine-free BAS2 mutant complemented the histidine

requirement of a gcn4 bas2 double mutant (data not

shown). The ability of the Bas2p cys-free mutant to

activate the transcription of the ADE1, ADE17 and PHO5

genes under oxidative conditions was tested. Plasmids

expressing the wild-type or cysteine free Bas2p proteins,

in combination with plasmids expressing Bas1p wild-type

or cysteine free proteins, were transformed in a bas1bas2

double mutant. As shown in Fig. 6, the Bas2p cysteine

triple mutant, in combination with either Bas1p wild-type

or triple mutant, was unable to restore transcription of the

ADE1, ADE17 and PHO5 genes. These results clearly

indicated that the redox regulation of the ADE genes was

not strictly mediated through the cys residues of either

Bas1p or Bas2p.

Fig. 5. The effect of either BAS1 or oxidation on PHO5transcription.A. The effect of BAS1 on PHO5 transcription. Strain L3080 wastransformed with plasmids B836 (empty vector, lane bas1) or P79(wild-type BAS1). Transformants were grown in SD 1 casaminoacid medium to an OD600 of 0.5. RNA were extracted and treatedas in Fig. 1A.B. The effect of mutations in the thioredoxin genes on PHO5transcription. Strains EMY60 (TRX1 TRX2) or EMY63 (trx1 trx2)were grown in SD 1 casamino acid medium, containing 0.15 mMof adenine, to an OD600 of 0.5. Total RNA were extracted andtreated as in Fig. 1C.C, D. The effect of sodium selenite (C) or diamide (D) on PHO5transcription. Strain L4233 was grown in SD 1 casamino acidmedium supplemented with uracil to an OD600 of 0.5. Then, sodiumselenite (1 mM) or diamide (3 mM) were added to the medium,total RNA were extracted from cells harvested at indicated time,and treated as in Fig. 1A.

Fig. 6. Transcription of ADE1, ADE17 and PHO5 is still affected byoxidation in the Bas2p cysteine triple mutant. Strain L4233 wastransformed with plasmids P79 (BAS1 wild type) or P1163 (BAS1C82AC153VC206V mutant), both in combination with either P1262(BAS2 wild type) or P1454 (BAS2 C200AC282AC369S mutant).Transformants were grown in SC medium lacking uracil, leucineand adenine to an OD600 of 0.5. Then diamide (3 mM) was addedto the medium and total RNA were extracted and treated as inFig. 1A.



Interaction between Bas1p and Bas2p is affected by

oxidative stress

It has recently been suggested that Bas1p/Bas2p inter-

action could play an essential role in their ability to

activate their common target genes (Zhang et al., 1997).

We tested the possibility that oxidation could abrogate this

interaction between the two factors, thus leading to the

poor expression of the ADE genes under oxidative

conditions. To make activation by Bas1p and Bas2p

independent of their interaction, we took advantage of a

previously constructed chimera fusing the 1±705 N-

terminal residues of Bas1p to the entire Bas2p protein

(B. Pinson, T. L. Konsgrud, L. Johansen, B. Daignan-

Fornier and O. S. Gabrielsen, submitted paper). Such a

construct, carrying the BAS1±BAS2 fusion driven by the

BAS1 promoter, was able to complement the growth

defect of a bas1 bas2 double mutant (data not shown).

The expression of ADE and PHO5 genes was assayed in

a bas1bas2 strain, expressing the Bas1p±Bas2p fusion,

in the presence or absence of diamide. The results

(Fig. 7A) clearly show that the fusion between Bas1p and

Bas2p makes the ADE gene expression more resistant to

the oxidative treatment. In contrast, this fusion cannot

restore expression of the PHO5 gene under the same

conditions. Indeed, in the absence of diamide, the

expression of the PHO5 gene was found to be similar

for both strains expressing the wild-type Bas2p or the

Bas1p±Bas2p fusion protein. These results strongly

suggest that the ADE gene expression is inhibited in the

presence of oxidative conditions due to an impairment of

the Bas1p/Bas2p interaction. To substantiate this hypoth-

esis further, we used a two hybrid approach to monitor the

Bas1p/Bas2p interaction in vivo under oxidative condi-

tions (Fig. 7B). A LexA±BAS1 and a BAS2±VP16 fusion

were introduced in a bas1 bas2 mutant strain. As

expected, neither LexA±Bas1p nor Bas2p±VP16 alone

were able to activate transcription of the LEU2 reporter

gene controlled by the LexA operator. However, the

combination of both fusion proteins allowed transcription

of the reporter gene and this transcription was abolished

by diamide treatment. Finally, when cells were trans-

formed with a plasmid expressing a LexA±GAL4 fusion,

expression of the reporter gene was found to be

unaffected by both the Bas2p±VP16 chimera and the

diamide treatment. Therefore, this experiment clearly

establishes that interaction of Bas1p and Bas2p is

required for expression of the reporter gene and that

this interaction is abolished by oxidative stress as

suggested by the Bas1p±Bas2p fusion experiment.

However, the precise mechanism leading to the impair-

ment of Bas1p/Bas2p interaction during oxidative stress

remains to be elucidated.

Discussion

The extracellular environment of aerobic organisms is

predominantly oxidizing. Because oxidation can damage

macromolecules, cells have developed several mechan-

isms to maintain a reduced intracellular state to cope with

their oxidative environment. In this paper, we show that

purine biosynthesis genes are downregulated under

conditions of oxidative stress. Because this regulation

was observed after various oxidative treatments, i.e. use

of oxidizing agents such as diamide or sodium selenite or

use of a yeast mutant lacking thioredoxin (Muller, 1991;

1996), this regulation seems to be a broad response

rather than a specific one to a particular oxidizing agent.

We propose that the physiological reason for such a

regulation could be to diminish ribose-5-phosphate

(ribose-5P) consumption under conditions of oxidative

stress, and thus to allow regeneration of NADPH through

the pentose±phosphate pathway, the major source of

NADPH in yeast. It is noteworthy that total recycling

compared with total consumption of ribose-5P results in a

Fig. 7. Impairment of Bas1p±Bas2p interaction is responsible forthe redox sensitivity of ADE genes.A. The Bas1p±Bas2p fusion restores transcription of ADE genes butnot PHO5 gene under oxidative conditions. Strain L4233 wastransformed with either plasmids P79 (BAS1 wild type) and P1262(BAS2 wild type) or the BAS1±BAS2 fusion-carrying plasmid and theYCplac111 empty vector. Transformants were grown in SC mediumlacking uracil, leucine and adenine to an OD600 of 0.5. Then, diamide(3 mM) was added to the medium and total RNA were extracted atindicated times and treated as in Fig. 1A.B. Redox sensitivity of Bas1p±Bas2p interaction monitored by a twohybrid approach. RR88 yeast strain was transformed with a plasmidexpressing LexA fusions (pEG202, p2099 or pSH17-4), and with asecond plasmid expressing or not the Bas2p±VP16 chimera. Cellswere grown in SC medium lacking histidine, tryptophan and adenineto an OD600 of 0.5. Then, diamide (3 mM) was added to the mediumand total RNA were extracted at indicated times and treated as inFig. 1A.



sixfold increased production of NADPH (12 moles

compared with 2 moles of NADPH produced per mole of

glucose-6P consumed). Therefore, downregulation of

nucleotide biosynthesis could be an important way for

cells to resist oxidative stress by increasing NADPH

regeneration. NADPH is the reducing power utilized for

regenerating the two major cellular reducing agents:

glutathione and thioredoxin and therefore its regeneration

is a central issue for cells facing oxidative stress.

Interestingly, in Bacillus subtilis, oxidation of glutamine

phosphoribosylpyrophosphate amidotransferase (the first

step of purine de novo biosynthesis) has been shown to

be a critical step for in vivo degradation of this enzyme

(Grandoni et al., 1989). The iron sulphur cluster respon-

sible for this regulation in the B. subtilis enzyme is found in

glutamine PRPP amidotransferase from many species

including chicken (Zhou et al., 1990), rat (Iwahana et al.,

1993a), human (Iwahana et al., 1993b) or drosophila

(Clark, 1994) but does not exist in Escherichia coli (Tso

et al., 1982) or Saccharomyces cerevisiae (MaÈntsaÈ laÈ and

Zalkin, 1984). It is therefore tempting to speculate that, in

S. cerevisiae, transcriptional redox regulation of the ADE

genes could be an alternative to the oxidation-enhanced

degradation of the first enzyme of the pathway, both

mechanisms presumably resulting in a decreased flux

through the pathway and decreased consumption of

ribose-5P, as discussed above.

We investigated the molecular mechanism leading to

transcriptional redox regulation of the purine biosynthesis

genes. Binding of the transcriptional activator Bas1p to

DNA was severely affected by oxidizing agents in vitro.

This redox sensitivity involves the cysteine residues of

Bas1p. Surprisingly, the redox sensitivity of Bas1p, clearly

established in vitro, does not explain the redox regulation

of ADE genes observed in vivo. This suggests that in the

case of Bas1p, the impairment of DNA-binding activity

through oxidation of specific cysteine residues is not

sufficient to explain the lack of ADE gene expression

under oxidative conditions. Another example of a eukar-

yotic transcription factor reported to be redox regulated at

the level of DNA binding is the p53 protein (Verhaegh

et al., 1997). A recent study of in vivo activation by p53

carried out in yeast (Pearson and Merrill 1998) showed

that activation of a lacZ reporter by p53 is abolished in

yeast cells mutated in the trr1 locus encoding thioredoxin

reductase. Therefore, the lack of reduced thioredoxin in

the mutant somehow abolishes the capacity of p53 to

activate expression of its reporter gene. However,

activation of a LexAop±lacZ reporter by a LexA±p53

fusion was still regulated by the redox state (Merril et al.,

1999), indicating that this regulation does not operate only

at the DNA-binding level.

The role of Bas2p in the oxidative stress response of

ADE genes was also investigated. Our data suggest an

important role for Bas2p in this process because PHO5, a

gene activated by Bas2p but not by Bas1p, is down-

regulated in response to oxidative stress. However, it is

formally possible that downregulation of PHO5 expression

could be due to independent effects on Pho4p and not on

Bas2p (Pho2p). The physiological significance of this

redox regulation of a phosphate utilization gene is not yet

clear. Intriguingly, as in the case of Bas1p, a triple Bas2p

mutant deprived of cysteine residues does not abolish the

redox regulation of the PHO5 or ADE genes.

Finally, a Bas1p±Bas2p fusion, covalently linking the

two proteins, is far less responsive to oxidative stress,

thus suggesting that the redox regulation might occur at

the protein±protein interaction level. The results with the

Bas1p±VP16 fusion are in good agreement with this

hypothesis because the wild-type Bas1p±VP16 fusion,

which makes expression of the ADE genes totally

independent of Bas1p±Bas2p interaction, is totally insen-

sitive to oxidative treatment. This hypothesis was further

confirmed by the two hybrid experiment clearly showing

that interaction between Bas1p and Bas2p is required for

expression of the reporter gene and that this interaction is

abolished by the oxidative treatment. Interaction between

Bas1p and Bas2p has been proposed to play a central

role in the adenine downregulation of the ADE genes

(Zhang et al., 1997), but the precise mechanism leading

to this regulation is not yet elucidated. It is tempting to

speculate that adenine and redox regulation could be the

result of two signalling pathways converging on the level

of transcription by exploiting a similar mechanism of

molecular interactions.

Experimental procedures

Yeast strains and media

Yeast strains used in this study were L3080 (MATa gcn4-2ura3-52 bas1-2), L4233 (MATa leu2D2 ura3-52 gcn4-2 bas1-2 bas2-2), EMY60 (MATa lys2 ura3-1 ade2 ade3 trp1-1leu2,3-112 his3-11 can1-1), EMY63 (MATa lys2 ura3-1 ade2ade3 trp1-1 leu2,3-112 his3-11 can1-1 trx1::TRP1trx2::LEU2), and RR88 [MATa ura3-52 trp1 bas1-2 bas2-2his3 leu2::(lexAop)6-LEU2]. Yeast were grown in SD mediumor SC media as described in Sherman et al. (1986). SD±CASA medium is SD supplemented with 0.2% (w/v)casamino acids (Difco Laboratories).

Oligonucleotides

The following oligonucleotides were used for BAS1 site-directed mutagenesis: 70: 5 0-GAGGATGAGCAGCTCTTG-3 0;73: 5 0-TGGAGGAACAGAGGATCAANNNGCGAAA-AGG-TACATTGAA-3 0; 76: 5 0-TGTCGAATATAAGTACC-3 0; 91:5 0-CCTGGAAGAACAGAGGATCAGCGCGCGAAAAGGTA-CATTGAAGTG-3 0; 94: 5 0-CCTGATTGGTGGAGGTGC-3 0; 96:5 0-TGCAGCGGCCGC-3 0; 105: 5 0-AACATCCCAAGCGAT-



CGCTTTAGAAAGATG-3 0; 108: 5 0-GATCTCTAGA-3 0; 208:5 0-GCCATCTGTTACGTACCGTTAAACTTG-3 0. Oligonucle-otides used for BAS2 site-directed mutagenesis were asfollows:240 : 5 0-TTTTTTGACATTGCTAGCATTACTGTGGGA-3 0; 243 : 5 0-TTCTAAAGTTAGGCTAGCATTCGTGACTGA-3 0;292: 5 0-GAGAAATCAGATATCGACCACTGATTG-3 0. Oligo-nucleotides used for BAS1±BAS2 fusion construct were asfollows: B102 (5 0-CTCTTAGGATCCCCCGGGATGATGGA-AGAATTCTCGTACGAT-3 0) and B103 (5 0-TCCTGGATCCT-CATCATATCCATCTATGCTCG-3 0). The oligonucleotidesused for ADE1, ADE5,7, ADE16, ADE17, HIS5 and PHO5radiolabelled probes in Northern blot experiments were asfollows: 32: 5 0-CGCCCCGGGTTATTCATTG-GCCAGCTT-3 0;36: 5 0-CGCCCCGGGTTAGTGAGACCATTTAGA-3 0; B134:5 0-TTAAGAGAACCATTCTCC-3 0; B135: 5 0-ATATTGTTGAT-TAGTAAAGC-3 0; 41: 5 0-CGCAGATCTTAATGTCAATTACGA-AAGA-3 0; 51: 5 0-CGCGGATCCCTATGGTTTTTGA-TTTGA-3 0;53: 5 0-GCTGGTTGATGGAAAATA-3 0; 54: 5 0-TGCATGCA-CAGCAGGGTG-3 0; 55: 5 0-GGATCTGCAATCCCGTCC-3 0;56: 5 0-CACAGCGGCACCAGCTGG-3 0; 227: 5 0-ATAGAG-CAAGCAAATTCG-3 0; 228: 5 0-GGCGTTGTAATGAGTAG-3 0.For PCR amplification of the ADE5,7 probe used in gelretardation assays, the following oligonucleotides were used:125: 5 0-CGCCCCGTCGGTAG-3 0; 126: 5 0-AGTTCAAGCC-CATCGC-3 0.

Site-directed mutagenesis of BAS1

The plasmid used for Bas1p expression in yeast is namedP79 (Pinson et al., 1998). The control plasmid without BAS1is named B836 (G. Fink's laboratory collection). The mutantsdescribed below were constructed in the P79 plasmid andwere verified by sequencing. The construct of theC82AC153VC206V triple mutant was obtained in severalsteps. First, the three simple mutants were obtained using agap-repair method (Ma et al., 1987) in the L3080 strain. TheC82A Bas1p mutant was obtained by transformation of thecells with P871 (a derivative of P79 in which the oligonucleo-tide 108 was inserted in the BamHI site) opened with XbaIand a PCR fragment obtained with oligonucleotides 76 and105 (containing the mutation). The resulting gap-repairedplasmid (P1014) was extracted from clones able to grow inthe absence of histidine by the method of Robzyk and Kassir(1992), then amplified in E. coli and sequenced. The C153Vmutant was obtained as follows. First, a C153R mutant(P838) was obtained by transformation of yeast with the P79plasmid linearized by XhoI and with a PCR fragment obtainedusing P79 as a template and the oligonucleotides 91(containing the mutation and creating a BssHII site) and 94as PCR primers. The P838 plasmid carrying a genecorresponding to a non-functional Bas1p was then used toobtain the fully active C153V BAS1p mutant (P928). Theyeast strain was transformed with the P838 plasmidlinearized with BssHII and with a PCR fragment obtainedon P79 as a template and the oligonucleotides 73 (containinga randomized sequence at the cysteine codon) and 94 asPCR primers. The C206V (P1152) mutant was obtained withthe P712 plasmid (a derivative of P79 in which oligonucleo-tide 96 was inserted in the XhoI site) linearized with NotI andwith a PCR fragment obtained using P79 template and theoligonucleotides 70 and 208 (containing the mutation). The

C82AC153V double mutant (P1067) was then constructed byreplacement of the HindIII±XhoI fragment of plasmid P1014by the HindIII±XhoI fragment from plasmid P928. Then, theC82AC153VC206V triple mutant (P1163) was finallyobtained by replacement of the P1152 BamHI±XhoI fragmentby the BamHI±XhoI fragment from P1067.

Site-directed mutagenesis of BAS2

A HindIII±HindIII fragment carrying the entire BAS2 genewas inserted into the YCplac111 vector (Gietz and Sugino,1988). The resulting plasmid, named P1262, was linearizedat the unique NcoI site in BAS2 and co-transformed with aPCR fragment obtained with oligonucleotides 240 and 243.This PCR fragment carried mutations converting bothCys200 and Cys282 residues to Ala. The resulting gap-repaired plasmid (P1363) carried the two mutations as shownby sequencing and complemented the bas2 deletion. Thisplasmid was used, with oligonucleotides 240 and 292, toamplify a PCR product carrying the Cys200Ala, Cys282Alatogether with a mutation in the third cysteine residueCys369Ser. This PCR product was co-transformed in yeastwith P1363 linearized at NcoI. The resulting gap-repairedplasmid, named P1454, was shown by sequencing to carrythe triple Cys200Ala, Cys282Ala and Cys369Ser substitu-tions. This plasmid, containing the BAS2 gene deprived of allits cysteine residues, complemented the bas2 deletion (datanot shown).

BAS1±VP16 fusion constructs

The BAS1±VP16 fusions were constructed as follows. First,the SalI±ClaI fragment from either P79 or P1163 weretransferred to pBluescriptII opened by SalI±ClaI. Then, in theresulting plasmids, a BamHI±BglII cassette containing thestrong and constitutive transactivation domain VP16 from theHerpes simplex virus (residues 413±488) and a stop codon(from plasmid pDBD11-R2R3, Ording et al., 1996) wasintroduced to give plasmids B174 and B176. The finalplasmids containing BAS1 wild type (B184) and BAS1C82AC153VC206V mutant (B186) fused to VP16 wereobtained by replacing the SalI±ClaI fragment of P1016 bySalI±ClaI fragments from B174 and B176 respectively.

GST±HA±Bas1p fusion constructs

The plasmid used for expression of the wild-type GST±HA±Bas1p fusion (P841) has already been described (Pinsonet al., 1998). A plasmid carrying the C82AC153VC206V triplemutant version of the GST±HA±Bas1p fusion was con-structed by replacement of the BamHI±BbuI fragment ofP870 (Pinson et al., 1998) by the BamHI±BbuI fragment fromP1163.

GST±HA±Bas1p, Bas1p and Bas2p purification

The GST±HA±Bas1p was purified from E. coli on aglutathione-sepharose 4B resin as described previously(Pinson et al., 1998). The Bas1p and Bas2p proteins were



purified on heparin-agarose resins as described (Tice-Baldwin et al., 1989).

Protein separation and detection

SDS±PAGE, Western blot analysis and EMSA were per-formed as described previously (Pinson et al., 1998).However, for EMSA experiments dithiotreitol was omitted inthe sample buffer.

Northern blots

Yeast strains L3080, L4233, EMY60 and EMY63 weretransformed with the BAS1 and/or BAS2 carrying plasmidsand grown in SD with casamino acids, supplemented(EMY60 and EMY63) or not (L3080, L4233) with 0.15 mMadenine, to an OD600 of 0.5. For the two hybrid experiment,RR88 yeast strain was transformed with two plasmids, thefirst expressing the DNA-binding domain of lexA proteinfused or not to either full-length Bas1p or Gal4p proteins(plasmids pEG202, p2099 and pSH17-4 respectively;described in Zhang et al., 1997), and the second expressing(Hirst et al., 1994) or not (YCplac112) the Bas2p-VP16chimera. Cells were grown in SC medium to an OD600 of 0.5.RNA extraction, separation by electrophoresis, capillarytransfer on positively charged nylon membrane, hybridizationand dehybridization were performed as previously described(Denis et al., 1998). Each blot was probed with a radi-olabelled fragment specific for the gene studied. The probesused were as follows: for ADE1, ADE5,7, ADE16, ADE17 andHIS5 specific probes were amplified by PCR using yeastgenomic DNA as template and, respectively, the followingpairs of synthetic oligonucleotides: oligonucleotides 36 and41 for ADE1, B134 and B135 for ADE5,7, 53 and 54 for ADE16, 55 and 56 for ADE17 and 32 and 51 for HIS5. For ACT1and LEU2, the probes correspond to the ClaI±ClaI andBstEII±EcoRV internal fragments of the genes respectively.Radiolabelling of the probes was carried out using therandom priming procedure (Ready to go DNA labelingbeads, Amersham-Pharmacia Biotech).

Acknowledgements

We are grateful to Drs Fink, Goding, Muller and Rolfes forsending yeast strains and plasmids. We thank F. Borne forhelp in mutant constructs. This work was supported by grantsfrom ARC No. 9722, Conseil ReÂgional d'Aquitaine and CNRS(UPR9026), The Norwegian Research Council and TheNorwegian Cancer Society. B.P. was supported by a FEBSpost-doctoral long-term fellowship.

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