The ALD6 Gene Product Is Indispensable for Providing NADPH in Yeast Cells Lacking Glucose-6-phosphate Dehydrogenase Activity*

Dorota Grabowska and Anna ChelstowskaDagger

From the Department of Genetics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5A, 02-106 Warsaw, Poland

Received for publication, October 10, 2002, and in revised form, January 23, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Reducing equivalents in the form of NADPH are essential for many enzymatic steps involved in the biosynthesis of cellular macromolecules. An adequate level of NADPH is also required to protect cells against oxidative stress. The major enzymatic source of NADPH in the cell is the reaction catalyzed by glucose-6-phosphate dehydrogenase, the first enzyme in the pentose phosphate pathway. Disruption of the ZWF1 gene, encoding glucose-6-phosphate dehydrogenase in the yeast Saccharomyces cerevisiae, results in methionine auxotrophy and increased sensitivity to oxidizing agents. It is assumed that both phenotypes are due to an NADPH deficiency in the zwf1Delta strain. We used a Met- phenotype displayed by the zwf1Delta strain to look for multicopy suppressors of this deletion. We found that overexpression of the ALD6 gene coding for cytosolic acetaldehyde dehydrogenase, which utilizes NADP+ as its cofactor, restores the Met+ phenotype of the zwf1Delta strain. Another multicopy suppressor identified in our screen, the ZMS1 gene encoding a putative transcription factor, regulates the level of ALD6 expression. A strain bearing a double ZWF1 ALD6 gene disruption is not viable. Thus, our results indicate the reaction catalyzed by Ald6p as an important source of reducing equivalents in the yeast cells.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Glucose-6-phosphate dehydrogenase is a housekeeping enzyme, encoded in mammals by the G6PD1 gene located on the X chromosome (1).2 It has important functions in intermediary metabolism since it catalyzes the first step in the pentose phosphate pathway, which supplies the cell with the reductive potential in the form of NADPH, required for a variety of biosynthetic pathways and redox reactions. Apart from generating NADPH, the pentose phosphate pathway contributes to the synthesis of ribose 5-phosphate, required for the biosynthesis of some amino acids, nucleotides, and coenzymes (3). Glucose-6-phosphate (G6PD) activity was reported in all organisms and cell types, and the enzyme structure is highly conserved.3 Moreover, we found recently that human G6PD can functionally replace this enzyme in Saccharomyces cerevisiae.4 G6PD deficiency is the most widespread human enzymopathy (5);2 still, a complete absence of G6PD activity has never been reported in mammals. Although it is possible to knock out the G6PD gene in the mammalian cell line, as it was shown in male mouse embryonic stem cells (6), it might be that at least residual G6PD activity is required at some step(s) in higher eucaryote development to such an extent that its total absence would be lethal. On the other hand G6PD-null mutants isolated in the unicellular organisms Escherichia coli and S. cerevisiae were both viable (7, 8). Inactivation of the ZWF1 gene encoding glucose-6-phosphate dehydrogenase in the yeast S. cerevisiae does not affect the cell growth in rich media supplemented with a variety of carbon sources, although it increases their sensitivity to oxidizing agents (8) and leads to methionine auxotrophy (9). It was suggested that the growth deficiencies are caused by an increased utilization of NADPH required for reductive assimilation of inorganic sulfur or for restoration of cellular pools of reduced glutathione and thioredoxin, which rapidly deplete under oxidative stress growth conditions (10). Due to surprisingly discrete phenotypes displayed by the zwf1-null mutant, the yeast S. cerevisiae was used as a model organism to clarify the contribution of the alternative routes of the NADPH synthesis, namely the reactions catalyzed by isocitrate dehydrogenases, to the overall pool of NADPH in the cell (11). There are three highly homologous but differentially compartmentalized isocitrate dehydrogenase isozymes in yeast encoded by the IDP1, IDP2, and IDP3 genes and localized in mitochondria, cytoplasm, and peroxisomes, respectively (12-14). The loss of either Idp1p and/or Idp2p activity produces no observable growth phenotype. The loss of Idp3p impairs growth on media containing unsaturated fatty acids as a carbon source, which indicates the role of this protein as a peroxisomal source of NADPH required for a double bond reduction (14, 15). Combinations of double, triple, and quadruple deletions of ZWF1, IDP1, IDP2, and IDP3 indicated that only ZWF1 and IDP2 have partially overlapping functions leading to the enhancement of the zwf1Delta idp2Delta mutant phenotype in comparison with single zwf1Delta or idp2Delta knockouts (11). However, the ability of the zwf1Delta idp1Delta idp2Delta idp3Delta strain to grow under conditions including the use of glucose, glycerol, ethanol, or acetate as carbon sources indicates that the reactions catalyzed by G6PD and cytosolic isocitrate dehydrogenase, postulated to be the major contributors of biosynthetic reducing equivalents in eucaryotic cells, are apparently not essential under many growth conditions (11). Here we present our evidence of another, as yet unidentified, activity contributing to the pool of NADPH in the yeast S. cerevisiae cells.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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Strains, Media, and Growth Conditions-- Yeast strains used in this study are listed in Table I. The DG zwf1Delta strain was derived from CD101-1A by inserting the kanMX4 selectable marker into the URA3 gene to restore the uracil auxotrophy lost by the disruption of the ZWF1 gene (9). The BY ald6Delta and the BY zms1Delta haploid strains were isolated after sporulation and tetrad dissection of diploid strains obtained from the EUROSCARF collection. The strains with a double deletion (zwf1Delta ald6Delta and zwf1Delta zms1Delta ) were obtained by mating the BY ald6Delta with the DG zwf1Delta or the BY zms1Delta strain with CD101-1A strain, respectively, and by subsequent sporulation and tetrad dissection, for which standard media and procedures were used (16). Cells were grown on standard rich YP medium with 2% glucose as a carbon source (YPD) or on minimal YNB medium (0.67% yeast nitrogen base without amino acids) supplemented with all the required amino acids and nucleotides, and 2% glucose (YNBD) or 2% ethanol (YNBE) as a carbon source. 2% agar was added to solidify the media. Ura- derivatives of the analyzed strains were obtained by selection with 0.1% 5-fluoro-orotic acid added to minimal medium. Oxidative stress growth conditions were obtained by incubating the plates in a dessicator jar refilled with pure oxygen every 24 h.


                              
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Table I
Yeast strains used in this study

Library Screening-- A yeast genomic library in the pFL44L high copy number plasmid, provided by Dr. F. Lacroute (17), was transformed into the DG zwf1Delta strain displaying methionine auxotrophy. Ura+ transformants were selected and analyzed as described under "Results."

DNA Manipulations-- Routine DNA manipulations such as plasmid preparation, subcloning, E. coli transformation, and agarose gel electrophoresis were carried out as described (18). Yeast transformations were performed by the lithium acetate procedure (19). Plasmid DNA from yeast, used subsequently to transform E. coli, was isolated as described (20). DNA sequencing reactions were analyzed on an ABI310 Genetic Analyzer (PerkinElmer Life Sciences).

Constructing the Multicopy Plasmid Containing the IDP2 Gene-- A 2109 bp DNA fragment comprising the IDP2 gene with 496 bp of its promoter and 362 bp of its terminator sequence was amplified by PCR using the primers: 5'-GGTACCAGCTTGGAACTAACACGAACC-3' and 5'-GGATCCTAGTGTCAGTGGAAGCACCTG-3', to which the KpnI and BamHI sites (underlined) were added, and cloned in the pGEM-T Easy vector (Promega) to obtain the pGEM-IDP2 plasmid. PCR was performed on genomic DNA of the S288C strain with the Expand high fidelity PCR system (Roche Applied Sciences) according to the manufacturer's instructions. The pGEM-IDP2 plasmid was then cut with the BamHI and KpnI, and the fragment containing the IDP2 gene together with flanking sequences was cloned into the pFL44L vector. A smaller, 1698-bp DNA fragment comprising the IDP2 gene with only 71 bp of its promoter and 362 bp of its terminator sequence was cut out from the pGEM-IDP2 plasmid with a SalI and cloned into the pYPGE2 expression vector, containing the PDK1 promoter (21).

RNA Isolation and Northern Blot Analysis-- Total yeast RNA was isolated from 50 ml of logarithmic phase cultures as described previously (22). For Northern blot analysis, 25 µg of the total yeast RNA was fractionated on 1.3% agarose gels containing 6.4% formaldehyde and transferred to a HybondTM-N membrane (Amersham Biosciences). Hybridizations were performed using a Rapid-hyb buffer (Amersham Biosciences). Prehybridization was carried out at 65 °C for 1h. Radiolabeled probes were added and hybridized overnight at 65 °C. 32P-labeled DNA probes for hybridization were prepared by the random oligonucleotide-primed synthesis using the HexaLabel PlusTMDNA labeling kit (Fermentas) and [32P]dATP (3000 Ci/mmol, Amersham Biosciences). The 0.6-kb ClaI internal fragment of the yeast actin (ACT1) gene and a 0.95-kb HindIII-EcoRI fragment of the ALD6 gene were used as probes. Hybridization signals were quantified with a Amersham Biosciences PhosphorImager.

Preparation of Cell Extracts and Determination of Isocitrate Dehydrogenase Activity-- The zwf1Delta deletion strain transformed with the pYPGE2 (21) or the pYPGE2-IDP2 plasmid was grown to A600 0.9-1.0 in selective YNBD medium supplemented with required amino acids. The cells were then pelleted, washed once with the extraction buffer (a 10 mM potassium phosphate buffer, pH 7.2, 5 mM MgCl2; 1 mM 2-mercaptoethanol, 1 mM phenylmethylsulphonyl fluoride), repelleted, resuspended in the above buffer and, subsequently, disrupted by shaking 6 × 30 s at 1-min intervals on ice in a BIOSPEC homogenizer in the presence of an equal volume of glass beads (0.45 mm in diameter). The suspension was centrifuged at 10,000 × g for 10 min at 4 °C; the supernatant was collected for the enzyme activity analysis performed as described by Haselbeck and McAlister-Henn (12).

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

ALD6 and ZMS1 Are Multicopy Suppressors of zwf1Delta Strain Growth Deficiencies-- The yeast strain bearing the null alleles of the ZWF1, IDP1, IDP2, and IDP3 genes, encoding glucose-6-phosphate dehydrogenase and three NADP+-dependent isocitrate dehydrogenases, respectively, was shown to be viable under most cultivation conditions (11). The above enzymes catalyze reactions presumed to be the major sources of biosynthetic reducing equivalents. Another activity, which can contribute to the provision of NADPH, is the malic enzyme reaction, identified in yeast (23). However, as reported by Minard et al. (11), the MAE1 gene deletion did not enhance the phenotype of the strains with different combinations of the zwf1Delta , idp1Delta , idp2Delta , and idp3Delta alleles. There is also no transhydrogenase activity in yeast, which would allow for the direct transhydrogenation of NADH to NADPH. Taken together, these results are strongly suggestive of another source of NADPH, sufficient to complement the shortage of reducing equivalents under most growth conditions.

To identify new genes coding for proteins, whose function is to provide reduced equivalents in yeast cells, we chose to complement the methionine auxotrophy phenotype of the zwf1Delta mutant by overexpressing the yeast genomic library. It is well established that there is some variation among laboratory yeast strains of different genetic backgrounds reflected, for example, in differences between mutant or deletion phenotypes in different strains; at its extreme, genes reported as essential in a strain of one genetic background were shown to be dispensable in another (24, 25). We checked whether two yeast strains, W303-1B and BY4742, widely used to obtain defined deletions of yeast open reading frames, display the same phenotype linked to the deletion of ZWF1 as described previously for other strains with presumably different genetic backgrounds (8, 9). As shown in Figs. 1 and 4, A and B, the methionine auxotrophy related to the zwf1Delta genotype is markedly pronounced in both W303-1B and BY4742 strains. The ability to grow in the atmosphere of high oxygen tension leading to oxidative stress is also impaired, although it is less affected in the deletion mutant obtained in the W303-1B than in the BY4742 strain (Figs. 1 and 4, A and C). The sod1 strain lacking the activity of cytoplasmic superoxide dismutase, which was reported to be unable to grow in the presence of high oxygen tension (26), was always used as a control to monitor the growth conditions (data not shown).


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Fig. 1.   Overexpression of the ALD6 and ZMS1 genes suppresses phenotypes displayed by the zwf1Delta strain. A and B, strains grown in atmospheric oxygen. C and D, strains grown in hyperoxia. A wild-type (WT) W303-1B strain or its derivative DG zwf1Delta transformed with the pFL44L plasmid (strain zwf1Delta ), pFL44-ALD6 (strain zwf1Delta + ALD6), pFL44L (strain zwf1Delta +ZMS1), of pPGK-IDP2 (strain zwf1+Delta IDP2) was grown overnight in selective YNBD medium supplemented with required amino acids. Cells were spun down and resuspended in water to a final concentration of 2 × 107 and 10-fold serially diluted. 5 µl of each dilution was spotted onto plates with methionine (A and C) or without methionine (B and D). Plates were incubated for 4 days at 28 °C.

The yeast genomic library in the pFL44L multicopy vector was used to transform the deletion mutant strain DG zwf1Delta (see "Materials and Methods"), and all the Ura+ transformants were screened for the ability to grow on minimal medium with appropriate auxotrophic supplements but lacking methionine. Fourteen Ura+ Met+ transformants were isolated and grown on medium with 5-FOA to remove the plasmid. Each isolated Ura+ Met+ transformant became Ura- Met- after growth on the plate with 5-FOA, thus showing that an acquired methionine prototrophy was plasmid-linked. The zwf1Delta strain was retransformed with plasmids isolated from the primary Ura+ Met+ transformants. All the Ura+ transformants acquired methionine protoprophy. The DNA sequence analysis revealed that the plasmids isolated from seven primary transformants contained the ZWF1 gene, the plasmids from four transformants encompassed the fragment of yeast genomic DNA coding for the ALD6 gene, and the inserts of plasmids isolated from three other transformants were derived from the part of chromosome X coding for open reading frames YJR127c and YJR128w. The subcloning experiment showed that the suppression was due to a gene YJR127c overdosage.

The ALD6 (YPL061w) gene encodes cytosolic aldehyde dehydrogenase, which utilizes NADP+ as its cofactor and contributes to the oxidation of acetaldehyde to acetate (27-29). Open reading frame YJR127c encodes a 1380-amino-acid protein named Zms1, which has two tandem zinc finger domains and is similar to the Adr1p transcription factor.5 This structural feature allows the prediction that Zms1p may also function as a transcription factor.

When the ALD6 gene expressed on the on high copy plasmid was introduced into the zwf1Delta strain, both phenotypes displayed by the mutant were restored (Fig. 1). The same was observed for the ZMS1 gene expressed on the on high copy plasmid, although under the most challenging conditions tested, i.e. when all the strains were grown in a high oxygen atmosphere on the medium without methionine, the ALD6 suppression effect was more potent than that of ZMS1 (Fig. 1D).

ZMS1 Gene Overexpression Influences the Level of the ALD6 Transcript-- As the ZMS1 gene encodes a putative transcription factor,5 it was justifiable to expect that ZMS1, when overexpressed, would augment the level of the ALD6 expression. In fact, as can be seen in Fig. 2, the level of the ALD6 transcript is increased in cells with a higher level of the ZMS1 expression. The finding that the second suppressor isolated in our screen acts by changing the level of the ALD6 transcript indicates that the reaction catalyzed by Ald6p must be a metabolic source of NADPH that can substitute for the lack of the Zwf1p activity. The ZWF1 gene inactivation does not increase the level of the ALD6 expression (Fig. 2), which may suggest that this alternative pathway, providing the reducing equivalents in yeast cells, is not induced when the level of NADPH is diminished.


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Fig. 2.   Overdosage expression of the ZMS1 gene leads to increased expression of the ALD6 gene. The DG zwf1Delta and DG zwf1Delta +ZMS1 strains, both derivatives of the W3031B wild-type (WT) strain, were grown to mid-log phase in minimal medium with 2% glucose as a carbon source. Total RNA was isolated from each strain and analyzed by Northern blotting using probes specific for ALD6 and actin as described under "Materials and Methods."

High Dosage of IDP2 Is Not Able to Restore Methionine Protoprophy in the zwf1Delta Strain-- In search for other sources of reduced NADPH in yeast cells, the reaction catalyzed by cytosolic NADP+-dependent isocitrate dehydrogenase, encoded by the IDP2 gene, was indicated as the one that can serve as an alternative source of reduced equivalents (11, 31). The IDP2 gene was not isolated in our suppressor screen, although the ALD6 and ZMS1 suppressors, as well as the ZWF1 gene itself, were isolated several times. We could not rule out the possibility that the intact IDP2 gene was not represented in the genomic library used for transformation. Therefore, we directly checked the ability of IDP2 to rescue the zwf1Delta strain phenotypes by expressing it on the multicopy plasmid from its native promoter or PGK1 promoter, whose activity is not affected by glucose repression (21). After PCR amplification, IDP2 was cloned into the pFL44L or pYPGE2 plasmid, respectively, and transformed into the zwf1Delta strain. The phenotypes of the IDP2 transformants were checked alongside with the phenotypes of the ALD6 and ZMS1 transformants. The IDP2 expression from the multicopy plasmid did not affect the zwf1Delta strain ability to grow under the conditions tested, whereas the multidosage of ALD6 and ZMS1 restored the zwf1Delta growth (Fig. 1).

The Idp2 activity was measured in cell-free extracts prepared from yeast transformants bearing the IDP2 gene under the control of the PGK1 promoter and the empty pYPGE2 plasmid as described under "Materials and Methods." The overexpression of IDP2 resulted in an ~10-fold increase in specific activity of isocitrate dehydrogenase, which rose from 0.04 units, as measured for the zwf1Delta strain transformed with an empty plasmid, to 0.35 units for the strain transformed with the pYPGE2-IDP2 plasmid; however, the zwf1Delta phenotype was not suppressed.

Increased ALD6 mRNA Expression Results in a Met+ Phenotype of the zwf1Delta Strain Grown on Ethanol-- The Met- phenotype of the zwf1Delta strain was observed on minimal medium containing glucose as a carbon source, whereas the same strain did not require methionine when grown on medium with ethanol as a carbon source. We reconfirmed the same phenomenon for W303 zwf1Delta (Figs. 1B and 3A). It was postulated that the Met- phenotype observed only on the glucose-containing medium was due to the glucose repression of the IDP2 expression, hence the absence of an alternative NADPH source. A higher expression of the IDP2 gene on ethanol medium would result in a restored Met+ phenotype of the zwf1Delta strain (31). However, the ability of the double zwf1Delta idp2Delta mutant to grow without methionine on medium with ethanol a carbon source (31) indicates yet another NADPH source in derepressed cells. Since our multicopy suppressors search results indicate the Ald6p activity as that which may contribute toward the pool of NADPH, we checked whether the restored methionine prototrophy might be due to the increased ALD6 expression in ethanol-grown zwf1Delta cells. Northern blot analysis of total cellular RNA isolated from cells grown on media with glucose or ethanol as a carbon source shows a 2-3-fold higher level of the ALD6 gene transcript in both wild-type and zwf1Delta cells grown on ethanol (Fig. 3B), as compared with glucose-grown cells. The increased level of the ALD6 transcript in the ethanol-containing medium versus the glucose medium was comparable with the rate of the ALD6 mRNA increase observed in the strain with an overexpressed ZMS1 gene grown on glucose medium (Figs. 2 and 3B). The result shows that the transcriptional induction of ALD6 is sufficient to maintain the growth of zwf1Delta on medium lacking methionine but containing ethanol as a carbon source.


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Fig. 3.   Increased expression of the ALD6 gene in yeast cells grown on ethanol-containing medium alleviates methionine requirement of the zwf1Delta strain. A, growth on medium with 2% ethanol used as a carbon source. The strains were grown overnight in selective YNBD medium supplemented with required amino acids. Cells were spun down and resuspended in water to a final concentration of 2 × 107 and 10-fold serially diluted. 5 µl of each dilution was spotted onto a YNBE plate without methionine. The plate was incubated for 6 days at 28 °C. B, Northern blot analysis of the ALD6 gene expression. The strains were grown to mid-log phase in minimal medium with 2% glucose or 2% ethanol used as a carbon source. Total RNA was isolated from each strain and analyzed by Northern blotting using probes specific for ALD6 and actin as described under "Materials and Methods." WT, wild type.

As the ZMS1 gene overexpression influences the ALD6 transcript level, we checked whether the observed methionine prototrophy of zwf1Delta grown on ethanol is ZMS1-dependent. The growth of the double zwf1Delta zms1Delta mutant is not distinguishable from the growth of the zwf1Delta strain (data not shown). This observation is in agreement with Northern blot analysis, which shows that ALD6 transcript is still induced in cells grown on ethanol although the ZMS1 gene is inactivated (Fig. 3B).

ALD6 and ZMS1 Deletion Strains Do Not Share the Same Phenotypic Defects with the zwf1Delta Strain-- The only reported phenotype associated with the disruption of ALD6 is its altered growth rate on ethanol media. Interestingly, in one study, the growth of ald6Delta on ethanol was reported as only marginally slower than the growth of the wild type (27), whereas in another case, the growth on ethanol was completely abolished (28). The zms1Delta phenotype was not studied at all.5 Since both ALD6 and ZMS1 genes were identified in our screen for suppressors of the zwf1Delta methionine auxotrophy and were also shown to alleviate zwf1Delta oxidative stress sensitivity, we checked whether strains deleted in the ALD6 or ZMS1 genes displayed any phenotypes similar to those found for zwf1Delta . No growth deficiencies were observed when the ald6Delta or zms1Delta cells were grown on minimal medium without methionine or in the presence of high oxygen tension (Fig. 4). Even when the challenging growth conditions were combined, i.e. cells were grown on medium without methionine and in an oxygen atmosphere, the growth rate of neither ald6Delta nor zms1Delta was affected (Fig. 4D).


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Fig. 4.   Strains bearing a disruption of the ALD6 or ZMS1 gene do not display any phenotype of the zwf1Delta strain. A and B, strains grown in atmospheric oxygen. C and D, strains grown in hyperoxia. The wild-type (WT) BY4742 strain or its derivatives carrying a deletion of the ZWF1, ALD6, or ZMS1 gene, as well as the zwf1Delta zms1Delta strain obtained by mating BY zms1Delta with DG zwf1Delta and isolating a double disruption bearing spore clone, were grown overnight in YNBD medium supplemented with required amino acids. Cells were spun down and resuspended in water to a final concentration of 2 × 107 and 10-fold serially diluted. 5 µl of each dilution was spotted onto plates with methionine (A and C) or without methionine (B and D). Plates were incubated for 4 days at 28 °C.

ALD6 and ZWF1 Deletion Are Co-lethal-- Although the strains with deletion of the ALD6 and ZMS1 genes do not display any phenotype characteristic of zwf1Delta that could show their involvement in supplying reducing equivalents in yeast cells, our results indicate the important role played by the product of the ALD6 gene in alleviating zwf1Delta growth deficiency. It was important to assess the existence of any additive effect of the deletion of the ZWF1 and the ALD6 genes, which encode proteins of putatively overlapping metabolic functions. The DG zwf1Delta strain, derivative of W303, was crossed to the BY ald6Delta . In the progeny of the above cross, no double disruption-bearing clones were isolated, which significantly suggests that the zwf1Delta ald6Delta strain is not viable. To prove that the combination of the ZWF1- and ALD6-null alleles leads to a synthetic co-lethality phenotype, the zwf1 ALD6/ZWF1 ald6 diploid strain was transformed with the ALD6 gene expressed on the pFL44L URA3 plasmid. From the progeny of this cross, four viable spore clones were isolated from each dissected tetrad. The spore clones were tested for their ability to grow on plates containing 5-FOA, which requires a loss of the plasmid. No spore clone with the double zwf1Delta ald6Delta disruption was able to grow without the plasmid containing the ALD6 gene (Fig. 5). We tried to remove the pFL44-ALD6 plasmid from the zwf1Delta ald6Delta cells under diminished oxidative stress by growing the cells in anaerobiosis or by supplementing the media with glutathione, as in various experimental systems, enhanced glutathione levels and oxidoresistance were shown to be mediated by an increased glucose-6-phosphate dehydrogenase expression (30, 32). No growth of the zwf1Delta ald6Delta strain was observed on 5-FOA-containing plates under any of the above mentioned conditions (data not shown). On the contrary, the zwf1Delta zms1Delta double mutant is viable and does not reveal any additional growth deficiencies as compared with the ones displayed by the zwf1Delta -null allele (Fig. 4). This indicates that the product of the ALD6 gene is absolutely indispensable in yeast cells lacking glucose-6-phosphate dehydrogenase activity.


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Fig. 5.   The strain with a double disruption of the ZWF1and ALD6 genes is not viable. The ald6Delta ZWF1/ALD6 zwf1Delta diploid strain transformed with the pFL44L-ALD6 plasmid was sporulated, and the growth of spore clones on 5-FOA containing minimal medium was analyzed. A, the growth of spore clones from a representative TT tetrad on minimal medium. B, the growth of the same spore clones on minimal medium supplemented with 0.1% 5-FOA. WT, wild type.

NADPH is required for many reactions of reductive biosynthesis that provide the cell with deoxyribonucleotides for DNA replication, fatty acids, or reduced sulfur. NADPH also protects the cell against oxidative stress, mainly by regeneration of the intracellular pool of reduced glutathione. It can be assumed that when the cellular content of NADPH decreases to a critically low level, even under the conditions alleviating the oxidative stress, the NADPH pool is too low to fulfill the biosynthetic requirements of the cell. We think that this occurs in the case of the double zwf1Delta ald6Delta mutant strain. Although it is difficult to predict which biosynthetic activity may be particularly affected, it is possible to see an analogy with a strain with quadruple deletion of the TRX1, TRX2, GRX1, and GRX2 genes, encoding both yeast thioredoxins and glutaredoxins, respectively (2). Thioredoxin and glutaredoxin are physiological reducing agents of a ribonucleotide reductase in a multistep electron transfer pathway in which NADPH provides the reducing equivalents for nucleoside diphosphate reduction (4). The trx1Delta trx2Delta grx1Delta grx2Delta strain is not viable (32), which shows that at least one thioredoxin or glutaredoxin gene is essential in yeast. The phenotype of the strain in which the NADPH level is drastically diminished may mimic the phenotype of the strain in which the thioredoxin/glutaredoxin system is not functional. This may explain why the yeast cells lacking both Zwf1 and Ald6 proteins activities are not be able to grow.

    ACKNOWLEDGEMENTS

We thank Dr. Dominique Thomas for kindly providing the CD101-1A strain. We are grateful to Prof. Joanna Rytka for helpful advice and discussions. We also thank Dr. Marek Skoneczny for a critical reading of the manuscript.

    FOOTNOTES

* This work was supported by PW1 grant from Institute of Biochemistry and Biophysics, Polish Academy of Sciences and State Committee for Scientific Research Grant 3 P04A 03822.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 48-22-659-70-72; Fax: 48-22-658-46-36; E-mail: chelstowska@ibb.waw.pl.

Published, JBC Papers in Press, February 12, 2003, DOI 10.1074/jbc.M210076200

2 Online Mendelian Inheritance in Man (OMIMTM), McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD), www.ncbi.nlm.nih.gov/omim.

3 Molecular Modeling Data Base, www.ncbi.nlm.nih.gov/Structure.

4 D. Grabowska, manuscript in preparation.

5 Saccharomyces Genome Database, genome-www.stanford.edu/ Saccharomyces.

    ABBREVIATIONS

The abbreviations used are: G6PD, glucose-6-phosphate dehydrogenase; 5-FOA, 5-fluoroorotic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

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