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
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ABSTRACT |
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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 zwf1 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 zwf1 Strains, Media, and Growth Conditions--
Yeast strains used in
this study are listed in Table I. The DG
zwf1 Library Screening--
A yeast genomic library in the pFL44L
high copy number plasmid, provided by Dr. F. Lacroute (17), was
transformed into the DG zwf1 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 zwf1 ALD6 and ZMS1 Are Multicopy Suppressors of zwf1
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 zwf1
The yeast genomic library in the pFL44L multicopy vector was used to
transform the deletion mutant strain DG zwf1
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 zwf1 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.
High Dosage of IDP2 Is Not Able to Restore Methionine Protoprophy
in the zwf1
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 zwf1 Increased ALD6 mRNA Expression Results in a Met+
Phenotype of the zwf1
As the ZMS1 gene overexpression influences the
ALD6 transcript level, we checked whether the observed
methionine prototrophy of zwf1 ALD6 and ZMS1 Deletion Strains Do Not Share the Same Phenotypic
Defects with the zwf1 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 zwf1
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 zwf1 strain. We used a
Met
phenotype displayed by the zwf1
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
zwf1
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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
idp2
mutant phenotype in comparison with single zwf1
or
idp2
knockouts (11). However, the ability of the
zwf1
idp1
idp2
idp3
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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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 ald6
and the BY zms1
haploid strains were isolated after sporulation and tetrad dissection
of diploid strains obtained from the EUROSCARF collection. The strains
with a double deletion (zwf1
ald6
and zwf1
zms1
) were obtained by mating the BY ald6
with
the DG zwf1
or the BY zms1
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.
Yeast strains used in this study
strain displaying methionine
auxotrophy. Ura+ transformants were selected and analyzed
as described under "Results."
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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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
zwf1
, idp1
, idp2
, and idp3
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.
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
zwf1
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 zwf1 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 zwf1
transformed with the pFL44L plasmid (strain zwf1
),
pFL44-ALD6 (strain zwf1
+ ALD6), pFL44L (strain
zwf1
+ZMS1), of pPGK-IDP2 (strain
zwf1+
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.
(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
zwf1
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.
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).
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Fig. 2.
Overdosage expression of the ZMS1
gene leads to increased expression of the ALD6
gene. The DG zwf1 and DG
zwf1
+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."
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 zwf1
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 zwf1
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 zwf1
strain ability
to grow under the conditions tested, whereas the multidosage of
ALD6 and ZMS1 restored the zwf1
growth (Fig. 1).
strain transformed with an empty plasmid, to
0.35 units for the strain transformed with the pYPGE2-IDP2 plasmid;
however, the zwf1
phenotype was not suppressed.
Strain Grown on Ethanol--
The
Met
phenotype of the zwf1
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 zwf1
(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 zwf1
strain (31). However, the ability of the double zwf1
idp2
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 zwf1
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 zwf1
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 zwf1
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 zwf1 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.
grown on ethanol is
ZMS1-dependent. The growth of the double
zwf1
zms1
mutant is not distinguishable from the growth of the zwf1
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).
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
ald6
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 zms1
phenotype was not studied at all.5 Since both
ALD6 and ZMS1 genes were identified in our screen for suppressors of the zwf1
methionine auxotrophy and
were also shown to alleviate zwf1
oxidative stress
sensitivity, we checked whether strains deleted in the ALD6
or ZMS1 genes displayed any phenotypes similar to those
found for zwf1
. No growth deficiencies were observed when
the ald6
or zms1
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 ald6
nor zms1
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 zwf1 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
zwf1
zms1
strain obtained by mating BY
zms1
with DG zwf1
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.
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 zwf1
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 zwf1
strain, derivative of W303, was
crossed to the BY ald6
. In the progeny of the above
cross, no double disruption-bearing clones were isolated, which
significantly suggests that the zwf1
ald6
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
zwf1
ald6
disruption was able to grow without the
plasmid containing the ALD6 gene (Fig.
5). We tried to remove the pFL44-ALD6
plasmid from the zwf1
ald6
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 zwf1
ald6
strain
was observed on 5-FOA-containing plates under any of the above
mentioned conditions (data not shown). On the contrary, the
zwf1
zms1
double mutant is viable and does not reveal
any additional growth deficiencies as compared with the ones displayed by the zwf1
-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 ald6 ZWF1/ALD6 zwf1
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.
ald6
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 trx1
trx2
grx1
grx2
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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.
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ABBREVIATIONS |
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The abbreviations used are: G6PD, glucose-6-phosphate dehydrogenase; 5-FOA, 5-fluoroorotic acid.
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