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INTRODUCTION |
Aerobic organisms have to maintain a reduced cellular redox
environment in the face of the prooxidative conditions of aerobic life.
The incomplete reduction of oxygen to water during respiration leads to
the formation of redox-active oxygen intermediates such as the
superoxide anion radical (O
2), hydrogen peroxide
(H2O2), and the hydroxyl radical (for review
see Refs. 1-3). Redox-active oxygen intermediates are also produced
during the
-oxidation of fatty acids by exposure to radiation,
light, metals, and redox active drugs. Redox-active oxygen
intermediates perturbate the cell redox status and when present in high
levels can induce toxic damage to lipids, proteins, and DNA, eventually
leading to cell death. Living organisms constantly sense and adapt to
such redox perturbations. The exposure of the yeast Saccharomyces
cerevisiae to low doses of H2O2 or
O
2-generating drugs switches on within minutes a resistance to
toxic doses of these oxidants (4-6). The adaptive response to
H2O2 involves a change in the expression of at
least 167 proteins (7). Such a rapid and widespread genomic response
suggests the existence of specific control pathways.
In S. cerevisiae, the transcription factors Yap1 (8, 9) and
Skn7 (10, 11) have been implicated in a cellular pathway that controls
the oxidative stress response. Yap1 is a bZIP DNA-binding protein of
the AP-1 family (12) that binds the sequence T(T/G)ACTAA termed the
Yap1 response element (YRE) (9, 13,
14).1 Skn7 contains a
receiver domain found in the family of two-component signal
transduction systems of prokaryotes and a domain similar to the
DNA-binding domain of heat shock factor (Hsf1) (15, 16). Skn7 is also
capable of specific DNA binding, but its cognate DNA sequence has not
been identified precisely (11). Strains inactivated for either one of
these regulators are hypersensitive to killing by
H2O2 (9, 10, 11, 17). This oxidative stress phenotype is related to the role of Yap1 and Skn7 in controlling the
induction of several defense genes by H2O2.
Yap1 controls the expression of GSH1 (
-glutamylcysteine
synthetase) (13), TRX2 (thioredoxin) (9), GLR1
(glutathione reductase) (18), and YCF1 (yeast cadmium
factor, a glutathione S-conjugate pump)(19). More recently, Morgan
showed that the induction of TRX2 and TRR1 (thioredoxin reductase) by H2O2 requires a
co-operation between Yap1 and Skn7 (11). YAP1 function can be activated
by H2O2, diamide, and diethylmaleate (9, 20),
and this activation is attributed to a redox stress-imposed nuclear
redistribution of the protein involving the nuclear export receptor
Crm1 (Xpo1) (21, 22).
Yap1 is also important in cadmium tolerance because deletion of its
gene results in a cadmium-hypersensitive phenotype (19, 20, 23). This
function is attributed to the control by Yap1 of GSH1 (13)
and of YCF1 (19). In addition to its involvement in the
oxidative stress response, Skn7 is implicated in the control of cell
wall biosynthesis, cell cycle, and the osmotic stress response.
Overexpression of SKN7 can suppress the cell wall assembly mutation kre9 (24) and the growth defect associated with a
pkc1 null deletion (15). Overexpression of SKN7
also suppresses the lethality associated with loss of the
G1 transcription factors SBF and MBF (16). Skn7 is
modulated by the Sln1-Ypd1 osmosensor and contributes to regulation of
the HOG osmo-stress pathway (25). The involvement of Skn7 into such
diverse pathways raises the question of the possible connection between
these pathways.
We sought to further analyze the co-operative functions of Yap1 and
Skn7 in the control of the oxidative and cadmium stress responses and
found that these two regulators do not always act together in these
stress responses. Although both Yap1 and Skn7 are important for
resistance to H2O2, only Yap1 is important for cadmium resistance, whereas Skn7 is not only dispensable but appears to
negatively affect this response. The role of Yap1 and Skn7 in the
induction of defense genes by H2O2 was further
analyzed by two-dimensional gel electrophoresis. The data presented
here identify, within a large Yap1 stress response regulon, a gene subset that also requires Skn7 for its control. This partition of the
Yap1 regulon correlates with two distinct classes of defense genes.
Such a specialization within the oxidative stress response may explain
the dissociated function of Yap1 and Skn7 in
H2O2 and cadmium resistance.
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MATERIALS AND METHODS |
Yeast Strains, Growth Conditions, and Reagents--
All studies
were performed with the wild type strain YPH98 (26) (MATa
ura3-52 lys2-801amber
ade2-101ochre trp1-
1
leu2-
1) and its isogenic derivatives yap1
-1
(yap1::LEU2), skn7
-1
(skn7::TRP1), yap1
-1, and
skn7
-1 (yap1::LEU2,
skn7::TRP1). The composition of synthetic complete, rich
broth, and glucose selective media are described elsewhere (27).
Strains were transformed by electroporation as described (28).
H2O2, t-BOOH, and cadmium sulfate were
purchased from Sigma. The 12CA5 anti-HA and the 9E10 anti-Myc
monoclonal antibody were purchased from Roche Molecular Biochemicals.
Gene Disruptions and Plasmid Constructions--
Standard
protocols and buffers were used (29). Gene disruptions were performed
by the one-step gene disruption technique (28). pSKN7 is a
YEp351 plasmid carrying a 3.5-kb genomic fragment containing the entire
SKN7 gene (24). pSKN7-HA is a tagged version of
SKN7 in which the HA epitope was introduced at the
SKN7 PstI site 28 codons downstream of the ATG (15). The
skn7::TRP1 construct used to create
skn7
-1 lacks an internal 1.5-kb
PvuII-HincII fragment of the wild type
SKN7 gene, which has been replaced by the TRP1 gene (24). The yap1::LEU2 construct used to create
yap1
-1 was prepared by removing the YAP1
coding sequence from the BamHI site (+186) to the
KpnI site (+1650) relative to the ATG and replacing it with
the LEU2 gene. pYAP1 was constructed by
subcloning a 2.5-kb EcoRI DNA fragment carrying the entire
YAP1 gene (12) into pRS426. pMF6(X-H) is a PUC18 plasmid
lacking the AccI polylinker site and carrying the same
2.5-kb EcoRI YAP1 fragment. To generate pYAP1-9Myc, a polymerase chain reaction-amplified 390-bp
sequence encoding 9 Myc epitopes was first introduced in vector
pMF6(X-H) into the YAP1 AccI site located two codons
downstream of the ATG; The YAP1-9Myc fusion was then
subcloned at the EcoRI site of pRS426 to generate
pYAP1-9Myc. The functionality of the YAP1-9Myc
construct was evaluated by its ability to rescue the
H2O2-hypersensitive phenotype of
yap1
-1. The TSA1-lacZ gene fusions
were constructed as follows: a 4.5-kb DNA fragment spanning the entire
lacZ coding sequence from the BamHI site, two
codons downstream of the ATG to a KpnI site approximately 1 kb from the stop codon, was subcloned into pRS424 to generate pMT1. A
NaeII to KpnI 328-bp plasmid fragment containing
the
peptide lacZ sequence was then removed from pMT1 to
generate pMT11. TSA1 promoter fragments corresponding to
1000,
837,
403,
243, and
204 to +1 relative to the ATG were
amplified by polymerase chain reaction from genomic DNA and subcloned
between the XhoI and BamHI sites of pMT11.
Sensitivity Assays--
Patch assays were performed as follows:
10-µl aliquots containing approximately 2 × 103
cells of an overnight culture were spotted on rich broth or synthetic complete solid plates containing H2O2, t-BOOH,
or cadmium sulfate at the indicated concentration. Plates were
monitored after 3-6 days incubation at 30 °C.
Northern Blot Analysis--
Yeast cells from overnight culture
were diluted to an A600 of 0.01 in synthetic
complete medium and incubated with shaking at 30 °C until they
reached an A600 of 0.3. The cells were then aliquoted and incubated in the absence or in the presence of
H2O2 (0.2 mM) for 20 min. Total RNA
was prepared by the hot phenol method (30). For each condition tested,
20-µg RNA samples were loaded per lane on an agarose gel containing
formaldehyde, separated by electrophoresis, transferred to a nylon
membrane (Bio-Rad), and hybridized with the indicated random
primed (Roche Molecular Biochemicals) 32P-labeled DNA
probe. Hybridization of each blot with a small nuclear RNA
U3 (SNR17A) specific 32P-labeled DNA probe
served as a RNA loading control. Pre-hybridization, hybridization, and
washes were carried out as described (29). Hybridized membranes were
exposed for autoradiography.
Yeast Crude Extracts and Electrophoretic Mobility Shift
Assays--
Yeast crude extracts used in EMSAs were prepared as
follows. Cells were grown to an A600 of 0.3 and
were left untreated or were treated with H2O2
(0.6 mM) for 5 min and harvested. Extracts were prepared by
glass bead disruption as described previously (7), except for the use
of a modified breakage buffer containing 200 mM Tris-HCl,
pH 8.0, 10 mM MgCl2, 10% glycerol, complete
mixture inhibitor (Roche Molecular Biochemicals). The DNA binding
reactions were carried out in 1× TC buffer (25 mM
Tris-HCl, pH 7.5, 50 mM NaCl, 2 mM EDTA, pH 8, 5 mM MgCl2, 0.1% (v/v) CHAPS, 10% (v/v) glycerol) with 20 µg of crude yeast extracts, 5-15 fmol
[32P]ATP-labeled probe, 1 µg of poly(dI-dC) (Amersham
Pharmacia Biotech) in a total volume of 20 µl. The binding reaction
was incubated for 5 min at room temperature and subjected to
electrophoresis in a 6% polyacrylamide gel
(acrylamide/N,N'-methylenebisacrylamide weight
ratio, 27.5:1) in 45 mM Tris/45 mM boric acid/1
mM EDTA for 1 h at 200 V.
Measurement of the H2O2 Response by
Two-dimensional Gel Electrophoresis--
Measurement of the
H2O2 response was carried out as described
previously (7). Basically, mid-log cells (A600 = 0.3) were exposed or not to H2O2 (0.2 mM) for 15 min, pulse labeled with [35S]methionine for another 15 min, and harvested. An
equal aliquot of 3H-labeled cells was mixed to the
35S-labeled cells and served as an internal protein
concentration standard for each two-dimensional gel spot. Cell mixtures
were extracted and subjected to comparative two-dimensional gel
analysis. Accordingly, the previously identified 71 proteins of the
H2O2 stimulon were analyzed in wild type and
isogenic yap1
-1, and skn7
-1. Uninduced and
H2O2-induced synthesis rate indexes (ratio of
individual [35S]/[3H] spot ratios to the
Act1p [35S]/[3H] spot ratio) were
calculated in each strain for the 71 H2O2-stimulated proteins.
Computer Search of Yap1 and Skn7 Upstream Recognition
Sequences--
Specific DNA sequences were searched within 1 kilobase
from the initiation codon of identified genes with the Saccharomyces Genome Data Base package. Searches were done with the following query
sequences: T(T/G)ACTAA, which corresponds to the known Yap1 recognition
sequences (YRE) (9, 13, 14), and CAGCAGCCGAAAAGA, which correspond to a
23-bp TRX2 promoter sequence capable of binding Skn7
in vitro (11).
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RESULTS |
Yap1 and Skn7 Have Distinctive Roles in the Oxidative and Metal
Stress Responses--
Although strains carrying deletions of
YAP1 (yap1
-1)or SKN7
(skn7
-1) are both hypersensitive to killing by
H2O2 (9-11), skn7
-1 can tolerate
higher concentrations of H2O2 (Fig.
1A). The same phenotypic
profile is seen in the tolerance of t-BOOH (Fig. 1B).
Another functional distinction is the ability of YAP1 overexpression to partially rescue the skn7
-1
peroxide-hypersensitive phenotype, whereas SKN7
overexpression has no effect in yap1
-1 (Fig.
1B). Analysis of the cadmium tolerance further demonstrates the distinctive roles of Yap1 and Skn7 in controlling stress responses (Fig. 1C). Whereas yap1
-1 is hypersensitive to
cadmium (19, 20, 23), skn7
-1 is significantly more
resistant than the wild type strain to this toxic metal. The double
delete yap1
-1,skn7
-1 is hypersensitive to
cadmium, suggesting that the skn7
-1 cadmium hyperresistance phenotype is dependent upon YAP1. Therefore,
Yap1 and Skn7 have distinctive roles in peroxide stress tolerance and opposite effects upon cadmium tolerance, with Yap1 acting positively and Skn7 acting negatively. A negative role for Skn7 is also suggested by the decreased cadmium tolerance observed upon overexpression of
SKN7 (not shown). These results prompted us to evaluate the role of Yap1 and Skn7 in the control of known
H2O2-inducible target genes.

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Fig. 1.
Yap1 and Skn7 have distinctive roles in the
oxidative and metal stress responses. A, wild type
(WT) and isogenic yap1 -1 ( yap1),
skn7 -1 ( skn7), and
yap1 -1,skn7 -1
( yap1 skn7) strains were compared by the
patch assay for their ability to grow on rich broth solid medium
containing H2O2 at the indicated concentration.
For each strain, 10 µl of an overnight culture
(A600 = 2-3) diluted to 2 × 103 cells were spotted on tester plates. B, wild
type and isogenic yap1 -1 ( yap1) and
skn7 -1 ( skn7) not carrying (WT,
yap1, skn7) or carrying an episomal plasmid
containing either YAP1 (pYAP1) or SKN7
(pSKN7) were compared by the patch assay for their ability
to grow on rich broth solid medium containing t-BOOH at the indicated
concentration. C, wild type (WT) and isogenic
yap1 -1 ( yap1), skn7 -1
( skn7), and yap1 -1,skn7 -1
( yap1 skn7) strains were compared by the patch assay
for their ability to grow on synthetic complete solid medium containing
cadmium sulfate at the indicated concentration.
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Yap1 and Skn7 Do Not Always Function Together in the Activation of
H2O2-inducible Target Genes--
Yap1 and Skn7
co-operate to activate TRX2 and TRR1 in response
to H2O2 (11). We thus evaluated by Northern
blot the respective roles of Yap1 and Skn7 in the control of three
other known Yap1 targets, SSA1 (31), GSH1 (13),
and GLR1 (18), and of four other
H2O2-inducible genes, TSA1 (TSA or
peroxiredoxin), AHP1 (an alkyl hydroperoxide reductase)
(32), CCP1 (cytochrome c peroxidase), and
HSP82 (7) (Fig. 2). The genes
analyzed were all potently induced by H2O2 in
wild type cells, and this induction was abolished in
yap1
-1 (Fig. 2, A and B). In
skn7
-1 cells, the induction by
H2O2 was also abolished for TSA1,
CCP1, TRR1, HSP82, and SSA1
and significantly diminished for TRX2 and AHP1 (Fig. 2A). In contrast, induction of GLR1 and
GSH1 by H2O2 was actually stronger
in skn7
-1 than in wild type cells (Fig. 2B). However, in
yap1
-1,skn7
-1 double null cells, the
induction of GSH1 by H2O2 was
totally abolished, demonstrating that the
H2O2-superinduced levels seen in
skn7
-1 cells are dependent upon YAP1.
Therefore, Yap1 and Skn7 co-operate in the control of several
H2O2 target genes but have opposite effects in
the control of other H2O2 target genes, with
Yap1 acting positively and Skn7 acting negatively. These results may
explain, at least in part, the opposite functions of Yap1 and Skn7 in
cadmium tolerance.

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Fig. 2.
Distinct functions of Yap1 and Skn7 in the
activation of H2O2-inducible target genes.
A, Northern blot analysis of Yap1 and
Skn7-dependent target genes. Total RNA was isolated from
exponentially growing (A600 0.3) wild type
(WT) and isogenic yap1 -1 ( yap1)
and skn7 -1 ( skn7) cells, which had not been
treated or were treated for 20 min with H2O2
(0.2 mM) as indicated. The resulting RNA were subjected to
Northern blot probed for the indicated genes and for U3 as a
loading control, as described under "Materials and Methods."
B, Northern blot analysis of Yap1-dependent and
Skn7-independent target genes (same as in A). C,
total RNA was isolated from a wild type and isogenic
yap1 -1,skn7 -1 ( yap1 skn7)
strains (same as in A and B).
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Yap1 Controls a Large Regulon of Thirty-two
H2O2-inducible Proteins--
To further
dissect the intricate functions of Yap1 and Skn7 in the control of the
oxidative and metal stress responses, we searched for other target
genes by comparative two-dimensional gel electrophoresis of total
soluble yeast proteins. We recently identified with this method 71 proteins whose synthesis rate is significantly increased minutes after
exposure to H2O2 (7). We sought to identify
among these proteins those whose induction by
H2O2 would be lost or significantly diminished
in yap1
-1. Exponentially growing wild type and
yap1
-1 cells were pulse-labeled after exposure to
H2O2 for 15 min and then subjected to
two-dimensional gel electrophoresis (Fig.
3, A-C). Uninduced and
H2O2-induced synthesis rate indexes of the 71 H2O2 targets were calculated in
yap1
-1 and divided by those of the wild type strain
(yap1
-1/WT). 31 proteins with a
yap1
-1/WT-induced synthesis rate index ratio equal to or
below the value of 0.6 were considered as dependent upon Yap1 for their
induction by H2O2. Their synthesis rate indexes are represented in Fig. 4. These proteins
were sorted into functional classes (Table
I). The Yap1 regulon includes most of the
oxidant scavenging enzymes. These are, in addition to those mentioned above, cytosolic catalase (Ctt1p), copper/zinc and manganese superoxide dismutases (Sod1p and Sod2p), YDR453Cp, and YOL151Wp. YDR453Wp is an
AhpC/TSA family member, and YOL151Wp is similar to plant NADPH
isoflavonoid reductases shown to rescue the diamide hypersensitivity phenotype of a yap1 null strain (33). The Yap1 regulon also includes several carbohydrate metabolism enzymes, a few heat shock proteins and proteases, amino acid metabolism enzymes, and other unclassified or unknown proteins.

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Fig. 3.
Comparative analysis of the
H2O2 response by two-dimensional gel
electrophoresis in strains deleted for YAP1 and
SKN7. Autoradiograms of two-dimensional gels
performed with total yeast extracts from
[35S]methionine-labeled wild type (A and
B), yap1 -1 (C), and
skn7 -1 (D) cells as described under
"Materials and Methods." Extracts were prepared from control
untreated cells (A) or from cells exposed to
H2O2 (0.2 mM) for 15 min
(B-D). A central region of the autoradiograms was blown up,
and proteins were indicated by their name and black arrows
(Yap1 and Skn7-dependent proteins) or white
arrows (Yap1-dependent and Skn7-independent
proteins).
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Fig. 4.
Basal and
H2O2-stimulated synthesis rate indexes of the
proteins of the Yap1-controlled regulon. Histogram representation
of uninduced (black bars) and
H2O2-induced (white bars) synthesis
rate indexes calculated in wild type (bars 1),
yap1 -1 (bars 2), and skn7 -1
(bars 3) cells as described under "Materials and
Methods." For each protein spot, values were normalized to the wild
type uninduced level that was arbitrarily given the value of 1. The
names of proteins are indicated above the histograms.
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A Large Subset of the YAP1 Regulon Is Dependent upon
Skn7--
Skn7 target genes were similarly identified by comparative
two-dimensional gel electrophoresis (Fig. 3, A-C). Thirteen
proteins with a skn7
-1 to wild type
(skn7
-1/WT)-induced synthesis rate index ratio equal to
or below 0.6 were considered as dependent upon Skn7 for their induction
by H2O2 (Table I). Their synthesis rates
indexes are represented in Fig. 4. Most of these proteins were also
identified as Yap1 target genes. However, two of them were not
identified as Yap1 targets. Conversely, several Yap1 target genes were
still normally induced or even superinduced in the skn7 null
strain (Fig. 4). Hence, Skn7 appears to partition the Yap1 regulon into
three distinct gene subsets. The first subset consists of 13 Yap1- and
Skn7-dependent proteins. It includes most of the
Yap1-dependent oxidant scavenging enzymes and a few other
activities. The second subset contains 19 Yap1-dependent but Skn7-independent proteins and includes Glr1p, Zwf1p, Tal1p, Cys3p,
and, as shown by Northern blot, GSH1. The third subset consist of two Skn7-dependent but Yap1-independent targets.
There was an excellent correlation between protein and mRNA levels
for most of the genes analyzed. However, for Pgm2p and Dnm1p, in mutant
strains mRNA levels appeared normal despite altered protein
synthesis rates (not shown). These discrepancies between mRNA
and protein levels may indicate the existence of post-transcriptional control mechanisms.
Identification of Upstream Regulatory Sequences for Yap1 and
Skn7--
Approximately half of the Yap1 target genes contain a YRE
motif within 1 kb upstream from their translation start, whereas the
other half do not contain such a motif (Table I) (see "Materials and
Methods"). We thus analyzed one such YRE negative target, the
TSA1 gene, for the presence of a Yap1/Skn7-responsive
element. A translational fusion between a 1-kb region upstream of the
TSA1 translational start and the bacterial lacZ
gene was created, and its expression was analyzed by Northern blot
(Fig. 5A). This
promoter-lacZ fusion was inducible by
H2O2 in wild type but not in
yap1
-1 or in skn7
-1 cells, demonstrating
that it recapitulates the regulation of the TSA1 gene. A 5'
deletion analysis showed that a minimal promoter sequence spanning 204 bp from the translational start (
204/+1) retained the
H2O2 inducibility of lacZ gene
expression in wild type cells (Fig. 5B). Furthermore, this
induction was abolished in yap1
-1 and diminished in
skn7
-1. A higher constitutive expression from this
minimal promoter fragment was also observed. These data suggest that
the
204/+1 minimal promoter fragment retains the Yap1- and
Skn7-dependent H2O2 induction of
the TSA1 gene but may have lost a repression mechanism
imposed on the full TSA1 promoter.

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Fig. 5.
Identification of a Yap1- and
Skn7-controlled, H2O2-responsive element in the
TSA1 gene. Northern blot analysis of 1000/+1
(A) and 204/+1 (B)
TSA1-lacZ fusions. Schematic representations of
the two TSA1-lacZ fusion constructs are
represented below each graph. Total RNA was
isolated from exponentially growing (A600 = 0.3)
wild type (WT), and isogenic yap1 -1
( yap1), and skn7 -1 ( skn7)
cells, carrying TSA1-lacZ fusions as indicated.
Cells were not (black bars) or were treated for 20 min with
H2O2 (0.2 mM) (shaded
bars). The resulting RNA were subjected to Northern blot probed
with a lacZ probe and with U3 as a loading
control. Autoradiograms were quantified with the ImageQuant software.
The lacZ specific signals were normalized to the
U3 control and represented in a histogram.
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To examine whether Yap1 and Skn7 affected the expression of the
TSA1 gene by direct binding to its promoter, we performed EMSA with a probe corresponding to the TSA1 promoter
204/+1 fragment. A H2O2-inducible slow
migrating binding complex (complex a) was seen with wild type but not
with yap1
-1,skn7
-1 cell extracts (Fig.
6A) or with
yap1
-1 or skn7
-1 cell extracts (not shown). The same but more intense shifted complex was already seen prior exposure to H2O2 with YAP1 and
SKN7 overexpressing cell extracts, suggesting that it
contains Yap1 and Skn7 (Fig. 6B, lanes 7 and 8). Note that H2O2 treatment
resulted in a slight change in the mobility of complex a. The presence
of Yap1 and Skn7 in complex a could be demonstrated with the use of
specific monoclonal antibodies (mAb). When the EMSAs were performed
with extracts expressing a Myc epitope-tagged version of Yap1, adding
the anti-Myc mAb to the binding reaction resulted in the decrease and
supershift of complex a (Fig. 6C, lanes 1 and
2). Similarly, adding the anti-HA mAb to EMSAs performed
with extracts expressing a HA epitope-tagged version of Skn7 (Fig.
6D, lanes 1 and 2) or to extracts
overexpressing both Yap1 and the HA-tagged Skn7 (lanes 5 and
6) resulted in the complete disappearance of complex a.

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Fig. 6.
Direct binding of Yap1 and Skn7 to the
TSA1 promoter. All EMSAs were performed with the
204/+1 TSA1 probe. A, EMSA using crude extracts
prepared from uninduced (lanes 1 and 3) and
H2O2-induced (lanes 2 and
4) wild type (lanes 1 and 2) and
yap1 -1,skn7 -1 (lanes 3 and
4) cells as described under "Material and Methods." The
H2O2-inducible complex containing Yap1 and Skn7
is indicated with an arrow as a. B,
EMSA performed with crude extracts from uninduced (lanes 1,
3, 5, and 7) and
H2O2-induced (lanes 2, 4,
6, and 8) wild type (lanes 1 and
2), skn7 -1 carrying pYAP1
(lanes 3 and 4), yap1 -1 carrying
pSKN7 (lanes 5 and 6), and wild type
cells carrying both pYAP1 and pSKN7 (lanes
7 and 8). Complexes sensitive to the presence or the
absence of Yap1 are indicated with arrows as a,
b, and c. Complexes sensitive to the presence or
the absence of Skn7 are indicated with arrows as
a and d. C, EMSA performed with
extracts prepared from H2O2-induced wild type
(lanes 1 and 2) and skn7 -1 carrying
pYAP1-9Myc (lanes 3 and 4). The 9E10
anti-Myc monoclonal antibody was added to the binding reaction in
lanes 2 and 4. D, EMSA performed with
extracts prepared from H2O2-induced wild type
(lanes 1 and 2), yap1 -1 carrying
pSKN7-HA (lanes 3 and 4) and wild type
cells carrying both pYAP1 and pSKN7-HA. The 12CA5
anti-HA monoclonal antibody was added to the binding reaction in
lanes 2, 4, and 6.
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Extracts from skn7
-1 cells overexpressing Yap1 did not
generate complex a, but instead two faster migrating complexes
(complexes b and c) (Fig. 6B, lanes 3 and
4). These two complexes were also seen, in addition to
complex a, with extracts from cells overexpressing both Yap1 and Skn7
(lanes 7 and 8). We conclude that they both contain Yap1 because they could be supershifted with anti-Myc mAb (Fig.
6C, lanes 3 and 4). Extracts from
yap1
-1 cells overexpressing Skn7 also did not result in
the formation of complex a but instead resulted in the formation of a
faster migrating complex (complex d) (Fig. 6B, lanes
5 and 6). Complex d contains Skn7, as demonstrated by
its disappearance when anti-HA mAb are added to the binding reaction
(Fig. 6C, lanes 3 and 4). We conclude
that both Skn7 (complex d) and Yap1 (complex b and c) can bind directly
and independently to the TSA1 promoter fragment and can also
form a ternary complex (complex a) with this fragment. Identical EMSA
were observed with a 400-bp-long TSA1 promoter fragment
(
400/+1) and with a probe corresponding to the TRX2
promoter (
300/+1) (not shown).
 |
DISCUSSION |
The respective contributions of the Yap1 and Skn7 transcription
factors to the control of the S. cerevisiae response to
oxidative stress and metal toxicity were analyzed. Yap1 controls a
large regulon of at least 32 proteins, accounting for approximately half of the 71 identified proteins of the H2O2
stimulon (7). As shown for TRX2 and TRR1 (11), 15 of these proteins also require the presence of both Yap1 and Skn7 for
their induction by H2O2. Hence, two subsets of
the Yap1 regulon can be delineated. Interestingly, these two gene
subsets distinguish the antioxidant scavenging enzymes from the
metabolic pathways that regenerate the main cellular reducing power,
GSH and NADPH. These two gene subsets appear to carry specialized
defense functions as suggested by the fact that although both Yap1 and
Skn7 are essential for resistance to peroxides (H2O2 and t-BOOH, a synthetic alkyl
hydroperoxide), only Yap1 is required for resistance to cadmium.
The Genes Required for Resistance to Peroxides Are Specified in the
Entire Yap1 Regulon--
The Skn7-dependent subset of the
Yap1 regulon specifies genes important for peroxide tolerance. However,
the more severe H2O2-hypersensitive phenotype
of yap1
-1 versus skn7
-1
indicates that, in addition to the Skn7-dependent gene
subset, activities of the Skn7-independent Yap1 regulon play a role in
peroxide tolerance.
The partition of the Yap1 regulon by Skn7 correlates with two distinct
classes of defense activities: (i) The Skn7-dependent gene
subset includes most of the known yeast redox-active oxygen intermediate scavenging activities. These are cytosolic catalase (CTT1), cytochrome c peroxidase
(CCP1), Tsa1p (TSA1) or peroxiredoxin (34) and
its homologue YDR453Cp (7), an alkyl hydroperoxide reductase
(AHP1) also related to the AhpC/TSA family (32), thioredoxin 2 (TRX2), thioredoxin reductase (TRR1),
copper/zinc (SOD1), and manganese (SOD2)
superoxide dismutases. Therefore, three yeast proteins of the AhpC/TSA
peroxiredoxin family, which are believed to reduce
H2O2 with electrons donated by NADPH via
thioredoxin (TRX2) and thioredoxin reductase
(TRR1) (32, 35, 36), together with their essential cofactors
Trx2p and Trr1p, are coordinately controlled by the same
H2O2-inducible control mechanism. Consistent with their H2O2 inducibility, the participation
of CTT1, TRX2, TSA1, AHP1,
and TRR1, in yeast peroxide protection is demonstrated by
the increased sensitivity of the corresponding null mutants to killing
by these oxidants (9, 32, 35-38). In contrast, the presence of Sod1p
and Sod2p in the H2O2-inducible Yap1 regulon is
not expected, because both sod1 and sod2 null
strains, although hypersensitive to killing by O
2-generating
drugs, retain a proper resistance to H2O2 (40,
41). (ii) The Skn7-independent gene subset comprises several activities
of glutathione and pentose phosphate pathways. Cystathionine
lyase
(CYS3) generates cysteine, a precursor of glutathione (GSH),
from cystathionine.
-Glutamyl cysteine synthase (GSH1) is
the rate-limiting enzyme in GSH biosynthesis, and
NADPH-dependent glutathione reductase (GLR1)
acts to recycle oxidized GSH (GSSG). Glucose-6-phosphate dehydrogenase
(ZWF1) regulates the carbon flow through the pentose
phosphate pathway by catalyzing its first oxidative step, and
transaldolase (TAL1) catalyzes the interconversion of
pentose phosphates that have been generated in the oxidative part of
this pathway (42). A set of four other proteins of this gene subset
could be also involved in the maintenance of the pyrimidine redox
balance. These are a putative NADPH dehydrogenase (Oye3p), two proteins
with similarity to alcohol dehydrogenase (YMR318C and YNL134C), and
YDR032C, which encodes a member of a new family of flavodoxin-like
proteins (43). The GSH pathway is most likely involved in peroxide
resistance by the reduction of peroxides by glutathione peroxidase
using the pentose phosphate pathway as the primary source of NADPH, the
electron donor system for glutathione and thioredoxin reductases. The
importance of GSH1, GLR1, ZWF1, and
TAL1 in the yeast cellular tolerance of
H2O2 is demonstrated by the increased
sensitivity of their null mutants to this oxidant (18, 44-48). Other
activities are also part of this gene subset (Table I). Their role in
the tolerance of H2O2 has been recently
discussed (7).
The yeast peroxide stress response is different from that of
Escherichia coli and Salmonella tiphymurium,
which involves a H2O2-inducible regulon
controlled by the regulator OxyR (49). The OxyR-controlled regulon
includes katG (hydroperoxidase I), ahpCF (an
alkyl hydroperoxide reductase of the AhpC/TSA family), dps
(a nonspecific DNA-binding protein), gorA (glutathione
reductase), and grxA (glutaredoxin) (50, 51). However, in
contrast to the Yap1 regulon, the OxyR regulon excludes both
sodA (manganese superoxide dismutase) and zwf
(glucose 6 phosphate dehydrogenase), which are part of the
O
2-inducible regulon under control of the SoxR/S regulator
(52).
The Genes Required for Cadmium Tolerance Are Mostly Confined in the
Skn7-independent Subset of the Yap1 Regulon--
GSH represents a
first line of defense against cadmium toxicity (53). GSH acts through a
cadmium detoxification pathway that involves the yeast cadmium factor
Ycf1p, a vacuolar GSH S-conjugate pump of the ABC
superfamily of transporters (54-56). Interestingly, YCF1 is
also a target of Yap1 (19). In this pathway, cadmium is conjugated to
GSH, and the GS-cadmium conjugates are transported to the vacuole by
Ycf1p. Glutathione reductase (GLR1) probably acts as a
cadmium tolerance factor by recycling oxidized GSH, which accumulates
upon cadmium exposure (39) and is known to inhibit Ycf1p (55). The role
of GSH1 (39), GLR1, and ZWF1 in the
yeast tolerance of cadmium is demonstrated by the increased sensitivity
of their respective null mutants to this toxic metal (Spector,
unpublished observations). A few activities of the
Skn7-dependent gene subset also play a role in cadmium
tolerance. These are alkyl hydroperoxide reductase (AHP1)
and thioredoxin reductase (TRR1), as shown by the
cadmium-hypersensitive phenotype associated with deletion of their gene
(32) (Spector, unpublished observations). However, neither
peroxiredoxin (TSA1), nor thioredoxin 2 (TRX2) are important in the tolerance of this toxic metal (not shown).
A Co-operative Gene Control Exerted by Yap1 and Skn7--
As shown
previously (11), Yap1 and Skn7 exert a dual control on the expression
of oxidative stress defense genes. However, the data presented here
also show that these two regulators do not always function together. In
addition, Skn7 is not only dispensable but has a negative effect upon
cadmium tolerance and upon the expression of several Yap1 target genes
such as GSH1 and GLR1. This may suggest that Skn7
acts as a repressor for the Skn7-independent Yap1 gene subset.
Alternatively, the defective expression of several oxidant scavenging
enzymes in skn7
-1 could create cellular pro-oxidative conditions resulting in a sustained activation of Yap1. It is also
possible that, as recently proposed for OxyR (51), one of the Skn7
target genes participates in a negative autoregulatory loop interrupted
in skn7
-1, thus leading to a deregulation of Yap1 activity.
Approximately half of the genes of the Yap1 regulon do not contain a
YRE. Still, one such gene, TSA1, carries a Yap1- and Skn7-responsive element within a 200-bp proximal region of its promoter
and directly binds Yap1 and Skn7. This strongly suggests that
TSA1 and very likely most of the other YRE negative genes are directly controlled by Yap1 and Skn7. They also indicate the existence of another Yap1 DNA recognition sequence in addition to the
YRE. Efforts are being made to characterize this sequence, as well as
the sequence recognized by Skn7.
Conclusions--
The H2O2-inducible Yap1
transcription factor controls a major yeast stress response regulon,
which is partitioned into two distinct classes of defense genes by the
differential requirement for the Skn7 transcription factor. Such an
organization may provide the cell with the ability to use two
overlapping sets of genes in distinct stress responses. However, like
H2O2, cadmium can induce both gene subsets of
the Yap1-controlled regulon (not shown). Conditions under which Yap1 is
active and Skn7 is inactive would result in the differential induction
of these two gene subsets. The function of Skn7 can be modulated by
phosphorylation of the conserved aspartate of the receiver domain (15,
16, 25). However, although important for the contribution of Skn7 to
cell cycle control, cell wall metabolism, and modulation of the
osmo-stress HOG pathway, this aspartate is dispensable in the oxidative
stress response (10, 11). Another post-transcriptional modification might exist that affects the ability of Skn7 to co-operate with Yap1.