From the Service de Biochimie et
Génétique Moléculaire, Bât. 142, Commissariat
à l'Energie Atomique, F-91191 Gif-sur-Yvette Cedex, France,
Department of Pharmacology and Toxicology, College of
Pharmacy, Rutgers University, Piscataway, New Jersey 08855, § Graduate Program in Molecular Genetics and Microbiology,
University of Medicine and Dentistry of New Jersey, Graduate School of
Biomedical Sciences, Piscataway, New Jersey 08854
Received for publication, October 27, 2000
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ABSTRACT |
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In an attempt to elucidate the essential function
of glutathione in Saccharomyces cerevisiae, we searched for
suppressors of the GSH auxotrophy of Glutathione (GSH) is a broadly conserved tripeptide with a highly
reactive thiol and a very low redox potential of about In Saccharomyces cerevisiae and in probably all other
GSH-containing organisms, GSH is synthesized in two steps beginning with the action of We wished to understand the basis for the differential requirement of
GSH between eukaryotes and prokaryotes by attempting to identify which
of the functions of GSH might determine its essential role in yeast. To
this end, we investigated whether mutations capable of suppressing the
Yeast Strains and Growth Conditions--
Y252 (MATa
ura3-52 lys2-801amber
ade2-101ochre trp1- Ethylmethanesulfonate Mutagenesis and Mutant
Selection--
Approximately 2 × 108 cells from an
overnight culture were exposed to 3% ethylmethanesulfonate (Sigma) for
25-30 min at 30 °C, followed by a 4 h recovery in SD.
Ethylmethanesulfonate-treated cells were spread on SD plates lacking
GSH at a density of 5 × 106 cell/plate to select for
mutants suppressing the GSH auxotrophy.
Genomic Library Construction--
Genomic DNA was isolated from
the ySOG1-2 strain with the Qiagen blood and cell culture
DNA kit and partially digested with Sau3AI.
8-15-kilobase-size fragments of the digested DNA were purified with
the Qiagen QiaexII Gel extraction kit after separation on an agarose
gel and ligated into the BamHI site of centromeric vector
Ycp50. 40% of the E. coli transformants contained an
insert, resulting in a library consisting of ~10,000 independent clones.
Gene Disruptions and Plasmid Constructions--
The
PRO2 allele isolated from the library was amplified by
polymerase chain reaction using the following primers,
(5'-CCCGGATCCATTTCTTTTGATGGGCGGC-3', 5'-CCCGGATCCTCGAGTCAGCTCAAATCGCAT-3') and ligated into the
BamHI site of Ycp50 to create pPRO2-1. The wild
type PRO2 expression vector, PRO2, and
PRO1 disruption cassettes were gifts from Dr. Brandriss
(23). The Sensitivity Assays--
For the patch assay, cells were grown
overnight to saturation in YPD, diluted in distilled H2O,
and patched (2000 cells/10 µl) on plates containing various
concentrations of H2O2 or t-butyl hydroperoxide. Plates were incubated for about 72 h at 30 °C. For the challenge assay, exponentially growing cells
(A600 = 0.3-0.5) in SD were exposed to
various concentrations of H2O2 for 1 h and then plated on YPD to determine the number of colony-forming units.
Measurement of GSH by a Microbiological Assay--
Cells (50 ml)
grown in SD to an A600 = 2 were collected by
centrifugation, resuspended in distilled H2O (2 ml), and
lysed by alternately placing in boiling water 10 min and freezing in dry ice/ethanol for three cycles. Samples were centrifuged and the
supernatant, which contained the total free cellular GSH (which included the cytosolic, endoplasmic reticulum and vacuolar pools) was
analyzed for its ability to complement the growth of the
Analysis of GSH by Thin Layer Chromatography--
For the
measurement of GSH in DNA Content Determination by Fluorometry--
To examine
growth-arrested cells, Phenotypes of the gsh1 Null Strain--
The Specific Mutations in PRO2 Can Suppress GSH
Auxotrophy--
Given the apparently essential nature of GSH, we
wished to determine whether it was possible to isolate mutations that
suppressed the GSH requirement of the Suppression by Alleles of PRO2 Requires
We wished to determine whether any suppressors of GSH auxotrophy
existed independently of the proline pathway. To this end, we
performed a mutagenesis in the
Suppression by Alleles of PRO2 Involves the Synthesis of Trace
Amounts of GSH--
Since the suppression of the GSH auxotrophy by
alleles of PRO2 could involve the synthesis of GSH, the
GLR1 Is Not Essential for the Suppression Phenotype--
Since the
High Levels of GSH Are Essential for Acute but Not Chronic
Oxidative Stress--
Previous examinations of the contribution of GSH
to the tolerance to oxidative stress could have been limited by the GSH
requirement of the
We also examined the tolerance to H2O2 by a
challenge assay in liquid medium. In contrast to the plate assay,
Decreased GSH Synthesis Rate during Adaptation to
H2O2--
As another means of evaluating the
importance of GSH during the H2O2-adaptive
response, we measured its biosynthesis rate by pulse-labeling cells for
5 min after exposure to 0.6 mM H2O2 (Fig. 5A). Surprisingly,
compared with untreated cells, the amount of labeled GSH was
significantly lower both 20 and 50 min after H2O2 exposure, suggesting either a decrease in
the GSH biosynthesis rate or a release of GSH in the extracellular
milieu. However, the GSH pool, estimated by labeling cells for 2 h
before exposure to H2O2, was similar in
untreated cells and in cells that had been exposed for 20 min to
H2O2 (Fig. 5B). As another control, exposure to 0.6 mM H2O2 during
either 20 or 50 min did not increase the GSH levels in the
extracellular milieu (Fig. 5C), demonstrating that the
decrease of 5-min pulse-labeled GSH after exposure to low
H2O2 concentrations is most likely due to a
decreased synthesis rate. Since GSH1 is induced by
H2O2 (30, 31), the decreased synthesis rate of
GSH after exposure to H2O2 could be related to
a decreased availability of cysteine, as this amino acid can be
limiting for GSH synthesis (32). The addition of cysteine to the medium
indeed significantly increased to the same extent the GSH synthesis
rate of untreated and H2O2-treated cells (Fig. 5D), suggesting that this amino acid is limiting for GSH
biosynthesis, especially under oxidative stress conditions.
The essential nature of GSH in yeast (14-16) and in mammals (17)
but not in prokaryotes provides an interesting puzzle. To address this
difference, we designed a genetic selection for mutations that could
bypass the requirement for GSH in yeast. We found that mutations in the
proline biosynthetic pathway could suppress this requirement but
actually did so by restoring the synthesis of GSH. These mutations were
unique, since a second screen after ablation of the proline pathway did
not yield any other suppressor under either aerobic or anaerobic
conditions. Therefore, no single gene mutation can suppress the GSH
growth requirement, suggesting that more than one mutation might be
needed or, alternatively, that GSH is absolutely essential in yeast,
and there is no genetic condition that can suppress this requirement.
Abduction of the Proline Pathway for GSH Biosynthesis--
The
suppression by alleles of PRO2 involves the biosynthesis of
small quantities of GSH. Although not very efficient, the modified
proline pathway provides about 0.5-1% wild type GSH content, which is
therefore a quantity compatible with the viability of the strain. What
could then be the GSH biosynthetic mechanism by the modified proline
pathway? Deletion of either PRO1 or PRO2 abolishes the suppression phenotype, demonstrating that formation of
The Essential Role of GSH--
In bacteria, only the simultaneous
inactivation of both the GSH or the thioredoxin pathways results in
aerobic inviability due to defects in protein thiol maintenance and/or
ribonucleotide reduction (33, 34). However, anaerobic conditions
restore growth because of the presence of an E. coli
alternative ribonucleotide reductase that is independent of disulfide
reduction (35) and also by the likely decrease in non-native disulfide
bond formation. In contrast, GSH is essential in yeast (Refs. 14-16
and this work). This essential function is not related to
ribonucleotide reduction since The Role of GSH in the Protection against Oxidative
Stress--
Yeast cells depleted of GSH were shown to be
hypersensitive to peroxides and to superoxide-generating drugs (13, 15,
16, 32). This together with the inducibility of GSH1
expression by oxidants suggested that GSH provides an important defense
mechanism against oxidative stress (3, 8). We reevaluated this question with gsh1, a strain
lacking the rate-limiting enzyme of glutathione biosynthesis. We found
that specific mutations of PRO2, the second enzyme in
proline biosynthesis, permitted the growth of
gsh1 in
the absence of exogenous GSH. The suppression mechanism by alleles of
PRO2 involved the biosynthesis of a trace amount of
glutathione. Deletion of PRO1, the first enzyme of the proline biosynthesis pathway, or PRO2 eliminated the
suppression, suggesting that
-glutamyl phosphate, the product of
Pro1 and the physiological substrate of Pro2, is required as an
obligate substrate of suppressor alleles of PRO2 for
glutathione synthesis. A mutagenesis of a
gsh1 strain
also lacking the proline pathway failed to generate any suppressor
mutants under either aerobic or anaerobic conditions, confirming that
glutathione is essential in yeast. This essential function is not
related to DNA synthesis based on the terminal phenotype of
glutathione-depleted cells or to toxic accumulation of non-native
protein disulfides. Analysis of the suppressor strain demonstrates that
normal glutathione levels are required for the tolerance to oxidants
under acute, but not chronic stress conditions.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
240 to
250
mV. Its elevated concentration, up to 10 mM, and the fact that its reduced state is efficiently maintained by
NADPH-dependent glutathione reductase (reviewed in Refs.
1-3) confers to this small molecule the properties of a cellular redox
buffer. As a redox buffer, GSH is thought to be a major determinant in
maintaining a reducing cellular thiol-disulfide balance. GSH is also
important as an electron donor for several enzymes that have a reducing step in their catalytic cycle, such as ribonucleotide reductase (4). In
these situations, electrons from GSH are generally transduced to their
substrates through glutaredoxins. GSH may protect protein sulfhydryls
from irreversible oxidation by glutathionylation (5, 6). It also
participates in the detoxification of peroxides, either directly or
indirectly as a cofactor of glutathione peroxidase (1, 7, 8) and of
several chemicals such as cadmium (9, 10).
-glutamyl cysteine synthetase (GSH1)
(11, 12), which catalyzes the condensation of glutamic acid to
cysteine, in the rate-limiting step of this biosynthetic pathway. The
product of this reaction is
-glutamylcysteine, which is combined
with glycine through the action of glutathione synthase
(GSH2) (13) to form GSH. Null mutations in GSH1
result in GSH auxotrophy (14-16), demonstrating that GSH is essential
in yeast. In mammals, GSH is also essential, as demonstrated by the
embryonic lethality resulting from disruption of
-glutamyl cysteine
synthetase and by the inability of blastocysts isolated from such
knock-out strains to grow, except in the presence of GSH or the GSH
substitute N-acetyl cysteine (17). The reason for the
essential requirement of GSH in eukaryotes is not known. In contrast,
GSH is not essential in prokaryotes since strains of Escherichia
coli lacking gshA, the homologue of GSH1,
can grow in minimal media without GSH supplementation (18-20).
gsh1 auxotrophy exist in yeast. We found that mutations
of the proline pathway are the only suppressors of the lethal phenotype
of the
gsh1 strain under both aerobic and anaerobic
conditions. However, this suppression involves the biosynthesis of a
small amount of GSH through a modified proline biosynthesis pathway,
demonstrating that there is no single gene mutation able to bypass the
yeast GSH requirement. Our data indicate that this requirement is not
related to ribonucleotide reduction or to preventing toxic accumulation
of non-native protein disulfide, leaving open this question. We used
this strain as a model to study the contribution of GSH to oxidative
stress tolerance.
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
1
leu2-
1) and Y253 (MAT
ura3-52
lys2-801amber ade2-101ochre
his3-
1 leu2-
1) are derived from
YPH98 (21) and were used throughout this study. The
yap1,
ahp1, and
tsa1 strains were described (22).
Cells were grown in YPD1 (1%
yeast extract, 2% peptone, and 2% glucose) or in minimal media
(SD) (0.67% yeast nitrogen base w/o amino acids, 2% glucose) with casamino acid, tryptophan, uracil, adenine, and GSH supplements as
appropriate. For anaerobic growth, plates containing SD supplemented with ergosterol (30 mg/liter) and Tween 80 (0.2%), were incubated in
an anaerobic chamber in which the oxygen had been removed with a Merck
Anaerocult A cassette, as confirmed by an indicator strip color change.
With the exception of
gsh1PRO2-2/
glr1, the
strains used in these assays were all respiration-competent, as shown by their ability to grow on YPG media that contains glycerol as the
sole carbon source (not shown).
gsh1 null mutant was created by replacing 180 base pairs upstream and 500 base pairs downstream of the
GSH1 ATG with the LEU2 gene. Disruption was
confirmed by Southern blot. Oligonucleotide-mediated gene replacement
was used to replace the entire coding sequence of GLR1 with
the Kluyveromyces lactis URA3 gene and of GPX3
with the TRP1 gene.
gsh1 strain previously depleted of its GSH content by
overnight growth in GSH free media.
gsh1PRO2-1, cells were grown in SD
to an A600 = 0.4 and pulse-labeled with
[35S]Met (150 µCi) for 1 h or for 3 h with a
second [35S]Met pulse at t = 1 h 30 min, centrifuged, resuspended in 30 µl of distilled H2O,
and heated for 10 min at 99 °C. Cell debris was centrifuged, and the
supernatant, which contained the metabolic pool, was mixed with an
equal volume of performic acid (90% formic acid, 10%
H2O2) to resolve GSH into a single oxidized
spot. 0.2 µl of each sample was dropped on a thin layer
chromatography plate and allowed to migrate twice 6-7 h in the
presence of 60% butanol, 10% acetic acid. The dried plate was exposed
to autoradiography. Measurement of the GSH pool and GSH biosynthesis
rate was performed similarly except that cells were grown in synthetic
medium (SM) with glucose (5 g/liter), tryptophan, uracil, lysine,
leucine, histidine, and adenine instead of casamino acids
(A600 = 0.4) and labeled with
[14C]glucose (150 µCi/ml) or
[14C]glutamate for the indicated time. For the
measurement of GSH export, 1 mCi of [35S]Met was added to
cells and grown in SM (1.2 ml, A600 = 0.4) for
60 min. Cells were washed and resuspended in SM/distilled H2O (1:9, vol/vol) supplemented with 2% glucose and amino
acids, and the supernatant was analyzed for GSH content at the
indicated times after evaporation in a SPEEDVAC to
gsh1 was depleted of GSH by
overnight growth in GSH-free SD medium. Cells were fixed with 70%
ethanol, washed in phosphate-buffered saline pH 7.4, treated with 1 mg/ml RNase A for 1 h, stained with propidium iodide, and analyzed
by flow cytometry on a FACScalibur. Exponentially growing wild type and
gsh1 supplemented with GSH were used as controls.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gsh1
strain is a glutathione auxotroph on minimal (SD) but not on rich media
(YPD) (14-16), which contains GSH. After GSH withdrawal, the
auxotrophic strain underwent growth arrest after about seven
generations, forming large unbudded cells followed by a loss of
viability after 3-4 days (not shown). GSH, whether in the reduced (14,
15) or oxidized form, supported growth of the auxotrophic strain at a
concentration as low as 0.5 µM, as did the reduced thiol
dithiothreitol (15) at the mM range concentration (Fig.
1A). However, anaerobic
conditions did not rescue the GSH auxotrophy but only delayed growth
arrest by several generations (not shown). Similarly, cysteine only
delayed growth arrest of a
gsh1 strain that had not been
depleted of GSH and had no effect upon cells that had been previously
depleted of GSH by overnight growth in SD media lacking GSH (Fig.
1A). GS-CH3, a GSH derivative with a methyl
group replacing the sulfhydryl, did not support growth either (not
shown). Microscopic analysis showing a predominance of unbudded cells
after GSH withdrawal (not shown) and the analysis of the cellular DNA
content by fluorocytometry (Fig. 1B) suggested that cells
arrested predominantly at the G1 phase of the cell cycle.
This observation and the inability of anaerobic conditions to rescue
the GSH auxotrophy strongly suggest that the essential requirement of
GSH is not related to ribonucleotide reduction or to preventing a toxic
accumulation of non-native protein disulfides (see
"Discussion").
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Fig. 1.
Phenotypic consequence of GSH depletion.
A, the growth of gsh1 is rescued by GSH or
dithiothreitol (DTT) but not by cysteine or methionine. The
gsh1 strain not depleted (
gsh1) or depleted
of GSH by overnight growth in SD media devoid of GSH
(
gsh1 depleted) was patched on SD medium containing GSH,
dithiothreitol, cysteine, or methionine at the indicated
concentrations. B,
gsh1 arrests in
G1 after GSH withdrawal. The DNA content of exponentially
growing wild type,
gsh1 grown in medium containing GSH
(
gsh1), and
gsh1 growth-arrested due to GSH
depletion (
gsh1 depleted) was measured by flow cytometry
after propidium iodide staining.
gsh1 strain. Using
ethylmethanesulfonate mutagenesis, about 40 mutants able to grow on SD
without GSH were isolated in each mating type of the
gsh1
strain and labeled SOG (suppressor of
GSH auxotrophy). Complementation analysis showed that nine
of the mutants were dominant and that all of the recessive mutations
were in the same locus. Two dominant (ySOG1-1,
ySOG1-2) and 1 recessive mutant (ysog1-3) were
further analyzed. After failing to clone the recessive mutant by
library complementation, a genomic library was constructed from the
dominant mutant ySOG1-1. Several plasmids able to suppress
the GSH auxotrophy of the
gsh1 strain were isolated from
the ySOG1-1-derived genomic library, each containing a
related genomic insert. Sequencing one of these inserts showed that it
contained an allele of the PRO2 gene, which encodes
-glutamyl phosphate reductase. The suppression phenotype was further
mapped to the PRO2 locus by the ability of the
PRO2 allele polymerase chain reaction amplified from the
isolated genomic library clone and referred to as PRO2-1 to
confer the suppression phenotype to the
gsh1 strain and
by the loss of the ySOG1-1-suppressed phenotype after null
disruption of PRO2. The sog recessive suppressor mutations were also mapped to the PRO2 locus by the loss of
the suppression phenotype of ysog1-3 upon either expression
of wild type PRO2 or deletion of the corresponding locus.
Sequencing of PRO2-1 and of the PRO2 alleles of
ySOG1-2 and ysog1-3 showed that each contained
a single base substitution corresponding to S124P, G148S, and A226V,
respectively. The suppressor mutation was established in a fresh
background by integrating the PRO2-1 allele at the PRO2 locus in
gsh1, resulting in strain
gsh1PRO2-1.
-Glutamyl
Phosphate--
PRO2 encodes
-glutamyl phosphate
reductase, which catalyzes the second step of the proline biosynthetic
pathway. The first step of this pathway is catalyzed by
-glutamyl
kinase (PRO1), which converts glutamate to
-glutamyl
phosphate (23). The PRO2 gene product then converts
-glutamyl phosphate to L-glutamate-
-semialdehyde, the
precursor of L-
1-pyrroline-5 carboxylic
acid, which is converted to proline by the PRO3 gene
product. To further examine the role of this pathway in the suppression
phenotype, we disrupted PRO1 in both
gsh1PRO2-2 and ysog1-3. This restored GSH
auxotrophy in both strains, demonstrating that the mechanism of
suppression requires
-glutamyl phosphate as an obligate substrate of
the suppressor alleles of PRO2. Note that none of the
PRO2 suppressor alleles analyzed had the proline auxotrophy
of the null mutations of this gene (23), demonstrating that their
proline biosynthetic pathway remained functional (not shown).
gsh1
pro1PRO2-1 background. This
mutagenesis failed to isolate any new suppressor under either aerobic
or anaerobic conditions, leading to the conclusion that this pathway is
probably the only mechanism that can suppress the GSH auxotrophy of the
gsh1 strain.
gsh1PRO2-1 strain was analyzed for its GSH content. This
strain grew slightly slower than the wild type strain in SD, but the
slow growth rate was corrected by the addition of GSH (Fig.
2A). Unlike the wild type strain, which can support the growth of the
gsh1 strain
when grown in its vicinity, presumably by releasing GSH into the
medium,
gsh1PRO2-1 failed to cross-feed
gsh1 (Fig. 2B). These data indicate that if
gsh1PRO2-1 contains GSH, it is present in very limiting quantity. We more directly analyzed the presence of GSH by thin layer
chromatography (TLC) of the yeast metabolic pool after
[35S]methionine metabolic labeling (Fig. 2C).
GSH was visible as a discrete spot in wild type but not in
gsh1 cell extracts. In
gsh1PRO2-1 cells,
glutathione was not visible after a 1-h labeling but could be detected
after a 3-h labeling. To further quantify the presence of GSH in
gsh1PRO2-1, we used an assay based upon the ability of
cell extracts to support the growth of
gsh1 (Fig. 2D).
gsh1PRO2-1 cell extracts could support
the growth of
gsh1 with an efficiency similar to that of
wild type extracts diluted 150-fold. This confirmed the presence of GSH
in
gsh1PRO2-1 at levels about 0.5-1% that of wild type
levels. The growth support provided by this extract could not be due to
cysteine, because cysteine is not a GSH substitute (see Fig.
1A). This demonstrates that the mechanism of suppression by
alleles of PRO2 involves the synthesis of small amounts of
GSH.
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Fig. 2.
gsh1PRO2-1 contains
low but measurable levels of GSH. A, growth curves of
wild type (WT, black diamond) and
gsh1PRO2-1 (gray square) grown in SD
and
gsh1PRO2-1 grown in SD containing GSH (gray
triangle). B, the wild type strain but not
gsh1PRO2-1 can support growth of the
gsh1
GSH auxotrophic strain by releasing GSH in the medium.
gsh1 was spotted (2000 cells/10 µl) on a SD plate
adjacent to the wild type (wt) or to the
gsh1PRO2-1 strain. Growth was recorded after 3 days of
incubation at 30 °C. C, autoradiogram of a TLC plate that
separated the metabolic pool of the wild type (wt,
lanes 1 and 2),
gsh1 (lanes
3 and 4), and
gsh1/PRO2-1 (lanes
5 and 6) strains labeled with
[35S]methionine for 1 (lanes 1, 3,
and 5) or 3 h (lanes 2, 4, and
6). Extracts were oxidized with performic acid to resolve
GSH into a single oxidized spot (see the brackets).
D, measurement of GSH by a microbiological assay. Cells from
a culture of
gsh1 or
gsh1PRO2-1 were
spotted (2000 cells/10 µl) as indicated on the figure onto SD plates
containing 3, 5, 20, 100, 500, or 1000 µl of crude extracts prepared
from the wild type (wt) or the
gsh1PRO2-1
strains. Growth was recorded after 3 days of incubation at
30 °C.
gsh1PRO2-1 strain contains very low levels of GSH, we
considered that its ability to reduce GSSG might become essential for
cell viability. Therefore, we disrupted the GLR1 gene in
wild type,
gsh1, and
gsh1PRO2-1. Despite
the absence of glutathione reductase, the
gsh1PRO2-1
glr1 strain retained the ability
to grow on minimal media without GSH supplement. Interestingly, we also
observed that the growth of
gsh1
glr1 could
be supported by GSSG (data not shown), suggesting that either an
alternate mechanism for GSSG reduction exists in yeast or, less likely, that GSSG can somehow perform the essential role of GSH in yeast.
gsh1 strain. The
gsh1PRO2-1 strain seemed in this regard an appropriate
model. On YPD plates (Fig. 3,
A and B), the
gsh1PRO2-1 strain
had a wild type H2O2 (A) tolerance
but was slightly sensitive to t-butyl hydroperoxide
(B, t-BOOH). On SD plates (C and
D), the H2O2 (C) but not
the t-BOOH (D) tolerance was slightly decreased (Fig. 3,
C and D).
glr1 is hypersensitive to
peroxide (24), and its deletion in
gsh1PRO2-1
(
gsh1PRO2-1
glr1) further decreased the
peroxide-sensitive phenotype of both mutations (A-D). To
further evaluate the contribution of the GSH pathway to the defense
against peroxide, we compared the H2O2
tolerance of
gsh1PRO2-1 to that of strains lacking
either the oxidative stress response regulator Yap1 (25, 26), the thiol
peroxidases Tsa1 (27), Ahp1 (22, 28), or the GSH peroxidase Gpx3 (29).
In all conditions tested,
gsh1/PRO2-1 was the
least sensitive to peroxide among these strains, clearly demonstrating
that normal levels of GSH are not critical in this response as measured
by the plate assay.
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Fig. 3.
GSH is not essential to peroxide tolerance on
solid medium. A-D, tolerance to peroxide of wild type
(wt), gsh1PRO2-1,
glr1,
gsh1PRO2-1
glr1,
yap1,
tsa1,
hyr1, and
ahp1 was
evaluated by the patch assay on YPD (B and D) and
on SD plates (C and E) containing the indicated
amounts of H2O2 (B and C)
or t-butyl hydroperoxide (t-BOOH) (D
and E).
gsh1PRO2-1 was considerably more sensitive than wild
type (Fig. 4). Since the challenge assay
measures the cell tolerance to an acute exposure to high levels of
H2O2, whereas the plate assay examines the
growth adaptation to a moderate concentration of this oxidant, these results might indicate that GSH is more important for acute stress rather than for the adaptation to H2O2.
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Fig. 4.
GSH is important to peroxide tolerance in
liquid medium. The tolerance of wild type (wt,
black diamond), gsh1PRO2-1 (black
square),
glr1 (gray triangle), and
gsh1PRO2-1
glr1 (gray
circle) was evaluated by the challenge assay in SD medium.
Exponentially growing cells (A600 = 0.3-0.5) in
SD were exposed to various concentrations of
H2O2 for 1 h and then plated on YPD to
determine the number of colony-forming units.
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Fig. 5.
The GSH biosynthesis rate decreases after
H2O2 treatment. A, measurement
of the GSH biosynthesis rate. Exponentially growing wild type cells in
SM (A600 = 0.4) not exposed (lanes 1 and 3) or exposed to H2O2 (0.6 mM) (lanes 2 and 4) at
t = 0 were pulse-labeled with
[14C]glucose at t = 5 min and harvested
at t = 20 min (lanes 1 and 2) or
t = 50 min (lanes 3 and 4). Cells
were lysed as indicated in the method, and the extract was separated on
a TLC plate. B, measure of the GSH pool as described in
A except that cells had been labeled with
[14C]glucose at t = 0 and not exposed
(lane 1) or exposed to H2O2
(lane 2) at t = 2 h and harvested at
2 h 15 min. C, measurement of GSH export after
H2O2 treatment. Cells grown in SM
(A600 = 0.4) and labeled during 1 h with
[35S]Met were subsequently not exposed (lanes
1 and 3) or exposed (t = 0)
(lanes 2 and 4) to H2O2
(0.6 mM). The supernatant of each sample was analyzed for
its GSH content at t = 20 min (lanes 1 and
2) and t = 50 min (lanes 3 and
4). Purified labeled GSH was used as a control of GSH
migration (lane 5). D, cysteine is limiting for
GSH biosynthesis in the presence of H2O2. Cells
were grown in SM in the absence (lanes 1 and 2)
or in the presence of cysteine and glycine (400 µM)
(lanes 3 and 4) and labeled with
[14C]glutamate. 3 min after labeling, cells were not
exposed (lane 1 and 3) or exposed (lanes
2 and 4) to H2O2, and the GSH
content was analyzed at 20 min.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glutamyl phosphate is required for the suppression as an obligate
substrate for the modified PRO2 allele. It is thus probable that the PRO2 suppressor alleles allow condensation of
cysteine to the enzyme-bound
-glutamyl phosphate to produce
-glutamylcysteine. However, it is not clear how the addition of
cysteine to
-glutamyl phosphate occurs, whether spontaneous,
catalyzed by mutant Pro2p, or perhaps by a transpeptidase. The
suppression of the GSH auxotrophy by alleles of PRO2
illustrates how a biosynthetic pathway can be genetically diverted from
its natural role to fulfill another function.
gsh1 arrests in
G1 with predominantly unbudded cells after GSH withdrawal,
whereas a strain with a defect in ribonucleotide reduction and,
therefore, DNA synthesis would be expected to arrest with large buds in
S phase (36). In addition, the failure of anaerobic conditions to
rescue the viability of
gsh1 indicates that
its lethal phenotype is probably not related to oxidative stress or to
toxic accumulation of non-native protein disulfides. Furthermore, in
the latter case, it is expected that thioredoxin would compensate for
the lack of GSH. The GSH auxotrophy is also not due to a defect in
sulfate assimilation since methionine and cysteine cannot rescue the
growth defect of
gsh1. What could then be the essential
function of GSH in yeast? This essential function is clearly dependent
on the redox properties of GSH, since it can be replaced by a thiol
such as dithiothreitol (15) but not by a GSH analog with a methyl group
instead of a thiol. Since GSH is the reductant for glutaredoxin and
glutaredoxin is also essential (37), their essential function(s) might
be the same. One or more essential enzymes requiring the reduction of a
disulfide bond to complete their catalytic cycle might be exclusively dependent on the GSH reductive pathway in yeast. A search for the
cellular targets of glutaredoxin might help us understand the essential
requirement for GSH in yeast.
gsh1PRO2-1 and observed that it was indeed
sensitive to H2O2 by the 1-h challenge assay in
liquid medium. However, when measured by the plate assay,
gsh1PRO2-2 had a near wild type H2O2 tolerance on YPD (Fig. 3A) and
was just slightly sensitive to this oxidant on SD (Fig. 3C).
Still,
gsh1PRO2-1 was moderately sensitive to
t-butyl hydroperoxide but not as sensitive as strains lacking the Yap1 regulator or the peroxidases encoded by
GPX3 or AHP1. The difference regarding the
peroxide tolerance of strains with low GSH intracellular levels between
the reported literature and this study might be accounted for by the
fact that the
gsh1 strain used in those studies was
respiratory-deficient, which by itself lowers the tolerance to
oxidative stress (38), whereas
gsh1PRO2-1 was
respiration-proficient. We conclude that high levels of GSH are
important for surviving acute peroxide stress, as measured by the
challenge assay, but not for growth adaptation under moderate peroxide
concentrations, as measured by the patch assay. An inverse situation
was observed with a strain lacking the regulator of the
H2O2-adaptive response Yap1, which was
hypersensitive to H2O2 in the patch assay but
had a wild type resistance to this oxidant in the challenge assay (16).
In addition, we observed that, although GSH1 transcription
is induced by H2O2 (30, 31), the GSH synthesis
rate decreases upon exposure to H2O2 when
cysteine is limiting, suggesting that an increase in GSH might not be
essential during adaptation to H2O2. The
importance of GSH during an acute exposure to
H2O2 might be related to its redox buffering
properties. However, during adaptation to H2O2,
the GSH levels might not be limiting for its function as a cofactor for
glutathione peroxidase, allowing nearly normal detoxification of
H2O2 provided that it is kept reduced by
glutathione reductase. Indeed, deletion of GLR1 in
gsh1PRO2-2 resulted in a drop in
H2O2 tolerance which demonstrated that, despite
very low levels, GSH could still function in the scavenging of
H2O2 in this strain.
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ACKNOWLEDGEMENTS |
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We thank Dr. Brandriss for kind gifts of plasmids, André Sentenac for continuous support, and Carl Mann and Christophe Leroy for helpful suggestions and help with flow cytometry. We thank members of the Toledano group for enthusiastic discussions and advice.
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FOOTNOTES |
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* This work was supported by a grant from Associatíon de Recherche Centre de Cancer (9575) and from the Ministère de l'Éducation (to M. B. T.).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.
¶ Recipient of a National Institutes of Health predoctoral fellowship and of a Bourse de Formatíon Doctorale pour Érrangers/ Commissariat à l'Energie Atomique (Saclay, France) fellowship.
** To whom correspondence should be addressed. Tel.: 331-69088244; Fax: 331-69084712; E-mail: toledano@jonas.saclay.cea.fr.
Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M009814200
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ABBREVIATIONS |
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The abbreviations used are: YPD, yeast extract, peptone, and glucose; SM, synthetic medium; SD, minimal medium.
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