From the Service de Biochimie et Génétique Moléculaire, Bât 142, CEA-Saclay, F-91191, Gif-sur-Yvette Cedex, France
Received for publication, September 22, 2000, and in revised form, November 2, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cadmium is very toxic at low concentrations, but
the basis for its toxicity is not clearly understood. We analyzed the
proteomic response of yeast cells to acute cadmium stress and
identified 54 induced and 43 repressed proteins. A striking result is
the strong induction of 9 enzymes of the sulfur amino acid biosynthetic pathway. Accordingly, we observed that glutathione synthesis is strongly increased in response to cadmium treatment. Several proteins with antioxidant properties were also induced. The induction of nine
proteins is dependent upon the transactivator Yap1p, consistent with
the cadmium hypersensitive phenotype of the YAP1-disrupted strain. Most of these proteins are also overexpressed in a strain overexpressing Yap1p, a result that correlates with the cadmium hyper-resistant phenotype of this strain. Two of these
Yap1p-dependent proteins, thioredoxin and thioredoxin
reductase, play an important role in cadmium tolerance because strains
lacking the corresponding genes are hypersensitive to this metal.
Altogether, our data indicate that the two cellular thiol redox
systems, glutathione and thioredoxin, are essential for cellular
defense against cadmium.
Heavy metals represent major environmental hazards to human
health. In particular, cadmium is very toxic and probably carcinogenic at low concentrations. However, the biological effects of this metal
and the mechanism of its toxicity are not yet clearly understood. It
has been proposed that Cd2+ ions might displace
Zn2+ and Fe2+ in proteins (1), resulting in
their inactivation and in the release of free iron, which might
generate highly reactive hydroxyl radicals (OH·) (2). In support
of this hypothesis, a major effect of cadmium is oxidative stress (3),
particularly lipid peroxidation (1). However, it is not known whether
these effects are responsible for the extreme toxicity of the metal.
Living organisms use several mechanisms to counter cadmium toxicity. In
bacteria, efflux pumps are able to export toxic ions outside the cell
(4). In higher eukaryotes, Cd2+ is sequestered by
metallothioneins through their high cysteine content (5). Cadmium can
also be detoxified by chelation to GSH or to phytochelatin, a
glutathione polymer of general structure ( Yap1p and Skn7p are yeast transcription factors that regulate the
adaptive response to oxidative stress (9-11). Strains lacking either
transcription factor are sensitive to H2O2 and
are defective in the induction by H2O2 of
several enzymes with antioxidant properties (9). Yap1p is also
important in cadmium tolerance because yap1-deleted strains
are very sensitive to cadmium, and strains overexpressing YAP1 are hyper-resistant to this toxic metal (12). The
contribution of Skn7p to the cadmium response is more complex, because
skn7-deleted strains are hyper-resistant to cadmium (9),
suggesting that Skn7p is not only dispensable for this response but
might also repress genes that are important for cadmium tolerance.
Using two-dimensional gel electrophoresis, we have identified several
proteins induced by cadmium in Saccharomyces cerevisiae, providing a framework to the mechanisms of Cd2+ toxicity
and cellular protection against this toxic metal. In particular, our
results highlight the importance of both glutathione and of the
thioredoxin/thioredoxin reductase system in the cellular defense
against cadmium.
Strains and Growth Conditions--
Studies were performed with
the wild-type strain YPH98 (13) (MATa ura3-52
lys2-801amber ade
2-101ochre trp1 Survival Assays--
Aliquots from an exponential growth culture
(A600, 0.5) were serially diluted in water
(10-fold at each step). Ten µl were spotted onto rich broth (YPD)
plates containing different cadmium sulfate concentrations: 0, 25, 50, 75, 100, 150 and 200 µM. Plates were incubated at
30 °C for 2 to 5 days, and the colony-forming units were counted.
35S Labeling--
Ten-ml cultures were inoculated
with a colony and cultivated overnight aerobically at 30 °C with
shaking. Two-ml aliquots of mid-log phase culture
(A600, 0.4) were withdrawn and treated with 0, 0.5, 1, or 1.5 mM cadmium sulfate. 15 or 45 min after cadmium addition, cells were labeled with [35S]methionine
(200 µCi) for 15 min for analysis of protein expression or for 40 min
for analysis of glutathione synthesis rate.
Analysis of Glutathione Synthesis
Rate--
[35S]methionine-labeled cells (1 ml) were
collected by centrifugation, washed in 200 µl of water, and
resuspended in 50 µl of water. Cells were then boiled for 5 min to
extract metabolites (15) and centrifuged to obtain the
35S-labeled metabolites in the supernatant.
For analysis, the metabolites were oxidized by adding equal volume of
supernatant and performic acid (16). This treatment converts both
oxidized and reduced forms of glutathione to glutathione-sulfonic acid.
Samples (0.1 µl) were then applied on cellulose thin layer chromatography in the following solvent system: butanol-1/acetic acid/water (90:15:33). The 35S-labeled metabolites were
quantified by phosphor technology (Phosphor-Imager, Molecular Dynamics).
Analysis of Protein Expression--
Protein extraction and
two-dimensional gel electrophoresis were performed as previously
described (17) with a Millipore Investigator apparatus. The radioactive
gels were recorded by PhosphorImager and were analyzed with
two-dimensional gel analysis software (Melanie II, Bio-Rad). The spot
intensities were obtained in pixel units and normalized to the total
radioactivity of the gel. The cadmium stimulation index was calculated
as the ratio of spot intensity between cadmium and standard conditions.
Proteins with a stimulation index of higher than 1.8 are reported in
Table I. Proteins with a stimulation index lower than 0.60 (repressed proteins) are: ACS2, ADH1, ADH2, ADK1, ALD6, ARG1, BEL1, COF1, CPA2,
EFB1, EFT1, EGD1, EGD2, FBA1, GDH1, GLN1, GPP1, HIS4, HOM2, ILV2, ILV3,
ILV5, KRS1, LYS9, LYS20, PDC1, PUB1, PRO2, RPA0, RPA2, RPL45, RPS5,
SSB1, SSB2, TDH2, TIF1, TIF45, TIF51A, TPI1, VMA1, YEF3, YJL200C, and
YKL056C. The other analyzed proteins with stimulation indices lower
than 1.8 and higher than 0.60 are not reported in Table I (with the
exception of CTT1, TRR1, and SOD1). Mutant and wild-type strains were
compared by their ratio of spot intensities under different cadmium
conditions. The experiments were performed at least twice with similar
results, and the mean value was reported here. The S.D. of the analysis
ranged from 20 to 25%.
Identification of Protein Spots on Two-dimensional
Gels--
Proteins induced by cadmium were all identified by matching
two-dimensional maps of cadmium-treated cell extracts with a reference gel containing more than 450 previously identified proteins (17, 18,
19) with the exception of 3 new spots. These previously uncharacterized
proteins were identified by a peptide mass mapping approach using
matrix-assisted laser desorption ionization-time of flight mass
spectrometry (20).
Gene Disruptions--
Standard protocols and buffers were used
(21). Strains gre2
The ahp1 RNA Analysis--
Quantitative reverse transcriptase-PCR
analysis was performed essentially as previously described (19).
ACT1 reverse transcriptase-PCR products were used as
internal standards.
The Yeast Genomic Response to Cadmium--
We analyzed the
proteomic response to cadmium to identify activities, which could be
potentially important for the resistance to this toxic compound.
Exponentially growing cells were untreated or treated with 1 mM cadmium for 60 min, pulse-labeled with
[35S]methionine, and analyzed by comparative
two-dimensional gel electrophoresis (Fig.
1). The changes in spot intensity between untreated and treated cells were quantified by phosphorimager and
software analysis (see "Materials and Methods"). More than 50 proteins were induced by a factor greater than 2 after cadmium treatment (Table I). Concomitantly, about
40 other proteins were significantly repressed. A lower dose of cadmium
(0.5 mM) or shorter period of treatment (15 min) gave a
similar pattern of protein expression, but with lower induction levels.
Proteins induced by cadmium included enzymes with antioxidant
properties, heat shock proteins, proteases, enzymes of the sulfur amino
acid biosynthesis pathway, carbohydrate metabolism enzymes, and other
unclassified proteins or with unknown function. Conversely, proteins
repressed by cadmium were mainly components of the translational
apparatus and metabolic enzymes.
Induction of Glutathione Synthesis--
The particularly high
induction level of enzymes of the sulfur amino acid pathway (Fig.
2) suggested an increased synthesis of
cysteine and perhaps of GSH, which is essential in the cellular detoxification of cadmium. To test this hypothesis, we directly measured GSH synthesis rate by TLC after [35S]methionine
pulse labeling. After cadmium treatment, the synthesis rate of GSH was
increased more than 4-fold (Fig.
3A, lanes 1 and 2). As expected, in extracts from cells lacking
GSH1 (encoding the rate-limiting enzyme of GSH
biosynthesis), no GSH was produced (Fig. 3A, lanes
3 and 4). The production of another
35S-labeled compound was also augmented in response to
cadmium treatment, but we could not identify this molecule.
The stimulation of GSH biosynthesis correlated with a more than 10-fold
increase of GSH1 mRNA level after 1 h of cadmium
treatment (not shown and Ref. 24). As metallothioneins are
cysteine-rich and potentially important for cadmium detoxification, we
also analyzed CUP1 mRNA levels and found no increased
expression of this gene in response to cadmium, but rather a slight
repression. These results strongly suggest that the cysteine
biosynthesis pathway was induced to allow enhanced glutathione synthesis.
The Cadmium Response Is Altered in Regulatory Mutants--
Strains
lacking the transcriptional activator Yap1p are very sensitive to
cadmium and conversely, strains overexpressing this regulator are
hyper-resistant (12). However, strains lacking the transcriptional
activator Skn7p are hyper-resistant to this toxic metal (9). We
therefore analyzed the cadmium proteomic response in strains lacking
either YAP1 (yap1
Given the importance of GSH in cellular cadmium protection, we also
measured the biosynthesis of GSH in these mutants as we did in the
wild-type strain. The GSH biosynthesis rate was significantly decreased
in yap1 The Thioredoxin/Thioredoxin Reductase System Is Essential for
Cadmium Tolerance--
Proteins with both a defective cadmium
induction in yap1
One of the main Cd2+ toxic effects is believed to be the
generation of toxic lipid peroxides. It was therefore surprising to find that the strain deleted of AHP1, the gene encoding an
important alkyl hydroperoxide reductase, is not defective in cadmium
tolerance. Because another enzyme, the glutathione peroxidase Gpx3p, is
also active against organic peroxides (25), we analyzed the cadmium resistance of the GPX3-disrupted strain. Although this
mutant is clearly sensitive to tert-butyl hydroperoxide
(t-BOOH, Ref. 25 and data not shown), it showed a normal
cadmium resistance (Fig. 5).
To gain insight into the biological effects of Cd2+
and to identify activities relevant to its detoxification, we have
analyzed the proteomic response to this toxic metal. Fifty-seven
proteins were found to be induced by Cd2+, including
enzymes of the cysteine and glutathione biosynthesis pathway and
proteins with antioxidant properties. In addition, the induction of
several of these proteins is controlled by the transcription factor Yap1p.
Cadmium Stimulates the Biosynthesis of Cysteine and
Glutathione--
The proteomic response to cadmium has revealed a
strong induction of eight enzymes of the sulfur amino acid and GSH
biosynthesis pathways, particularly the last enzyme of the cysteine
biosynthesis pathway, Cys3p, and the rate-limiting enzyme of
glutathione biosynthesis, GSH1. This increased gene
expression was correlated with a strong stimulation of GSH biosynthesis.
These results are consistent with the notion that GSH acts as a first
line of defense against cadmium toxicity by chelation and sequestration
of the toxic metal. In yeast, cadmium is sequestered in the vacuole
upon transport of Cd2+·(GSH)2 complexes by
the membrane ATP-binding cassette Ycf1 (6). The importance of this
detoxification system is demonstrated by the hypersensitive phenotype
resulting from the deletion of either YCF1 (26) or
GSH1 (27). The cysteine biosynthetic pathway is also
important as demonstrated by the cadmium hypersensitive phenotype of
strains deleted for CYS4 (data not shown). This pathway is
probably essential for the increased production of GSH under these
stress conditions.
The coordinated regulation of GSH1 expression with enzymes
of the sulfur amino acids pathway may suggest that a common
transcriptional mechanism is involved in these inductions. Consistent
with this idea, a recent work (28) has shown that the GSH1
induction by cadmium is dependent upon the transcription factors Met4p,
Met31p, Met32p, and Cbf1p, which belong to the transcriptional complex of MET genes. It was also found that the GSH1
promotor contains functional elements typical of MET genes
(28).
Interestingly, the sulfur amino acid pathway is not induced but rather
repressed under most of the other stress conditions examined, including
the oxidative stress (19), the osmotic stress (29), and the heat shock
response (30). Furthermore, it is remarkable that among the enzymes
involved in amino acid metabolism, only those of the sulfur amino acid
biosynthetic pathway are induced, which is consistent with a very
specific control mechanism.
Does Cadmium Cause Oxidative Stress?--
Cd2+ is not
a redox active metal ion. However, it could cause oxidative stress and
lipid peroxidation, perhaps by displacing protein-bound
Fe2+, allowing this ion to become available for the Fenton
reaction. This hypothesis is based on previous studies showing an
increase in lipid peroxidation products after exposure to
Cd2+ (1, 31-34). In addition, yeast strains deleted for
cytosolic copper and zinc and mitochondrial manganese superoxide
dismutases (SOD1 and SOD2) are hypersensitive to
cadmium (3). The observation that several antioxidants such as Ahp1p,
Ccp1p, Tsa1p, and Sod2p are induced by cadmium is consistent with this
hypothesis. We also found that strains deleted for AHP1 or
GPX3, which encode two main organic peroxide-scavenging
activities, are not sensitive to cadmium, which does not support the
idea that cadmium toxicity is related to the cellular generation of
lipid peroxidation products. However, we cannot rigorously rule out the
possibility that yeast tolerance to cadmium-generated lipid
peroxidation products involves specific activities other than Ahp1p or
Gpx3p, which have not yet been elucidated.
It is also possible that cadmium indirectly contributes to oxidative
stress by affecting the cellular thiol redox balance. We indeed found
that Trx2p is significantly induced by cadmium and that strains deleted
for both TRX1 and TRX2 or for TRR1 are hypersensitive to this metal. In support of such a mechanism, a recent
work has shown that cadmium inactivates thioredoxins, thioredoxin
reductases, and glutathione reductases (35). Therefore, given the
essential nature of these systems (36), thiol transferase inactivation
could be the primary deleterious effect of cadmium. In addition, the
inactivation of the thiol transferase pathway could also result in an
increase in cellular lipid peroxidation products as a consequence of
their unavailability of the main electron donors for the thiol and GSH peroxidases.
Yap1p Target Genes--
The observation that YAP1-deleted strains
were hypersensitive to cadmium (9, 12) and strains overexpressing YAP1
were hyper-resistant (27), suggested that this transcriptional
activator might control genes important for cadmium tolerance.
YCF1 and GSH1 are two of them (27, 8). Given the
importance of the Ycf1p-GSH detoxification system, these two genes are
probably the primary targets by which Yap1p exerts a control of cadmium tolerance. In accord with this notion, we found that cadmium-induced GSH synthesis is controlled by Yap1p. We also discovered that nine
other genes required the presence of Yap1p for their proper induction
by cadmium. These mostly include antioxidant defense genes also induced
by H2O2 in a Yap1-dependent manner
(9). Several other proteins of the Yap1p
H2O2-inducible regulon (Ccp1p, Ssa1p, Hsp78p,
Hsp82p, Mpr1p, Uba1p, Cys3p, Tal1p, Zwf1p, Dak1p, and Tps1p) were still
induced by Cd2+ in yap1
Interestingly, most of the cadmium-inducible
Yap1p-dependent proteins were also overexpressed in a
YAP1 multicopy strain after cadmium treatment. This
YAP1 gene dosage effect, which was not observed in the
H2O2
response3 correlate well with
the hyper-resistance of the YAP1-overexpressing strain
toward Cd2+ but not toward
H2O2.
Skn7p Acts As a Repressor in Cadmium Response--
Skn7p is an
important regulator of the H2O2 response that
cooperates with Yap1p to activate the expression of several genes in
response to H2O2 (9). However, although Yap1 is
also important for the cadmium response, the function of Skn7p in this
response is more complex because skn7
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Glu-Cys)n-Gly
synthesized from GSH in plants and in the yeast Schizosaccharomyces pombe. Cd2+-phytochelatin
and Cd2+·(GSH)2 complexes are transported
into the vacuole by ATP-binding cassette transporters (6-8).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 leu2-
1). All
disruptants and strains used in this study were isogenic derivatives of
YPH98. The strain overexpressing YAP1 and the mutant
skn7
::TRP1 were previously described (9). Strains tsa1
::TRP1,
trx1
::TRP1,
trx2
::LEU2,
trx1
::TRP1-trx2
::LEU2 and trr1
::TRP1 were previously described (14).
Strains yap1
::TRP1, gre2
::TRP1,
ahp1
::TRP1 and
gpx3
::TRP1 were constructed in this work. The
strain gsh1
Pro2-2 has been
described.1 The strain
overexpressing YAP1 (mcYAP1) was obtained by transformation of strain YPH98 by a 2-µm plasmid pRS425 carrying a 2.5-kilobase EcoRI genomic YAP1 fragment. Cells were grown in
liquid medium containing 0.67% yeast nitrogen base without amino acids
(YNB, Difco), 2% glucose supplemented with uracil, adenine, lysine, tryptophan, and leucine at a final concentration of 30 mg/liter. The
YAP1-overexpressing strain was grown in the same medium but without leucine.
::TRP1 and
gpx3
::TRP1 were prepared by a one
step PCR2 amplification
protocol that replaced the entire GRE2 or GPX3 open-reading frame by the TRP1 gene (22). The mutant
yap1
was created by a one-step gene disruption technique
(23), which removed the YAP1 coding sequence from the
BamHI site (+186) to the KpnI site (+1650)
relative to the ATG and replaced it with the TRP1 gene.
::TRP1 strain used in the
present work was generated by one back-cross of the previously
described ahp1
::TRP1 (14) with a
wild-type strain.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (80K):
[in a new window]
Fig. 1.
Two-dimensional gel electrophoresis analysis
of total yeast proteins expressed in response to cadmium.
Autoradiograms of two-dimensional gel electrophoresis of total yeast
extracts from [35S]methionine-labeled wild-type cells.
35S labeling and two-dimensional gel electrophoresis were
performed as described under " Materials and Methods." Extracts
were prepared from control untreated cells (A) or cells
treated with 0.5 mM cadmium for 15 min (B) or
for 1 h (C) or treated with 1.5 mM cadmium
for 1 h (D). A central region of the autoradiograms was
blown up, and the induced proteins are indicated with black
arrows.
Identification of proteins induced by 1 mM Cd and effects
of Yap1p and Skn7p on its induction
View larger version (29K):
[in a new window]
Fig. 2.
Induction of the sulfur amino acid pathway in
response to cadmium. Induction factors are indicated in
brackets near the protein names.
View larger version (64K):
[in a new window]
Fig. 3.
Glutathione synthesis rate is increased after
cadmium treatment. Autoradiograms of thin layer chromatography of
intracellular metabolites from [35S]methionine-labeled
cells. A and B, the wild-type and the mutant
strains were left untreated ( ) or treated with 0.5 mM
Cd2+ for 30 min (+) and labeled with
[35S]methionine for 40 min. Cells were further
processed as described under "Materials and Methods." The
radioactive spots observed on the figure are the sulfonic acid forms of
cysteine and glutathione.
) or SKN7
(skn7
) or overexpressing YAP1
(mcYAP1) (Fig. 4). The
comparative measure of the proteomic response between these mutants and
the wild-type cells is given in Table I. The yap1
strain
had a weaker induction of 9 of the 57 responsive proteins, by a factor
of 2 or more. These included four antioxidant defense proteins (the
superoxide dismutase Sod2p, the alkyl hydroperoxidase Ahp1p, the
thioredoxin Trx2p, and the thioperoxidase Tsa1p) and five other
proteins (Sam1p, Oye3p, Gre2p, YNL134C, and YNL274C). Except for Sam1p,
all these proteins were significantly overexpressed in response to
cadmium in mcYAP1. Additionally, most of the heat shock
proteins and proteasome subunits were also overexpressed in
mcYAP1 (Table I). In skn7
, only two proteins
(Cdc48p and Shm2p) were not properly induced by cadmium. However,
surprisingly, Ctt1p, Ccp1p, Sod1p, Oye3p, Gre2p, YNL134C, and several
heat shock proteins and proteasome subunits were overexpressed in
response to cadmium.
View larger version (90K):
[in a new window]
Fig. 4.
Comparative analysis of cadmium response in
regulatory mutant strains. A, autoradiograms of
two-dimensional gel electrophoresis of total yeast proteins from
[35S]methionine-labeled strains treated with 1 mM cadmium for 1 h. Extracts were prepared from
wild-type (A1), yap1 (A2),
skn7
(A3), and YAP1 multicopy
strains (A4). A region of the autoradiograms was blown up,
and the proteins that were no longer induced in the yap1
strain are indicated by black arrows. LEU2 is the
marker of the plasmid that overexpresses YAP1. B,
histogram representation of noninduced (white bar) and
Cd2+ induced (black bar) synthesis rate indexes
calculated in yap1
(bar 1), wild-type
(bar 2), YAP1-overexpressing strain (bar
3) and skn7
(bar 4) cells as described
under "Materials and Methods." For each protein spot, values were
normalized to the wild-type noninduced level that was arbitrarily given
the value of 1. The protein names are indicated above the
histograms.
and augmented in mcYAP1(Fig.
3B, lanes 5-7). However, GSH biosynthesis rate
was not modified in skn7
.
and an increased expression in
mcYAP1 could be important for Cd2+ protection. 5 of 9 proteins matching this criteria were therefore evaluated for their
role in Cd2+ tolerance (Fig.
5A). Deletion of
TRR1 encoding the thioredoxin reductase or of both
TRX1 and TRX2 resulted in a significant decreased tolerance to cadmium (Fig. 5). However, the deletion of either thioredoxin does not affect the tolerance to cadmium, showing that
these two proteins can substitute for each other in this function. In
contrast, strains carrying a deletion of either GRE2, TSA1,
or AHP1 had a wild-type tolerance to cadmium (data not
shown). Note that the ahp1
strain has a wild-type
tolerance contradicting an earlier report (14) that demonstrates
ahp1
is sensitive to this metal. However, the cadmium
phenotype of this strain was shown related to an additional
unidentified mutation (data not shown).
View larger version (13K):
[in a new window]
Fig. 5.
Cadmium survival assay. Strains
gpx3 , trr1
, trx2
,
trx1
, trx1
trx2
, and the
wild-type strain were tested for cadmium resistance as described under
"Materials and Methods." The experiments were performed at least
twice with similar results; a typical error bar is
represented at the bottom left corner of the figure.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(see Table I),
suggesting the existence of other control pathways for the cadmium
response. This pathway does not involve the Yap1p homologous factor
Yap2p, because a strain deleted for both YAP1 and
YAP2 had the same proteomic response to cadmium than the
yap1
strain (not shown).
is hyper-resistant
to this metal (9). This resistance phenotype could not be explained by
a defective Cd2+ intracellular transport or alternatively,
by the overproduction of metallothioneins (data not shown) or
glutathione (this work). However, this could be explained by the
observation that several proteins of the cadmium stimulon were
superinduced in the skn7
mutant, supporting the idea that
Skn7p may act to repress these genes upon cadmium treatment. This was
particularly striking for heat shock proteins and for some
Yap1p-dependent proteins of unknown function (YNL134C,
Gre2p, or Oye3p). Interestingly, the YAP1 multicopy strain
overexpresses nearly the same set of proteins, suggesting a correlation
between the cadmium hyper-resistance phenotype and the overexpression
of these proteins. Some of these proteins, yet to be studied, might be
important for cadmium resistance.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank A. Sentenac for encouragement and
critical discussions, D. Thomas for providing cys4
strain, J. Lee for mRNA analysis, and J. Garin and S. Kieffer for
mass spectrometry analysis.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Ministère de l'Education Nationale de la Recherche et de la Technologie.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.
Supported by a National Institutes of Health Predoctoral
Fellowship and a Bowse de Formation Doctorale pour Etrangers
(BFDE/CEA).
§ To whom correspondence should be addressed. Tel.: 33-1-69-08-22-31; Fax: 33-1-69-08-47-12; E-mail: labarre@jonas.saclay.cea.fr.
Published, JBC Papers in Press, November 14, 2000, DOI 10.1074/jbc.M008708200
1 D. Spector, J. Labarre, and M. Toledano (2000) J. Biol. Chem. 276, in press.
3 C. Godon, J. Lee, J. Labarre, and M. Toledano, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviation used is: PCR, polymerase chain reaction, WT, wild-type.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Stohs, S. J., and Bagchi, D. (1995) Free Radic. Biol. Med. 18, 321-336[CrossRef][Medline] [Order article via Infotrieve] |
2. | Halliwell, B., and Gutteridge, J. M. C. (1984) Biochem. J. 219, 1-14[Medline] [Order article via Infotrieve] |
3. | Brennan, R. J., and Schiestl, R. H. (1996) Mutat. Res. 356, 171-178[CrossRef][Medline] [Order article via Infotrieve] |
4. | Silver, S., and Phung, L. T. (1996) Annu. Rev. Microbiol. 50, 753-789[CrossRef][Medline] [Order article via Infotrieve] |
5. | Hamer, D. (1986) Annu. Rev. Biochem. 55, 913-951[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Li, Z. S.,
Lu, Y. P.,
Zhen, R. G.,
Szczypka, M.,
Thiele, D.,
and Rea, P. A.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
42-47 |
7. |
Ortiz, D. F.,
Ruscitti, T.,
McCue, K. F.,
and Ow, D. W.
(1995)
J. Biol. Chem.
270,
4721-4728 |
8. |
Wemmie, J. A.,
Szczypka, M. S.,
Thiele, D. J.,
and Moye-Rowley, W. S.
(1994)
J. Biol. Chem.
269,
32592-32597 |
9. |
Lee, J.,
Godon, C.,
Lagniel, G.,
Spector, D.,
Garin, J.,
Labarre, J.,
and Toledano, M. B.
(1999)
J. Biol. Chem.
274,
16040-16046 |
10. |
Morgan, B. A.,
Banks, G. R.,
Toone, W. M.,
Raitt, D.,
Kuge, S.,
and Johnston, L. H.
(1997)
EMBO J.
16,
1035-1044 |
11. | Toone, W. M., and Jones, N. (1999) Curr. Opin. Genet. Dev. 9, 55-61[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Wu, A. L.,
Wemmie, J. A.,
Edgington, N. P.,
Goebl, M.,
Guevara, J. L.,
and Moye-Rowley, W. S.
(1993)
J. Biol. Chem.
268,
18850-18858 |
13. |
Sikorski, R. S.,
and Hieter, P.
(1989)
Genetics
122,
19-27 |
14. |
Lee, J.,
Spector, D.,
Godon, C.,
Labarre, J.,
and Toledano, M. B.
(1999)
J. Biol. Chem.
274,
4537-4544 |
15. | Ramos, F., Thuriaux, P., Wiame, J. M., and Bechet, J. (1970) Eur. J. Biochem. 12, 40-47[Medline] [Order article via Infotrieve] |
16. | Kumari, K., Khanna, P., Ansari, N. H., and Srivastava, S. K. (1994) Anal. Biochem. 220, 374-376[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Maillet, I.,
Lagniel, G.,
Perrot, M.,
Boucherie, H.,
and Labarre, J.
(1996)
J. Biol. Chem.
271,
10263-10270 |
18. | Boucherie, H., Sagliocco, F., Joubert, R., Maillet, I., Labarre, J., and Perrot, M. (1996) Electrophoresis 17, 1683-1699[Medline] [Order article via Infotrieve] |
19. |
Godon, C.,
Lagniel, G.,
Lee, J.,
Buhler, J. M.,
Kieffer, S.,
Perrot, M.,
Boucherie, H.,
Toledano, M. B.,
and Labarre, J.
(1998)
J. Biol. Chem.
273,
22480-22489 |
20. | Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Anal. Chem. 68, 850-858[CrossRef][Medline] [Order article via Infotrieve] |
21. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular cloning, a laboratory manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
22. | Baudin, A., Ozier-Kalogeropoulos, O., Danouel, A., Lacroute, F., and Cullin, C. (1993) Nucleic Acids Res. 21, 3329-3330[Medline] [Order article via Infotrieve] |
23. | Guthrie, C., and Fink, G. R. (1991) Methods Enzymol. 194, 273-362[Medline] [Order article via Infotrieve] |
24. | Stephen, D. W., and Jamieson, D. J. (1997) Mol. Microbiol. 23, 203-210[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Inoue, Y.,
Matsuda, T.,
Sugiyama, K.-i.,
Izawa, S.,
and Kimura, A.
(1999)
J. Biol. Chem.
274,
27002-27009 |
26. |
Szczypka, M. S.,
Wemmie, J. A.,
Moye-Rowley, W. S.,
and Thiele, D. J.
(1994)
J. Biol. Chem.
269,
22853-22857 |
27. | Wu, A. L., and Moye-Rowley, W. S. (1994) Mol. Cell. Biol. 14, 5832-5839[Abstract] |
28. |
Dormer, U. H.,
Westwater, J.,
McLaren, N. F.,
Kent, N. A.,
Mellot, J.,
and Jamieson, D. J.
(2000)
J. Biol. Chem.
275,
32611-32616 |
29. |
Norbeck, J.,
and Blomberg, A.
(1997)
J. Biol. Chem.
272,
5544-5554 |
30. | Boy-Marcotte, E., Lagniel, G., Perrot, M., Bussereau, F., Boudsocq, A., Jacquet, M., and Labarre, J. (1999) Mol. Microbiol. 33, 274-283[CrossRef][Medline] [Order article via Infotrieve] |
31. | Gupta, S., Athar, M., Behari, J. R., and Srivastava, R. C. (1991) Ind. Health 29, 1-9[Medline] [Order article via Infotrieve] |
32. | Howlett, N. G., and Avery, S. V. (1997) Appl. Microbiol. Biotechnol. 48, 539-545[CrossRef][Medline] [Order article via Infotrieve] |
33. | Howlett, N. G., and Avery, S. V. (1997) Appl. Environ. Microbiol. 63, 2971-2976[Abstract] |
34. | Sarkar, S., Yadav, P., and Bhatnagar, D. (1998) BioMetals 11, 153-157[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Chrestensen, C. A.,
Starke, D. W.,
and Mieyal, J. J.
(2000)
J. Biol. Chem.
275,
26556-26565 |
36. | Muller, E. G. D. (1996) Mol. Biol. Cell 7, 1805-1813[Abstract] |