From the Department of Molecular Sciences, University of Tennessee, Memphis, Tennessee 38163
Received for publication, December 2, 2002, and in revised form, January 24, 2003
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ABSTRACT |
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Ure2, the protein that negatively regulates GATA
factor (Gln3, Gat1)-mediated transcription in
Saccharomyces cerevisiae, possesses prion-like
characteristics. Identification of metabolic and environmental factors
that influence prion formation as well as any activities that prions or
prion precursors may possess are important to understanding them and
developing treatment strategies for the diseases in which they
participate. Ure2 exhibits primary sequence and three-dimensional homologies to known glutathione S-transferases. However,
multiple attempts over nearly 2 decades to demonstrate Ure2-mediated
S-transferase activity have been unsuccessful, leading to
the possibility that Ure2 may well not participate in glutathionation
reactions. Here we show that Ure2 is required for detoxification of
glutathione S-transferase substrates and cellular oxidants.
ure2 Several neurodegenerative conditions derive from the same
pathogenetic mechanism, i.e. a change in protein
conformation, polymerization, and plaque formation. These conditions
have been called conformational diseases such as Alzheimer's and the
prionoses. Recent studies have demonstrated that a protein associated
with such disease, amyloid Ure2 was originally identified as a mutated genetic locus that permits
cells growing with ammonia as nitrogen source to transport the
pyrimidine precursor ureidosuccinate; wild type cells are unable to do
this (14, 15). The ure2 mutation was subsequently found to
possess a pleiotropic phenotype in which transcriptional repression of
many genes encoding proteins needed to transport and degrade poor
nitrogen sources becomes resistant to nitrogen catabolite repression
(NCR),1 i.e.
repression no longer occurs in the presence of a good nitrogen source.
When URE2 was cloned and sequenced, it was found to possess
homology to known glutathione S-transferases (16). Homology between Ure2 and glutathione S-transferases now extends to
the level of its crystal structure (17-19). Two additional structures were determined using crystals in which glutathione or two of its
analogues were bound to Ure2 (19). Despite these structural characteristics, multiple attempts to demonstrate that Ure2 catalyzes a
glutathione S-transferase reaction have been unsuccessful
(7, 16, 20). There are also characteristics of the Ure2
three-dimensional structure that prompt the question of whether it
would even be expected to possess S-transferase activity.
Most specifically a residue that participates in catalysis by known
glutathione S-transferases (i.e. cysteine or
histidine in Beta class and tyrosine or serine residues in
eukaryotic classes) is not present in Ure2 (17). This residue is
critical because it destabilizes the cysteine S-H bond, thereby
facilitating formation of the active thiolate anion (GS Whether or not Ure2 is a glutathione S-transferase is a
question that has remained open and tantalizing for 2 decades and is
increasingly important to future studies of Ure3 prion formation and
the impact of environmental and metabolic influences on it. Therefore,
we have investigated the possibility of additional Ure2 functions. To
circumvent repeatedly reported problems associated with in
vitro enzyme assays, we adopted a more genetic approach, i.e. asking whether ure2 Strains and Media--
The S. cerevisiae strains we
used were TCY5 (MAT
The rich medium was YEPD, and minimal media for plating cells
were Difco Yeast Nitrogen Base without amino acids or ammonium sulfate
(0.17%) to which was added the indicated nitrogen source at 0.1% if
other than 0.5% ammonium sulfate was used. Further additions
(added after media were autoclaved and cooled) of metal ions,
xenobiotics, etc. are indicated in the figure legends. Our standard
auxotrophic supplements were added where necessary. Cells were grown at
30 °C. Although photographs are largely presented of cells at single
times and for a single concentration of perturbant, in most cases, we
have collected images at multiple times and multiple perturbant
concentrations. This approach gives us a better appreciation of what
occurs during the experiment and increases our confidence that the
images presented here are representative of the effects we report.
During this work, we noted wild type strain-to-strain differences in
overall sensitivity to various perturbants. However, the patterns of
sensitivity in wild type versus mutant cells were always the
same. For this reason a wild type control was included on all Petri
plates so that wild type versus mutant comparisons could be
made directly.
Northern Blot Analyses--
TCY5 was grown in Difco Yeast
Nitrogen Base (0.17%)-ammonia (0.1%) or -glutamate (0.1%) containing
0.6 mM nickel sulfate to mid-log phase
(A600 = 0.5). Cycloheximide (0.01% final
concentration) was added to the cells and incubated for 10 min.
Cells were then harvested by centrifugation (4 °C), washed in cold
lysis buffer (0.5 M NaCl, 0.5 M Tris base (pH
7.5), 0.01 M EDTA) containing 0.005% cycloheximide, and
resuspended in cold lysis buffer. An equal volume of acid-washed glass
beads was added to the cells along with an equal volume of cold PCI
(phenol/chloroform/isoamyl alcohol (25:24:1), and the cells were lysed
by vortexing. After two more PCI extractions, an additional extraction
with cold chloroform/isoamyl alcohol (24:1) was performed. Total RNA
was ethanol-precipitated overnight at
Northern blot analyses were performed as described previously (24)
using the PCR-generated probes that were radiolabeled with the
Invitrogen RAD Prime DNA Labeling System. The primers used to generate
probe DNA were as follows: URE2 (5'-CAAGTGTCGAATCTCTCCAA-3' and 5'-TCTATCCACGACATTATTCC-3'), GAP1 (12), and
H3 (12). Nine micrograms of total RNA were added to each
lane for analysis.
Ure2 Is Required for Protection against Heavy Metals--
Ure2
possesses clear structural homology with Theta or Beta classes of
glutathione S-transferases. Unfortunately attempts in
multiple laboratories to demonstrate glutathione
S-transferase enzyme activity for Ure2 have been
unsuccessful (7, 16, 20). Although negative results are rarely reported
in detail, the lack of success might derive from inherent instability
in the enzyme, an observed characteristic of glutathione
S-transferases, or from performing the assay with substrates
that are not the preferred ones for the putative transferase (25-28).
The difficulties experienced in attempts to assay an enzyme activity
prompted us to take a step backward from in vitro assays and
ask more simply whether Ure2 is required for protection of cells
against the growth-inhibitory effects of compounds reported to be
glutathione S-transferase substrates in other organisms.
Assays unable to demonstrate Ure2-dependent glutathione
S-transferase were performed with the commonly used
substrate 1-chloro-2,4-dinitrobenzene (CDNB) (7, 20). Therefore, we
first determined whether ure2 mutants might be
hypersensitive to previously untested glutathione S-transferase substrates, for example, heavy metal ions such
as nickel and cadmium. In wild type S. cerevisiae, cadmium
ions are conjugated to glutathione, and the glutathionato-cadmium
conjugate is transported into the vacuole or out of the cell (29). Ycf1 is responsible for transport of the conjugate (30), but identity of the
enzyme catalyzing conjugation is not known. We compared growth of wild
type (TCY-5) and isogenic ure2
Although growth differences of wild type and ure2
To test whether it was the ure2 mutation in TCY-5 that was
specifically responsible for increased metal ion sensitivity, we complemented the mutation by transforming strains RR114
(ure2
In studies of this nature, one of the most significant problems is to
distinguish whether observed effects derive from direct or indirect
participation of the gene product being tested. This is an especially
serious consideration when that product is known, as Ure2 is, to
negatively regulate NCR-sensitive, Gln3- and Gat1-mediated transcription in the presence of a good nitrogen source (31-33). By
this reasoning, loss of metal ion detoxification in a
ure2
Since Ure2 regulation of Gln3/Gat1-mediated transcription influences
expression of GDH1 and GDH2, another conceivable
explanation of the Ure2 requirement for metal ion detoxification might
be that it alters the level of reducing equivalents (NADPH) required to
maintain glutathione in its active, reduced form. Although this
explanation was circumstantially addressed in Fig. 1 by comparing metal
ion sensitivity with glutamate versus ammonia as nitrogen source, it can be addressed more specifically by comparing metal ion
sensitivity of wild type and gdh1 Ure2 Potential Participation in Detoxification of Organic
Xenobiotics and Hydrogen Peroxide--
Most glutathione
S-transferases exhibit rather broad substrate specificities,
although clear substrate preferences exist. Given this characteristic
and the fact that all reported in vitro glutathione
S-transferase assays of Ure2 were performed using the
chromogenic xenobiotic CDNB as the acceptor molecule (7, 20), we
compared wild type and ure2
Mutants with defects in glutathione S-transferase genes are
often found to exhibit increased sensitivity not only to preferred S-transferase substrates but also to compounds that are not
detoxified by direct conjugation to glutathione. Two compounds in this
category are hydrogen peroxide and diamide. Hydrogen peroxide is
detoxified in two ways: by peroxidation, an activity found in some
mammalian Theta-class GSTs and in S. pombe GST3 or by GSH
conjugation of cellular products that are oxidized by hydrogen or other
peroxides (25, 26, 28). Diamide also oxidizes cellular proteins and other constituents but, in addition, depletes the reduced glutathione pool because it is detoxified via glutathione-dependent
reduction (36). ure2
Mutants lacking glutathione S-transferases have also been
reported to become hypersensitive to hydrogen peroxide (25). Therefore, we compared hydrogen peroxide toxicity in wild type and
ure2 Ure3 Is Capable of Protecting Cells from Heavy
Metals--
URE3 and ure2 mutants were
originally isolated from the same selection (14, 15). Therefore, except
for being a bit more leaky, it is not surprising that URE3
exhibits the same phenotype as ure2 mutations. Prior to this
work, the only known ure2 phenotype was resistance to NCR
(14, 15, 31-33). Identification of a new ure2 phenotype
prompts the question of whether URE3 strains possess a
similar phenotype, as is the case for negative regulation of
GATA factor-mediated transcription. The question is pertinent because
there is a strong correlation between Ure2-Gln3 and Ure2-Gat1 complex
formation and the ability of Ure2 to inhibit NCR-sensitive transcription in the presence of a good nitrogen source,
i.e. the Ure2 regulatory activity appears to be associated
with a stoichiometric reaction between Gln3 and itself. Since Ure2 in
its Ure3 prion form is a polymer, it is not too surprising that it
cannot simultaneously interact with itself and Gln3. Indeed portions of
the Ure2 molecule that interact with Gln3 also participate in prion
formation (37). If we assume that Ure2 does possess glutathione
S-transferase activity, it may not be as adversely affected
by Ure3 prion formation because this activity is catalytic rather than
stoichiometric. Consistent with this possibility, glutathione has been
reported to bind to the polymerized form of Ure2 (6).
To answer the above question, we used three strains generously provided
by Edskes and Wickner: YHE711 (wild type, ure-o), TIFY3
(ure2:G418), and YHE731 (strain YHE711 into which
URE3 was cytoduced). Since Ure3 can be lost from some cells
during storage in glycerol,2
we streaked out all three strains on YEPD and then scored the phenotypes of multiple isolates on glucose ammonia + ureidosuccinate (USA) medium. Wild type ure-o cells (all strains are
ura2, a prerequisite of the plate assay) will not grow in
this medium, whereas ure2 and ure3 mutants will.
We indeed found that a few of the "ure3" isolates had become
identical to the wild type, i.e. no longer able to grow on
ammonia + USA medium. We assayed the metal ion sensitivity of seven
randomly chosen URE3 clones using wild type and
ure2 mutants as controls. As shown in Fig.
8, URE3 and ure2 clones grew similarly in glucose ammonia + USA medium but exhibited opposite phenotypes in the presence of metal ions. The
URE3 clones were just as resistant as the wild type to both
environmental insults. Note that there was some cross-feeding of the
"wild type" (ura2) strain. This did not occur when the
streaked cells were more distantly separated.
Effect of Heavy Metals on URE2 and NCR-sensitive
Transcription--
Given Ure2 regulation of NCR-sensitive
expression, we determined whether heavy metal ion treatment affects
URE2 expression or the ability of Ure2 to regulate GATA
factor-mediated transcription. We used 0.6 mM nickel
sulfate rather than cadmium chloride as the perturbant here because
cadmium ions have such a drastic effect on cell growth. URE2
expression increased about 2-fold in minimal medium containing nickel
sulfate (Fig. 9). Similar results were observed whether glutamate or ammonia was used as nitrogen source. Growth in the presence of nickel sulfate had little demonstrable effect
on NCR-sensitive gene expression using GAP1 as the
NCR-sensitive reporter gene. These data suggest that regulation of
URE2 expression is unlikely to be induced at moderate levels
of perturbant and that the protection of Ure2 cells from toxic
compounds does not demonstrably diminish its ability to regulate
NCR-sensitive gene expression.
This work identifies a new function for Ure2 in S. cerevisiae, i.e. participation in heavy metal ion and
oxidant detoxification. Deletion of URE2 results in
hypersensitivity to cadmium ions, hydrogen peroxide, and, to a lesser
extent, nickel ions. ure2 mutants are also slightly
hypersensitive to diamide, a glutathione cycling reagent, but only when
ammonia is used as sole nitrogen source. There is no convincing
hypersensitivity to diamide when glutamate is used in place of ammonia,
suggesting that hypersensitivity, if it exists, is seen only when NADPH
pools are diminished. Our data also offer a possible explanation of
negative results encountered in attempts to demonstrate Ure2-mediated
glutathione S-transferase activity using CDNB as substrate.
We found CDNB to be only slightly more toxic to ure2 Primary sequence and three-dimensional homology between Ure2 and known
glutathione S-transferases as well as the observed binding
of glutathione and glutathione S-transferase substrates to
crystallized Ure2 are consistent with the possibility that URE2 encodes a glutathione S-transferase. Our
work provides additional support for that argument, i.e. the
demonstration that Ure2 is required for metal ion detoxification and
repair or prevention of perturbant-generated cellular damage in
vivo. While present evidence argues in favor of Ure2 being a
glutathione S-transferase, it does not unequivocally
distinguish whether the ure2 mutant phenotype is a direct or
indirect effect of the loss of the protein. Our data do, on the other
hand, demonstrate that if the effect of Ure2 loss is indirect, it does
not occur through the only known function of Ure2, i.e.
negative regulation of Gln3 and Gat1. This possibility is eliminated by
the observation that increasing Gln3- and Gat1-mediated transcription,
the outcome of deleting URE2, does not increase sensitivity
to metal ions but rather slightly decreases it or leaves it unaffected
depending upon the strain tested.
Deletion of URE2 generates increased sensitivity to
multiple compounds including heavy metal ions, strong oxidants
(hydrogen peroxide), agents that deplete reduced glutathione pools
through oxidation (diamide), and perhaps those depleting the
glutathione pools per se through conjugation (CDNB).
However, the level of hypersensitivity generated by the
ure2 The fact that URE2 expression increased only 2-fold in the presence of
nickel sulfate suggests that cellular Ure2 levels are normally
sufficient for it to function effectively in protecting the cell from
heavy metal ions and strong oxidants. Consistent with this suggestion
is the fact that overexpression of URE2 (Fig. 3) did not result in
increased resistance to metal ions until their concentration had been
raised substantially. That the regulation of NCR-sensitive
GAP1 expression by Ure2 was not demonstrably affected by the
presence of metal ions may derive from one or more of several factors.
(i) Ure2 levels are sufficient for it to function in both
detoxification and GATA factor regulation. (ii) The effects of Ure2
levels on NCR are subtle and hence below detection in the assay we
used. (iii) If Ure2 functions both catalytically and
stoichiometrically, it would be unlikely for the catalytic function to
significantly interfere in the stoichiometric function. (iv) The
participation of Ure2 in glutathione S-transferase functions is indirect. Resolving these issues will require a more detailed understanding of the biochemistry of Ure2.
Finally, in addition to identifying a new function for Ure2, this work
has identified a phenotype that can be used along with growth in
ammonia + USA medium to distinguish wild type, ure2, and
URE3 alleles. URE3 strains are as resistant to
nickel and cadmium ions as wild type, while ure2 mutants
exhibit markedly increased sensitivity to these ions. Both
ure2 and URE3 strains, on the other hand, are
resistant to NCR, whereas the wild type is not. If Ure2 is a direct
participant in enzyme reactions involving glutathione, the shared and
distinguishable phenotypes of ure2 and URE3
mutations may derive from the physical requirements of the two
processes assayed, one of which requires stoichiometric participation
of Ure2, while for the other only catalytic participation is involved.
This ability to distinguish Ure2 and Ure3 in vivo will prove
highly useful in future studies of relationships between the two forms
of this fascinating protein.
mutants are hypersensitive to cadmium and nickel
ions and hydrogen peroxide. They are only slightly
hypersensitive to diamide, which is nitrogen source-dependent, and minimally if at all hypersensitive to
1-chloro-2,4-dinitrobenzene, the most commonly used substrate
for glutathione S-transferase enzyme assays. Therefore,
Ure2 shares not only structural homology with various glutathione
S-transferases, but ure2 mutations possess the
same phenotypes as mutations in known S. cerevisiae
and Schizosaccharomyces pombe
glutathione S-transferase genes. These findings are
consistent with Ure2 serving as a glutathione S-transferase
in S. cerevisiae.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-protein, protects neurons from
metal-induced oxidative damage (1). Neither the molecular basis for
this activity nor how it is affected by environment and neuronal
metabolism is yet known. The use of eukaryotic model systems, such as
the yeast Saccharomyces cerevisiae, has greatly facilitated
our acquisition of information about mammalian proteins, their
functions and interactions, and how their synthesis and activities are
regulated and integrated. In particular, the genetic study of prions
has been facilitated by Wickner's (2) discovery that S. cerevisiae Ure3 possesses prion-like characteristics. Ure3, the
prion form of the nitrogen-regulatory protein Ure2, has been well
studied in an attempt to gain further insight into the changes that
accompany polymerization and the cellular proteins that impact on that
process (3-7, 23). Significant emphasis has been placed on
determining whether metabolic activity influences Ure2
Ure3
conversion (8-13). Ure2
Ure3 conversion has been reported to
decrease in an mks1
strain, in a strain expressing
the constitutively active dominant Ras2Val19
allele (8), and in strains where the intracellular pool of glutamate is
enlarged (11).
).
The region of Ure2 that binds glutathione does contain an asparagine, Asn-124, that some, but not all, investigators suggest may be situated
at a location and distance that are consistent with permitting it to
function in destabilization of this critical S-H bond (19).
mutants exhibit
greater sensitivity than isogenic wild type strains to a range of
glutathione S-transferase substrates and compounds
generating oxidative stress in S. cerevisiae. We show that
Ure2 is indeed required for detoxification of glutathione S-transferase substrates and cellular oxidants. Ure2 shares
not only structural homology with various glutathione
S-transferases, but ure2 mutations exhibit
phenotypes similar to those of mutations in known S. cerevisiae and Schizosaccharomyces pombe glutathione S-transferase genes.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, lys2, ura3,
trp1), TCY1 (Mat
, lys2,
ura3), RR114 (MAT
, lys2,
ura3, trp1, ure2::TRP1), RR154 (Mat
,
lys2, ura3,
gdh1
::hisG- URA3-hisG), YHE711
(MAT
, ura2, leu2:hisG,
[ure-0]), YHE731 (MAT
,
ura2, leu2:hisG, [URE3]
{URE3 cytoduced into YHE711}), STCY32
(Mat
, lys2, ura3, trp1,
his3::hisG), TIFY3 (MAT
,
ura2, leu2:hisG,
ure2::G418), and BY4741
(Mata, his3 D1, leu2 D0,
met1 5D0, ura3 D0). The plasmids were pRA27 (21),
pEG202 (22), pRR529 (21), and YEp24 (New England Biolabs, Inc.).
20 °C, pelleted, resuspended
in diethylpyrocarbonate-treated water, ethanol-precipitated again, and
resuspended as before. RNA concentration was determined
spectrophotometrically (A260 nm), and the
samples stored at
80 °C until analyzed.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(RR114) strains in
glucose-ammonia or -glutamate medium containing nickel sulfate (0.6 mM) or cadmium chloride (0.1 mM). Both metal
ions markedly inhibited growth of the ure2
mutant
relative to wild type, although at the concentrations used, cadmium
ions were far more toxic (note differences in the times of incubation)
(Fig. 1). In addition, the
ure2 mutant phenotype was much tighter with cadmium than
with nickel ions. Equally important, growth was inhibited to roughly the same degree regardless of whether ammonia or glutamate was provided
as sole nitrogen source. This is a positive indication that observed
sensitivity did not derive indirectly from the influence of the
ure2 mutation on NADPH levels in the cell.
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Fig. 1.
Growth of wild type and
ure2 cells in the presence and absence
of heavy metal ions. Nitrogen sources and metal ions provided in
the medium are indicated. The times of incubation are indicated and
were the same for both ammonia and glutamate media. Minimal ammonia or
minimal glutamate medium used in the bottom two
panels did not contain any added heavy metal ions.
W.T., wild type.
strains
in Fig. 1 are marked, we were concerned that they might derive trivially from the fact that ure2 mutants in some strain
backgrounds grow a bit slower than wild type. Therefore, we asked, did
slow growth of the ure2
in the presence of nickel or
cadmium ions derive from loss of ability to detoxify the metal ions or
was it just a manifestation of the fact that ure2 mutants
grow slower? To assess these possibilities, we streaked the wild
type and ure2
strains on the same media used for
the metal ions toxicity test but in the absence of the metal ions. As
shown in the bottom panels of Fig. 1, the ure2
strain does grow a bit slower than wild type. Therefore,
ure2
hypersensitivity to nickel ions is not as great as
depicted in the top two panels but is still present and can be increased if the nickel ion concentration is raised to 0.9 mM (data not shown). For cadmium, the difference in growth
is dramatic. In fact, there was no growth of the ure2
mutant even after 4 days of incubation on YEPD-cadmium plates (Fig.
2).
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Fig. 2.
Growth of wild type and
ure2 mutant cells on YEPD medium in the
presence and absence of cadmium chloride or hydrogen peroxide.
W.T., wild type.
) with pRR529 (URE2). TCY5 (wild type)
transformed with vector YEp24 was the positive control. URE2
pRR529 restored the growth of strain RR114 in the presence of nickel or
cadmium ions to wild type levels (Fig.
3A). If Ure2 is itself
responsible for wild type detoxification of metal ions and is the
limiting entity in metal ion detoxification, one might expect to see
increased ability to cope with high levels of environmental metal ions
when URE2 is overexpressed. As shown in Fig. 3B,
overexpression of URE2 did not generate increased resistance
to 0.6 mM nickel sulfate relative to wild type. However, when the nickel ion concentration was increased to 0.9 mM,
overexpression of URE2 did result in detectably increased
protection relative to the wild type strain transformed with vector
YEp24.
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Fig. 3.
A, complementation of the
ure2 mutation by plasmid-borne
URE2. Metal ions and nitrogen sources are
indicated. B, effect of overexpressing URE2 on
the sensitivity of wild type cells to 0.6 and 0.9 mM nickel
sulfate. Bottom panels identify the strains and plasmids
with which they were transformed. W.T., wild type.
mutant might derive indirectly from increased
Gln3/Gat1-mediated transcription. To test this possibility, we
transformed wild type strain STCY32 with ADH1-GAT1 pRA27 or
vector control pEG202 and tested the metal ion sensitivity of the
transformants. Overexpression of GAT1 in this way has
been previously shown to result in both increased Gat1- and
Gln3-mediated transcription and to render that transcription largely
resistant to NCR (21, 34). In addition, we tested metal ion sensitivity
with glutamate as sole nitrogen source rather than ammonia since
glutamate generates much less NCR than ammonia in these strains, which
in turn would further increase Gln3/Gat1-mediated gene expression.
Rather than increasing metal ion sensitivity, as occurs when
URE2 is deleted, overexpression of GAT1 resulted
in greater nickel ion resistance than that observed when the wild type
strain was transformed with control pEG202 (Fig.
4). The effect of GAT1
overexpression, although visible with cadmium ions, was less striking.
When this experiment was repeated in strain BY4741, parent of the
consortium-generated set of yeast gene deletion strains, pEG202- and
pRA27-containing transformants grew indistinguishably in the presence
of cadmium chloride or nickel sulfate (data not shown). These data and
those in Fig. 4 argue against the possibility that metal ion
hypersensitivity in a ure2
mutant results from its
only demonstrated function, negatively regulating GATA factor-mediated
transcription.
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Fig. 4.
Effect of overexpressing GAT1
on the sensitivity of wild type cells (STCY32) transformed with
control YEp24 or ADH1-GAT1 pRA27 to cadmium and nickel
ions.
mutant cells. In
gdh1
mutants, ammonia is assimilated without the
consumption of NADPH because it occurs through the combined action of
the GLN1 (glutamine synthetase) and GLT1 (glutamate synthase,
GOGAT) gene products (35). As shown in Fig.
5, wild type and gdh1
strains are equally able to detoxify nickel ions. If anything, the
gdh1
mutant may grow slightly better than wild type.
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Fig. 5.
Nickel sulfate sensitivity of wild type and
gdh1 mutant in minimal ammonia and
minimal glutamate media. W.T., wild
type.
strain sensitivity to CDNB. As shown in Fig. 6, wild type colonies
are larger than a ure2
mutant after 54 h of
incubation in the presence of CDNB. At 68 h of incubation, the
difference in growth between the two strains, although still apparent,
is much less drastic. Similar results were observed with minimal
glutamate medium (Fig. 6 and data not shown). However, the growth
difference seen at 68 h is not convincingly different from that of
wild type and ure2
strains growing in the absence of
perturbant (Fig. 1, bottom two panels). Therefore, if loss
of Ure2 generates CDNB hypersensitivity it is modest at best. This
correlates well with the inabilities of multiple investigators, including ourselves, to detect in vitro glutathionation of
CDNB.
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Fig. 6.
Sensitivity of wild type and
ure2 cells to CDNB over time in minimal
ammonia and minimal glutamate media. W.T., wild type.
colonies grown in the presence of
diamide were smaller than wild type after 51 h of incubation in
minimal glutamate or minimal ammonia medium (Fig.
7, top two panels).
Differences in growth, however, were less marked after 72 h of
incubation (Fig. 7, middle two panels). Sensitivity of the
ure2
to diamide was greater than that observed for CDNB
but less than that observed for nickel ions when ammonia was used as
nitrogen source. In contrast, when glutamate was used as nitrogen
source, differences in growth were not different from those seen in the
bottom two panels of Fig. 1. It is important to note that
diamide was the only perturbant in which different growth patterns were
observed on glutamate versus ammonia medium. This difference
correlates with the facts that (i) ammonia assimilation places a
greater drain on the NADPH pool than does glutamate and (ii)
the only cellular constituent used in detoxifying diamide is NADPH.
Therefore, it is conceivable that apparent ure2
hypersensitivity to diamide derives indirectly from effects of the
mutation on NADPH metabolism or alternatively that hypersensitivity can
only be visualized at lower NADPH concentrations.
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Fig. 7.
Sensitivity of wild type and
ure2 cells to diamide over time and
hydrogen peroxide at 96 h in minimal ammonia and minimal glutamate
media. W.T., wild type.
strains. Hydrogen peroxide was more toxic to
ure2
mutants than were any of the other non-metal ion
perturbants (Fig. 7, bottom two panels). In contrast to what
occurred with diamide, hypersensitivity to hydrogen peroxide exhibited
by the ure2
strain was equivalent whether ammonia or
glutamate was provided as nitrogen source. Hypersensitivity was also
observed in hydrogen peroxide-containing YEPD medium (Fig. 2,
bottom panel). In sum, ure2 mutations, although most sensitive to heavy metal ions, exhibit the same pleiotropic hypersensitivity to oxidants, hydrogen peroxide being most toxic, whose
detoxification does not involve direct conjugation to glutathione as seen in gst1, gst2, and gst3
mutants of S. pombe (25).
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Fig. 8.
Sensitivity of wild type,
ure2 , and URE3 cells to
nickel sulfate and cadmium chloride. The top panel
depicts growth of the three strains in medium supplemented with USA
rather than uracil, which was used to supplement media depicted in the
lower two panels. W.T., wild type.
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Fig. 9.
Steady state levels of URE2 and GAP1 mRNA
in minimal glutamate medium in the presence or absence of 0.6 mM nickel sulfate. Conditions were as
described under "Materials and Methods."
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
cells than to wild type, suggesting that Ure2 may play only a
peripheral role at best in its detoxification.
varies quantitatively over a very wide range
depending upon the perturbant tested. How can Ure2 be a direct
participant in protecting cells from all of these compounds when the
enzyme mechanisms involved in detoxifying these agents are to varying
degrees different? Although this issue cannot be rigorously addressed
in the absence of detailed in vitro studies, using purified
proteins, varying hypersensitivity to a spectrum of compounds that are
detoxified in different ways is a commonly seen phenotype of mutations
in known glutathione S-transferase genes. Three glutathione
S-transferases (encoded by GST1, GST2,
and GST3) have been identified in S. pombe.
Single mutants in each of these genes result in increased sensitivity to diamide and hydrogen peroxide. Moreover, overproduction of Gst1,
Gst2, or Gst3 increased in vitro activity to catalyze the conjugation of CDNB and glutathione, while overproduction of only Gst3
increased glutathione peroxidase activity using cumene hydroperoxide as
substrate (25, 38). Two glutathione S-transferase genes have
been reported in S. cerevisiae, GTT1 and
GTT2 (20). Both genes were shown to catalyze the glutathione
S-transferase reaction using CDNB as substrate. When these
investigators similarly assayed Ure2 (20), the results were negative
just as they were for others and ourselves (data not shown). Finally,
Hynes and his co-workers (39) cloned the gstA gene from
Aspergillus nidulans and found its primary protein sequence
to be most homologous to Ure2. Among the compounds to which a
gstA deletion mutant was hypersensitive were nickel ions,
selenium, diamide, and CDNB (39). However, in contrast to S. cerevisiae Ure2, the A. nidulans GstA protein does not
participate in the regulation of GATA factor-mediated, nitrogen-responsive transcription.
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ACKNOWLEDGEMENTS |
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We thank Drs. H. Edskes and R. Wickner for strains, Dr. Kathleen Cox for many suggestions of pertinent literature, Tim Higgins for preparing the artwork, and the University of Tennessee Yeast Group for suggestions to improve the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM-35642.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 901-448-6179;
Fax: 901-448-8462; E-mail: tcooper@utmem.edu.
Published, JBC Papers in Press, January 31, 2003, DOI 10.1074/jbc.M212186200
2 H. Edskes, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: NCR, nitrogen catabolite repression; CDNB, 1-chloro-2,4-dinitrobenzene; GST, glutathione S-transferase; USA, ureidosuccinate.
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