From the Research Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011, Japan
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ABSTRACT |
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Methylglyoxal is a cytotoxic metabolite derived
from dihydroxyacetone phosphate, an intermediate of glycolysis.
Detoxification of methylglyoxal is performed by glyoxalase I. Expression of the structural gene of glyoxalase I (GLO1) of
Saccharomyces cerevisiae under several stress conditions
was investigated using the GLO1-lacZ fusion gene, and
expression of the GLO1 gene was found to be specifically induced by osmotic stress. The Hog1p is one of the mitogen-activated protein kinases (MAPKs) in S. cerevisiae, and both Msn2p
and Msn4p are the transcriptional regulators that are thought to be
under the control of Hog1p-MAPK. Expression of the GLO1
gene under osmotic stress was completely repressed in
hog1 disruptant and was repressed approximately 80 and
50% in msn2
and msn4
disruptants,
respectively. A double mutant of the MSN2 and
MSN4 gene was unable to induce expression of the
GLO1 gene under highly osmotic conditions. Glucose consumption increased approximately 30% during the adaptive period in
osmotic stress in the wild type strain. On the contrary, it was reduced
by 15% in the hog1
mutant. When the yeast cell is exposed to highly osmotic conditions, glycerol is synthesized as a
compatible solute. Glycerol is synthesized from glucose, and a
rate-limiting enzyme in glycerol biosynthesis is glycerol-3-phosphate dehydrogenase (GPD1 gene product), which catalyzes
reduction of dihydroxyacetone phosphate to glycerol 3-phosphate.
Expression of the GPD1 gene is also under the control of
Hog1p-MAPK. Methylglyoxal is also synthesized from dihydroxyacetone
phosphate; therefore, induction of the GLO1 gene expression
by osmotic stress was thought to scavenge methylglyoxal, which
increased during glycerol production for adaptation to osmotic
stress.
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INTRODUCTION |
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Several environmental stresses are known to trigger intracellular
alterations in organisms; e.g. synthesis of some
stress-inducible proteins or the cellular responses against
extracellular signals. Organisms of all types show the synthesis of
stress-inducible proteins, and the most advanced understandings of the
stress-inducible proteins have been obtained from the study of heat
shock protein (HSP).1 A
sudden increase in the temperature of the environments in which cells
are growing induces increased synthesis of a set of heat shock
mRNAs and proteins in the cells. Heat shock response in eukaryotes
is different from that in bacterial cells, although the basic
mechanisms are similar among eukaryotes. A heat shock transcription
factor, which is synthesized constitutively, is trimerized, modified,
and translocated by heat shock and binds to a cis-element
(the heat shock element) in the promoter region of HSP genes
to activate transcription (for review, see Ref. 1). Some HSP
genes in Saccharomyces cerevisiae, such as
HSP104, HSP26, and HSP12, have another
cis-element, termed stress response element (STRE)
(5-AGGGG-3
), in addition to the heat shock element (2, 3). In
addition to these HSP genes in S. cerevisiae, the
CTT1 gene encoding cytosolic catalase has also been known to
have the STRE in its 5
-flanking region (4). Expression of the
CTT1 gene is induced by a wide variety of stresses, such as
osmotic stress and oxidative stress, as well as heat shock, and these stress signals are thought to be focusing on the STRE (2). Stress
response mechanisms through the STRE are fairy broad; therefore, elucidation of molecular mechanisms for stress response, including screening an appropriate specific marker gene, is of considerable interest.
In addition to heat shock response, one of the well-known stress
response systems in S. cerevisiae is an osmotic stress
response (5-7). Increased osmolarity of environment surrounding the
cells induces rapid increase in expression of various kinds of genes. The osmosensing system has been extensively studied in a bacterial system, and existence of a two-component regulatory system was proved
(8, 9). S. cerevisiae also has a bacterial-like
two-component osmosensing system consisted of Sln1p and Ssk1p (10-13).
When the yeast cell is exposed to a highly osmotic environment, rapid
efflux of water from the cell shrinks cell and decreases its turgor
pressure. As one of adaptive responses to the osmotic stress, the yeast cell produces glycerol as a compatible osmolyte. A key enzyme in
biosynthesis of glycerol is glycerol-3-phosphate dehydrogenase, which
is encoded by the GPD1 gene. The GPD1 gene is
essential for survival under highly osmotic conditions (14). Expression of the GPD1 gene under highly osmotic conditions is under
the control of Hog1p (14). Hog1p is one of the mitogen-activated protein kinases (MAPKs) in S. cerevisiae. Both residues of
Thr174 and Tyr176 of the Hog1p are phosphorylated by a MAPK kinase
(MAPKK), Pbs2p. Pbs2p is phosphorylated by redundant MAPKK kinases,
Ssk2p and Ssk22p. The hog1 knockout mutant showed
lethality under highly osmotic conditions (2, 15).
The substrate for glycerol-3-phosphate dehydrogenase (Gpd1p) is
dihydroxyacetone phosphate, an intermediate of glycolytic pathway, and
the enzyme catalyzes reduction of dihydroxyacetone phosphate to
glycerol 3-phosphate in the presence of NADH. Some phosphatases, such
as Gpp2p, hydrolyze glycerol 3-phosphate to glycerol (16).
Dihydroxyacetone phosphate is also a substrate for methylglyoxal
synthase that converts dihydroxyacetone phosphate to methylglyoxal
(17-20). Methylglyoxal is also synthesized during the triosephosphate
isomerase reaction (-elimination) (21, 22). Methylglyoxal
(CH3COCHO) is a typical 2-oxoaldehyde in organisms, and it
has two carbonyl carbons, i.e. ketone and aldehyde. Methylglyoxal can react with various biological compounds in cells, such as protein, DNA, and RNA, to inactivate them (23, 24), and thus it
shows cytotoxicity. To avoid overaccumulation of methylglyoxal, the
cells from prokaryotes to higher eukaryotes have several defense systems. The glyoxalase system is a ubiquitous scavenging system for
methylglyoxal. The glyoxalase system consisted of two enzymes, i.e. glyoxalase I (EC 4.4.1.5), which converts methylglyoxal to S-D-lactoylglutathione in the presence of
glutathione, and glyoxalase II (EC 3.1.2.6), which hydrolyzes
S-D-lactoylglutathione to D-lactic
acid and glutathione. We have been systematically studying the
glyoxalase system in various microorganisms, and we have proved that
glyoxalase I is required for detoxification of methylglyoxal (25,
26).
We found that the GLO1 gene had two STREs in its 5-flanking
region, and expression of the GLO1 gene was specifically
induced by osmotic stress. In this paper, we also describe expression of the GLO1 gene in several gene disruptants, the gene
products of which are involved in the HOG (high osmolarity glycerol)
MAP kinase pathway, and discuss why the GLO1 gene is
expressed under highly osmotic conditions.
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MATERIALS AND METHODS |
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Strains and Plasmids--
The S. cerevisiae strains
used in this study are summarized in Table
I. Plasmids for disruption of
MSN2 gene (pt32-XB::HIS) (27), YAP1
gene (pSM27) (28), and GSH1 gene (pYOG1211) (29) were kindly
provided by Dr. F. Estruch (University of Valencia), Dr. W. S. Moye-Rowley (University of Iowa), and Dr. Y. Ohtake (Asahi Breweries),
respectively. pScCer1 (30) was kindly donated by Dr. T. Miyakawa
(Hiroshima University).
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Construction of GLO1-lacZ--
To construct the
GLO1-lacZ fusion gene, PCR primers were designed to amplify
the 5-flanking region of the GLO1 gene and 15 amino acid
residues from N-terminal methionine. The PCR primers used were
5
-CTGAAGGAGGTGCCCGGGGGATAAACCTAC-3
and
5
-ATCATTCCCGGGTTTCTCAATCTGAATTGG-3
. Both primers were
designed to contain the SmaI site (shown by italic letters).
A PCR fragment (992 bp) amplified using these primers was digested with
SmaI and then introduced to SmaI site of pMC1871,
which contained the lacZ gene of Escherichia coli without its original promoter region and N-terminal eight amino acids
(31). The resultant plasmid (pMCGlac18) was digested with SalI, and the GLO1-lacZ cassette was cloned to
the SalI site of the centromere plasmid pRS415 (32) to yield
pRSGlac415.
Construction of Disruptants--
The HOG1 gene with
its 5 and 3
regions was cloned by PCR using primers HOG1S
(5
-GTTGTTAGGAAAGCATGCTTTATCTCCAAG-3
) and HOG1R
(5
-CCTTTTATGGGATCCTAATTTCTTAAGGAG-3
). Both primers were designed to contain recognition site for SphI for HOG1S and
BamHI site for HOG1R, respectively; these sites are
indicated by italic letters. The PCR fragment (2340 bp) was cloned
between the SphI and BamHI sites of pUC19 to form
pUCHOG1. To construct the hog1
::URA3 mutant, pUCHOG1 was digested with BalI and
HincII, and a 400-bp fragment in the open reading frame of
the HOG1 gene was replaced with the URA3 gene to
yield pUHOG
Ura3. The resultant plasmid was then digested with
SphI and BamHI, and then the
hog1
::URA3 fragment was introduced
to S. cerevisiae YIT2 (cta1
) (33) to construct
the strain YYH1 (cta1
hog1
::URA3). The MSN2
gene of YIT2 was disrupted using a plasmid pt32-
XB::HIS as
described by Estruch and Carlson (27), and the disruptant was named
YYM2 (cta1
msn2
). The MSN4 gene
was cloned by PCR using primers MSN4S (5
-CGCCACACCAACATGCAACTTCTCCCAAGA-3
) and MSN4R
(5
-GCTCTTCCAACCAAGCCTCATTGCTCCTTG-3
). Primer MSN4S corresponded to
the region between 533 and 562 bp downstream from the ATG codon, and
the MSN4R corresponded to the region between 2653 and 2682 bp from the
ATG codon of the MSN4 gene. The PCR fragment (2150 bp) was
digested with SphI and EcoRI and then cloned
between the SphI and EcoRI sites of pUC19. The resultant plasmid (pUCmsn4) was digested with EcoRV and
AflII to delete the 593-bp fragment, which contained the
zinc-finger motif of Msn4p (27), and then replaced with the
URA3 gene to construct pUmsn4
Ura3. The plasmid was
digested with SphI and EcoRI, and the DNA
fragment containing the msn4
::URA3
cassette was introduced to strains YIT2 and YYM2 to construct YYM4
(cta1
msn4
) and YYM24 (cta1
,
msn2
, and msn4
), respectively. The GLO1 gene disruption plasmid (pUG
His3) was constructed as
follows. A plasmid containing the GLO1 gene, pE24GLO1 (26),
was digested with SphI, and the GLO1 gene was
recloned to the SphI site of pUC19 to form pUGLO1. The
plasmid was digested with EcoRV and HapI to
delete the 850-bp fragment containing the open reading frame of the
GLO1 gene, and it was replaced with the HIS3 gene to form pUG
His3. The pUG
His3 was digested with SphI,
and the glo1
::HIS3 fragment was
introduced to strain YGS1
(gsh1
::LEU2). The strain YGS1 was
constructed by using the GSH1-disruption plasmid pYOG1211
(29) and strain YPH250 as a host. The double mutant of the
GSH1 and GLO1 genes was named YGSL1
(gsh1
glo1
). The yap1
disruptant was constructed by disrupting the YAP1 gene of YPH250 using pSM27 (yap1
::HIS3)
(28). Disruption of each gene was verified by PCR or Southern analysis,
and transformation of yeast was carried out as described in our
previous paper (26).
Construction of the YAP1-overexpressing Strain-- A plasmid, pScCer1, carrying the YAP1 gene (30), was digested with EcoRV and ClaI, and the fragment with the YAP1 gene was cloned to the BamHI site of YEp13 after blunting each cohesive end with a Klenow fragment. The resultant plasmid, YEp-YAP1, was introduced into S. cerevisiae YPH250. Overexpression of the YAP1 gene was confirmed by monitoring the growth on SD (2% glucose, 0.67% yeast nitrogen base) minimal agar plate containing 0.2 µg/ml cycloheximide as reported by Hertle et al. (34).
Stress Experiments-- Each strain of S. cerevisiae carrying pRSGlac415 was cultured in a test tube containing 5 ml of SD medium (pH 5.5) with appropriate amino acids and bases at 28 °C with shaking for 30 h. A small portion of the culture was transferred to a 200-ml flask containing 50 ml of YPD medium (2% glucose, 2% peptone, 1% yeast extract; pH 5.5) and cultured at 28 °C with shaking for approximately 18 h. When the optical density of the culture at 610 nm (A610) reached approximately 1.0, several chemicals, such as H2O2, NaCl, and ethanol were added, and then culture was continued for another 1 h. For heat shock experiment, the flask containing the culture was transferred to an incubator preheated to 37 °C, and cultivation was continued for another 1 h. After stress treatment, cells were collected, and cell extracts were prepared as described below.
Preparation of Cell Extracts-- Cells were collected by centrifugation (500 × g at 4 °C for 10 min), washed twice with 0.85% NaCl solution, and suspended in 300 µl of 10 mM potassium phosphate buffer (pH 7.0). Cells were transferred to an Eppendorf tube containing an approximately equal volume of glass beads and then agitated with a vortex mixer at maximum speed for 3 min. Cell homogenates were centrifuged at 14,000 rpm for 15 min at 4 °C, and resultant supernatants were used as cell extracts.
Enzyme Assay--
Glyoxalase I activity was assayed as described
previously (35). One unit of the activity was defined as the amount of
enzyme forming 1 µmol of
S-D-lactoylglutathione per min at 25 °C.
Catalase activity was measured according to the method of Roggenkamp
et al. (36). One unit of the activity was defined as the
amount of enzyme decomposing 1 µmol of H2O2
per min at 25 °C. -Galactosidase activity was measured as
described by Miller (37). One unit of the activity was defined as the
amount of enzyme increasing A420 by 1000 per min
at 30 °C. Protein was determined by the method of Bradford (38).
Northern Blotting--
Cells of S. cerevisiae YPH250
were cultured in YPD medium until A610 reached
approximately 1.0, and then 0.5 M NaCl was added. After 30 min incubation, total RNA was prepared according to the method of
Schmitt et al. (39). To analyze the effect of the newly
synthesized protein for expression of the GLO1 gene in
osmotic stress response, cells were pretreated with 50 µg/ml
cycloheximide for 15 min before the addition of NaCl. RNA was separated
by an agarose gel containing formaldehyde as described by Sambrook
et al. (40). The GLO1 probe was prepared by
digesting pUGLO1 with BamHI, and the resultant fragment (837 bp) containing the open reading frame of the GLO1 gene was
purified by the low melting point agarose gel electrophoresis, and
labeled by [-32P]dCTP (Amersham Corp.) using a kit
(Takara).
Measurement of Glucose-- Cells of YIT2 and YYH1 were cultured in a 200-ml flask containing 50 ml of YPD medium until A610 reached approximately 1.0, and NaCl was added to bring the final concentration of the culture to 0.5 M. 1.5 ml of culture was withdrawn periodically, and the A610 and glucose concentrations of the culture were measured using a kit (Glucose B-test Wako).
Measurement of Methylglyoxal-- Preparation of cell extracts was essentially same as described above, except that cells were suspended in distilled water instead of 10 mM potassium phosphate buffer (pH 7.0). Cell extracts were used as a source of methylglyoxal in glyoxalase I reaction. The reaction mixture (1 ml) contained 100 µM potassium phosphate buffer (pH 7.0), 2 mM glutathione, 56 units/ml glyoxalase I (Sigma), and various concentrations of methylglyoxal, or cell extracts.
Chemicals--
Methylglyoxal and glyoxalase I was purchased from
Sigma. Glutathione was obtained from Kohjin (Tokyo, Japan). Restriction enzymes, DNA modification enzymes, and the DNA labeling kit were purchased from Takara Shuzo (Kyoto, Japan). [-32P]dCTP
(6000 Ci/mmol) and nylon membrane (Hybond-N) were obtained from
Amersham-Japan (Tokyo, Japan). The glucose assay kit was purchased from
Wako Chemicals (Kyoto, Japan). Plasmid pRS415 was purchased from
Stratagene (La Jolla, CA).
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RESULTS |
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Effect of Yap1p on Glyoxalase I Activity--
Yap1p is one of the
transcriptional regulators in S. cerevisiae, and it closely
correlates with glutathione metabolism as well as drug resistance
(41-45). Because detoxification of methylglyoxal is performed by
glyoxalase I in the presence of glutathione, we supposed that
expression of the GLO1 gene might be regulated by the Yap1p.
We then constructed both of the YAP1 overexpressing strain
and yap1 disruptant and measured the glyoxalase I
activity. However, glyoxalase I activity was not affected by the copy
number of the YAP1 gene product (wild type, 0.128 ± 0.015 units/mg protein; YAP1 overexpression, 0.145 ± 0.018 units/mg protein; yap1
, 0.142 ± 0.022 units/mg protein).
Expression of the GLO1 Gene Is Induced by Osmotic Stress--
By
an analysis of the 5-flanking region of the GLO1 gene, we
found two STREs 432 and 229 nucleotides upstream of the initiation (ATG) codon (Fig. 1). Several genes have
been reported to have the STRE, such as CTT1,
HSP104, HSP26, and DDR2, and
expression of such genes is induced by several stresses, such as heat
shock, oxidative stress, osmotic stress, ethanol stress, and so on (2). The CTT1 gene encodes cytosolic catalase, and it was widely
used as a marker gene for experiments for stress response analysis through STRE; therefore, we also used CTT1 catalase as a marker. Because S. cerevisiae has another catalase in peroxisome
that is encoded by the CTA1 gene (46), we disrupted the
CTA1 gene to specifically monitor the CTT1 catalase
activity. The GLO1-lacZ fusion gene was also constructed to
quantify the expression of GLO1 gene under several
environmental stresses. As shown in Fig. 2A, expression of
GLO1-lacZ was specifically induced by NaCl, and other
stresses that could induce expression of the CTT1 gene were
not effective for induction of GLO1-lacZ expression.
Glyoxalase I activity was also increased when the cells were treated
with 0.5 M NaCl, although other stresses did not affect the
enzyme activity (data not shown). As shown in Fig. 2B,
expression of GLO1-lacZ was induced by not only NaCl but
also by other osmotic stresses, including sorbitol. Therefore,
induction of GLO1-lacZ expression under high NaCl
concentrations was thought to be caused by osmotic stress rather than
salt stress.
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Northern Blotting--
To confirm that an increase of
-galactosidase activity from the GLO1-lacZ fusion gene
was due to the increased expression of the GLO1 gene,
Northern blot analysis was done. Because the optimal concentration of
NaCl for induction of GLO1-lacZ was 0.5 M (Fig.
3), S. cerevisiae YPH250 was treated with 0.5 M
NaCl, and the mRNA level of the GLO1 gene was measured.
As shown in Fig. 4, the GLO1
mRNA level increased by osmotic stress. Therefore, increased
activity of
-galactosidase from GLO1-lacZ by osmotic stress was confirmed to reflect the expression of GLO1 gene.
To investigate whether de novo synthesis of some factors is
required for induction of the GLO1 gene transcription, the
cells were pretreated with cycloheximide to block protein synthesis and
then exposed to osmotic stress. As shown in Fig. 4, mRNA level of
the GLO1 gene was also increased, even though protein
synthesis was blocked. Therefore, induction of GLO1 gene
expression was not dependent upon the newly synthesized protein(s)
during osmotic stress treatment.
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Involvement of GLO1 Gene Expression in HOG-MAPK
Pathway--
S. cerevisiae has been reported to have a
bacterium-like two-component system for sensing high osmolarity of
environments. Sln1p and Ssk1p are, respectively, a sensor kinase for
high osmolarity and a regulator to mediate the signal to the redundant
MAPKK kinases Ssk2p and Ssk22p. Activated MAPKK kinases phosphorylate
the Pbs2p MAPKK. The Pbs2p can also receive a high osmolarity signal
from the second osmosensor, Sho1p. The activated Pbs2p then
phosphorylates Hog1p-MAPK. Therefore, in both pathways, signals focus
on Hog1p. The Mig1p-like zinc finger proteins Msn2p and Msn4p are
thought to be under the control of Hog1p in osmotic stress response. We then disrupted HOG1, MSN2, MSN4, and
both MSN2 and MSN4 genes by gene replacement in
the cta1 background and measured expression of the
GLO1 and CTT1 genes under highly osmotic
conditions. As shown in Fig.
5A, induction of the
GLO1 gene expression under highly osmotic conditions was
almost completely repressed in the hog1
mutant (YYH1). In
the msn2
mutant (YYM2), induction was approximately 20%
compared with strain carrying the wild type MSN2 allele
(YIT2). On the other hand, the msn4
mutant (YYM4) could
induce approximately 50% more expression of the GLO1 gene than YIT2. In the case of double mutant of the MSN2 and
MSN4 genes (YMM24), induction of GLO1 expression
was not observed. These results strongly suggest that expression of the
GLO1 gene under highly osmotic conditions is controlled by
the HOG-MAPK pathway. On the other hand, expression pattern of the
CTT1 gene was different from that of the GLO1
gene (Fig. 5B). In the strain YYH1 (hog1
), induction of the CTT1 gene expression under highly osmotic
conditions was extremely reduced compared with that of YIT2, although
the CTT1 was still induced by osmotic stress. In the strains
YYM2 (msn2
) and YYM4 (msn4
), the
CTT1 gene expression was also induced as observed in the
case of GLO1-lacZ; however, the most conspicuous difference
was seen in the behavior of the msn2
msn4
double mutant (YMM24). The CTT1 gene was still induced in
the strain YMM24 when the cells were exposed to high osmotic
stress.
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Effect of Osmotic Stress on Consumption of Glucose--
S.
cerevisiae synthesizes glycerol as a compatible solute when the
cells are exposed to highly osmotic environments. Glycerol is
synthesized from glucose through dihydroxyacetone phosphate and
glycerol 3-phosphate. On the other hand, methylglyoxal is also
synthesized from dihydroxyacetone phosphate by methylglyoxal synthase
(17-20) or from triosephosphate isomerase reaction (-elimination) (21, 22) during catabolism of glucose. Therefore, glucose consumption
is expected to be increased to synthesize glycerol during the adaptive
response to highly osmotic environments. We measured the concentration
of glucose in culture medium to evaluate the consumption speed of
glucose by yeast cells. When the cells were exposed to highly osmotic
conditions, intracellular water efflux was increased, and it resulted
in causing cell shrinkage. Therefore, the A610
of the culture suddenly increased (Figs.
6, A and B);
however, it recovered during the adaptation period (5-30 min), and
growth was restarted (60-120 min). The generation time of the strain
YIT2 (HOG1) without osmotic stress was 295.6 ± 5.22 min (0-120 min), whereas it increased to 375.6 ± 13.7 min after recovery from cell shrinkage (60-120 min). In the case of strain YYH1
(hog1
), generation time (276.3 ± 7.74 min) without
osmotic stress during the 0-120-min period was similar to that of
YIT2. On the other hand, once the cells were exposed to high
osmolarity, generation time during the 60-120 min period increased to
589.6 ± 18.7 min. During the 5-30-min period, yeast cells seemed
to recover the cell size to adapt osmotic pressure. Glucose consumption speed of YIT2 under the osmotic conditions during the 5-30-min period
was approximately 30% higher than that during the same period without
osmotic stress (Fig. 6C). After recovery of the growth
(60-120 min), glucose consumption rate of YIT2 under highly osmotic
conditions was 10% higher than that of the same strain during the
60-120-min period. In the case of hog1
mutant (YYH1), the glucose consumption rate during the 5-30-min period was decreased approximately 15% under highly osmotic conditions compared with that
in the same strain in normal osmolarity. The glucose consumption rate
of the hog1
cells treated or not treated by NaCl during the 60-120-min period was similar (Fig. 6D).
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DISCUSSION |
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Regulation of GLO1 Gene Expression in Osmotic Response by the
HOG-MAPK Pathway--
The YAP1/PAR1/PDR4/SNQ3 gene product
is a transcriptional activator. A key enzyme for glutathione
biosynthesis is the GSH1 gene product, -glutamylcysteine
synthetase, and expression of the GSH1 gene is positively
regulated by the Yap1p. Glutathione recycling is also important for
cells to maintain the intracellular redox state, and the
GLR1 gene encoding glutathione reductase is also under the
control of Yap1p. Glutathione is involved in the glyoxalase I reaction.
Furthermore, overproduction of Yap1p renders yeast cells to resist
against many structurally unrelated drugs, such as 1,10-phenanthroline
(PAR1) (48), cycloheximide (PDR4) (49),
nitrosoguanidine (SNQ3) (34), cadmium (YAP1) (28), and H2O2 (YAP1) (30). Because
methylglyoxal is a metabolic cytotoxic compound in the cell, we thought
that expression of the GLO1 gene may also be regulated by
Yap1p. However, no alterations of glyoxalase I activity were observed
in the yeast cells overexpressing the YAP1 gene or in the
yap1
mutant. On the other hand, we found two STREs in the
5
-flanking region of the GLO1 gene, and expression of the
GLO1 gene was specifically induced when the cells were exposed to highly osmotic environments. Gounalaki and Thireos (50)
reported that the TPS2 gene encoding trehalose phosphate phosphatase had the STREs in its promoter region, and expression of the
TPS2 gene was induced by several stresses, such as heat shock, osmotic stress, and metabolic inhibitors. They also reported that Yap1p was required for transcriptional regulation of the TPS2 gene through the STRE. Yap1p is a functional homolog of
mammalian AP-1, and the consensus sequence for recognition site of the
Yap1p (Yap1p recognition element) was reported to be 5
-TTAGT(C/A)A-3
(51). The Yap1p recognition element sequence was not found in the
promoter region of the GLO1 gene, and the increased copy
number of the Yap1p in the cells did not affect the glyoxalase I
activity. Furthermore, glyoxalase I activity was increased in the
yap1
mutant by osmotic stress (data not shown).
Therefore, we concluded that expression of the GLO1 gene was
not dependent upon the Yap1p.
Physiological Significance for Induction of the GLO1 Gene in
Osmotic Stress Response--
As shown in Fig. 6, the glucose
consumption rate transiently increased by osmotic stress in the wild
type strain. Norbeck and Blomberg (52) reported that expression of both
of the HXT1 gene, encoding hexose transporter, and the
GLK1 gene, encoding glucokinase, was enhanced under highly
osmotic conditions. S. cerevisiae synthesizes glycerol as a
compatible solute when the cells are exposed to highly osmotic
environments. Glycerol is synthesized via glycolysis, and the
rate-limiting step for glycerol production is a glycerol-3-phosphate
dehydrogenase reaction. Glycerol-3-phosphate dehydrogenase is encoded
by the GPD1 gene, and the gene expression under highly
osmotic conditions is regulated by Hog1p (14). The gpd1
mutant cannot grow in a medium containing a high concentration of NaCl
(14). Expression of the GPP2 gene, encoding
glycerol-3-phosphate phosphatase, was also reported to be enhanced by
osmotic stress (16). To adapt to high osmolarity, S. cerevisiae cells produce glycerol from glucose, and
dihydroxyacetone phosphate is a precursor. On the other hand,
methylglyoxal is also synthesized from glycolysis, and dihydroxyacetone
phosphate is a substrate for methylglyoxal synthase (17-20).
Methylglyoxal is also inevitably produced from the triosephosphate
isomerase reaction (21, 22). Therefore, an increased flux of glucose to
glycolysis may cause the enhancement of intracellular methylglyoxal
content. Actually, we found that the steady state level of
methylglyoxal in the gsh1
glo1
mutant cells
treated by 0.5 M NaCl increased approximately 23% compared with that in the untreated cells. Glucose consumption was also increased approximately 30% by osmotic stress (Fig. 6C).
Therefore, the physiological purpose for increasing GLO1
gene expression under highly osmotic conditions may be to
scavenge methylglyoxal that is increased in the adaptive response to
high osmolarity (Fig. 7).
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ACKNOWLEDGEMENTS |
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We thank Drs. F. Estruch, W. S. Moye-Rowley, Y. Ohtake, and T. Miyakawa for providing
pt32-XB::HIS3, pSM27, pYOG1211, and pScCer1, respectively.
We thank S. Miyabe for construction of YEp-YAP1.
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FOOTNOTES |
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* 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.: 81-774-38-3773;
Fax: 81-774-33-3004; E-mail: inoue{at}food2.food.kyoto-u.ac.jp.
1 The abbreviations used are: HSP, heat shock protein; HOG, high osmolarity glycerol; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; STRE, stress response element; bp, base pairs; SOD, superoxide dismutase.
2 Y. Inoue, A. Kawamura, S. Izawa, and A. Kimura, unpublished data.
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