From the Institut de Biochimie et
Génétique Cellulaires, UMR 5095, 33077 Bordeaux Cedex,
France and the ¶ Department of Biochemistry, Biozentrum
of the University of Basel, CH-4056 Basel, Switzerland
Received for publication, September 25, 2000
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ABSTRACT |
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In yeast, the transition between the fermentative
and the oxidative metabolism, called the diauxic shift, is associated
with major changes in gene expression and protein synthesis. The zinc cluster protein Cat8p is required for the derepression of nine genes
under nonfermentative growth conditions (ACS1,
FBP1, ICL1, IDP2, JEN1,
MLS1, PCK1, SFC1, and
SIP4). To investigate whether the transcriptional control
mediated by Cat8p can be extended to other genes and whether this
control is the main control for the changes in the synthesis of the
respective proteins during the adaptation to growth on ethanol, we
analyzed the transcriptome and the proteome of a cat8 In Saccharomyces cerevisiae, the use of glucose by
fermentation leads to the production of ethanol in the medium. When
glucose is exhausted, a temporary arrest of growth occurs. During this phase, called the diauxic transition, cells switch from fermentative to
oxidative metabolism, and major changes in gene expression are
observed. Some of these alterations are due to the release of
glucose repression allowing the synthesis of a set of new proteins. Numerous metabolic functions are affected by this derepression, such as gluconeogenesis, the glyoxylate cycle, the tricarboxylic acid
cycle, and respiration (for reviews, see Refs. 1-4).
The changes in gene expression during growth on glucose and beyond were
characterized at the level of transcriptome using DNA microarrays (5).
The transcript levels for more than 1700 genes were altered by a factor
of at least 2. The changes in protein synthesis during the diauxic
shift were also investigated. Two-dimensional gel electrophoresis
showed that many proteins are affected (6, 7). These large scale
analyses showed the complexity of the regulations occurring at the
diauxic shift.
To understand how this reprogramming takes place in the cell, the role
of different transcriptional activators in the adjustment of the
pattern of gene expression was investigated. The induction of several
genes involved in the tricarboxylic acid cycle and in the respiratory
chain requires the HAP activator complex (see Ref. 8 for a
compilation of genes). Also, among all of the proteins induced at the
diauxic transition, 39 exhibited a reduced synthesis rate in a strain
msn2 msn4, both genes coding for homologous transcriptional
factors involved in the general stress response (9). But there are
still many genes whose induced expression during this shift is
not controlled by the HAP complex or Msn2/4p.
We have focused on the function of the transcriptional activator Cat8p.
The Cat8p zinc cluster protein is essential for growth of yeast on
nonfermentable carbon sources (10). Both expression of CAT8
gene and transcriptional activation by Cat8p are regulated by glucose
and require a functional Snf1p protein kinase (11). At least one
upstream activation sequence element, named carbon source-responsive element
(CSRE)1 was found in the
promoter of all the genes identified as Cat8p targets. The binding of
Cat8p on a CSRE motif has been recently shown (12).
The requirement of Cat8p for the expression of nine genes in the
absence of glucose and in the presence of nonfermentable carbon sources
was demonstrated. The proteins encoded by these genes are involved in
the production of acetyl-CoA from acetate (Acs1p), in gluconeogenesis
and glyoxylate cycle (Pck1p, Fbp1p, Icl1p, and Mls1p), and in the
import of succinate into mitochondria (Sfc1p) (10, 13-15). Recently,
IDP2 and JEN1 were identified as Cat8p targets
(16). The protein Jen1p is required for import of lactate into the cell
(17), and the product of the gene IDP2 is a
NADP-dependent isocitrate dehydrogenase. Last,
SIP4 is the only gene identified as target of Cat8p whose
product is a transcriptional activator that binds to the CSRE of the
genes FBP1 and ICL1 (18). Interestingly, no
phenotype was detected in a sip4 To determine its role during this shift, we used high density DNA
filters (miniarrays) to identify genes whose expression at the diauxic
transition is affected by the deletion of CAT8. As a
complementary approach, we undertook an analysis at the level of
proteome by two-dimensional electrophoresis, which allowed us to
visualize the effects of the cat8 null mutation on the
expression of the final gene products. With the identification of the
Cat8p-dependent genes, these two analyses provide
information concerning the consequences of the transcriptional
regulation by Cat8p (i.e. its repercussions on the
physiological state of the cell). Here we show that Cat8p has an
essential role during the adaptation of yeast on ethanol by controlling
the induction of many genes in response to the glucose depletion.
Knowing the functions of the proteins encoded by these genes and their
relationships in a biological pathway, it appears that these controls
take part essentially in the reprogramming of carbon metabolism
required for growth on ethanol.
Strains and Culture Conditions--
The wild-type strain
S. cerevisiae FY5 (MAT
Cultures were performed at 22 °C in a 500-ml Erlenmeyer flask
containing 50 ml of supplemented minimal medium YNBS (0.17% yeast
nitrogen base without ammonium sulfate and amino acids, 0.5% ammonium
sulfate, 2% (w/v) glucose, 25 µg/ml inositol, 85 mM
succinate/NaOH, pH 5.8) supplemented with 24 µg/ml of tyrosine to
stimulate [35S]methionine incorporation (20). Cultures
were shaken at 360 rpm, and growth was monitored by measuring their
absorbance at 600 nm (an A600 of 1 corresponds
to 107 cells/ml).
Glucose Measurement--
Glucose measurements were performed
with a Sigma Diagnostic kit (catalog no. 510-A).
RNA Isolation--
Total RNA was extracted as described
previously (21).
Miniarray Filter Hybridization--
2 µg of total RNA were
added to 2 µg of oligo(dT), heat-denatured for 10 min at 70 °C,
chilled on ice, and then used as template to synthesize
33P-labeled cDNA. The labeling conditions were 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 3.3 mM dithiothreitol, 1 mM dATP, 1 mM dTTP, 1 mM dGTP, 100 µCi of [ Northern Blot Hybridization--
15 µg of each RNA preparation
were electrophoresed under denaturing conditions (22) and blotted onto
nylon membranes (23). Hybridization probes used were the full-length
ORFs from Research Genetics or were polymerase chain reaction-amplified
with appropriate primers (sequences available upon request).
Hybridization signals were detected using a Molecular Dynamics
PhosphorImager, and quantification was achieved with the ImageQuant software.
Protein Labeling, Separation by Two-dimensional Gel
Electrophoresis, and Quantitative Analysis--
200 µl of culture
were harvested about 15 min after total glucose depletion in the
medium, and proteins were labeled in vivo during 10 min with
120 µCi of [35S]methionine (1000 Ci/mmol, 10 µCi/µl; ICN). Protein sample preparation and two-dimensional gel
electrophoresis were carried out as described previously (24).
After drying, gels were exposed to phosphor screens, which were scanned
with a Molecular Dynamics PhosphorImager. Quantification of spots and
comparative analyses were performed with the BioImage software. The
intensity of each spot was normalized to the actin spot. Normalization
to the global ratio of all matched spots on the gel gave similar
results. Three independent two-dimensional electrophoresis patterns
realized with different 35S-labeled protein extracts were
analyzed for each strain. A protein was considered as potentially
Cat8p-dependent when the spot displayed at least a 2-fold
difference between the mutant and the wild-type strains. Significant
differences were selected by using a statistical t test.
Protein Identification--
Protein identification was performed
by mass spectrometry. The method used was described previously
(25).
Kinetic of CAT8 Expression during Growth on Glucose Medium--
As
a preliminary to our studies on the involvement of CAT8 in
the adaptation of cells during the diauxic transition, it was necessary
to determine accurately at which stage of the culture these
investigations could be carried out. We therefore investigated the
kinetic of expression of CAT8 during the diauxic growth of a
wild-type strain. For this study, cells were grown in a synthetic medium containing 2% glucose as carbon source, and samples were harvested at different times of the culture for mRNA extraction. Under this culture condition, a first growth phase is observed corresponding to glucose consumption (Fig.
1). Once glucose is exhausted, a
transient arrest occurs, during which cells become capable of using the
ethanol produced by glucose fermentation. A second growth phase is then
observed corresponding to the use of ethanol. In a cat8
As shown in Fig. 1, CAT8 is not expressed during the growth
phase on glucose. This is consistent with our observation that the
growth rate, glucose consumption, and pattern of synthesized proteins
of a cat8 Comparison of the Global Pattern of Gene Expression during the
Diauxic Transition in a Wild-type Strain and in the Isogenic cat8
All of the genes already known to be controlled by Cat8p were
identified in this analysis (i.e. ACS1, FBP1,
ICL1, IDP2, JEN1, MLS1, PCK1, SFC1, and
SIP4) (Table I). Their
transcription levels were markedly decreased in the mutant strain.
Often the transcripts were barely detectable, and in no case was the
decrease less than 9-fold. The fact that all of the known
Cat8p-dependent genes were characterized confirms the
efficiency of the approach to discover new genes controlled by
Cat8p.
In addition, 22 other genes or ORFs were observed to have reduced
transcription levels in the cat8
Within the group of genes of unknown function, we found
YAR037W and YAR040C. These ORFs were spotted on
the filters but are no longer considered as true yeast ORFs. They were
mapped to positions 190,417-190,998 and 191,529-191,173,
respectively, on chromosome 1. These sequences are overlapping
YAT1 (190,183-192,243). This gene is a good candidate for
being regulated by Cat8p. A CSRE motif is present in its upstream
region, and its product, a carnitine acetyltransferase, may be involved
in the metabolism of acetate and ethanol (26). A possible explanation
is thus that the reduced hybridization level observed for
YAR037W and YAR040C was a consequence of a
decrease in the level of the YAT1 transcripts in the
cat8
Surprisingly, one ORF, YNR002C, was found to be more
expressed in the cat8 Characterization of Proteins Whose Synthesis Is Affected in the
cat8
42 spots were observed to have a significantly reduced intensity or
were not detected on the two-dimensional pattern of the cat8
Two spots more abundant on the protein map of the cat8 Comparison of Data from Transcriptome and Proteome
Analyses--
Nine genes were characterized as
Cat8p-dependent genes by both the transcriptome and the
proteome analyses: ACH1, ACS1, ALD6, DLD1, FBP1, ICL1, IDP2,
MLS1, and PCK1. To determine whether the changes
observed at the protein level in the cat8
In contrast, there was no correlation between mRNA levels and
protein synthesis for ADH2, CIT2, and
SDH1.
ADH2 seemed to be Cat8p-dependent only with the
proteome analysis. But ADH2 has 88% nucleotide identity
with ADH1. Therefore, cross-hybridization between the
radiolabeled cDNAs representing these two genes could have obscure
changes in their respective expression levels in the DNA filter
analysis. Using a probe specific to ADH2 in a Northern blot
analysis, the transcript level of ADH2 was observed to be
reduced in the cat8
The protein identified as Sdh1p was 3-fold less synthesized in the
absence of Cat8p, but the abundance of SDH1 transcripts did
not appear affected. This result led us to search for genes with
sequence highly related to SDH1. YJL045W and SDH1
share 76% nucleotide identity. A comparative analysis of the
expression of these two homologous genes in the cat8
The gene CIT2 was not selected in the transcriptome
analysis, whereas there is a 2.1-fold reduction in the synthesis of its corresponding protein in the cat8
Several genes have been identified as Cat8p targets by the
transcriptome analysis but not by the proteome analysis. The proteins encoded by these genes are hydrophobic, or have a pI higher
than 7, or are synthesized at a very low level. Protein with one of these characteristics cannot be separated or detected using standard two-dimensional electrophoresis conditions.
Conversely, 17 spots corresponding to unidentified proteins were found
to be Cat8p-dependent by proteome analysis. For at least half of these spots, the apparent pI and
Mr of ORFs are in good agreement with the
calculated pI and Mr of ORFs retained by the transcriptome analysis.
Promoter Analysis of the Genes Controlled by Cat8p--
A
functional upstream activation sequence element, designated
CSRE, has been identified in the promoter of all of the
Cat8p-dependent genes previously characterized, and a
consensus sequence has been defined (13). The binding of Cat8p to the
CSRE has been recently demonstrated (12). We screened the upstream
nontranslated region of the new Cat8p-dependent genes
identified here for the presence of CSRE-related sequences.
Four new Cat8p-dependent genes, MDH2,
YAT1, YCR010C, and YER024W,
contained a sequence matching perfectly the CSRE consensus sequence (Table III). Sequences distantly
related to CSRE were found in upstream regions of almost all of the
other genes. Similar distantly related sequences are present in the
promoters of SFC1, IDP2, and JEN1 and
have been shown to be functional CSRE sites (15, 16). Although the
in vivo function of the putative CSRE sites upstream of the
new Cat8p-dependent genes still remains to be demonstrated,
their presence argues in favor of a direct regulation by Cat8p or
indirectly through the binding of Sip4p.
Three genes characterized here as Cat8p-dependent genes do
not contain sequence related to CSRE (DAL81,
YGL128C, and TIM17). It is thus not excluded that
these genes are only indirectly regulated by Cat8p.
Temporal Expression Profiles of Genes Controlled by Cat8p during
the Diauxic Transition--
The expression of a large number of genes
was found to be controlled by Cat8p during the diauxic transition. To
confirm these results and determine whether the genes controlled by
Cat8p are similarly regulated, we compared their kinetics of expression during the diauxic shift in the wild-type strain and in the
cat8
For all of the genes, the results confirmed the requirement of Cat8p
for an optimum expression during the diauxic shift. In addition, in
agreement with the fact that Cat8p is a transcriptional factor involved
in the release of carbon catabolite repression, it appears that the
Cat8p-dependent genes are not expressed in the presence of
glucose or are only expressed at reduced levels as compared with the
levels observed in the absence of glucose. These genes are all markedly
induced when glucose is exhausted. There is one exception with
ALD6. This gene is already strongly expressed during growth
on glucose. Its expression slightly decreases before glucose exhaustion
and, once glucose is exhausted, only increases by a factor of 2.
These genes were classified in two groups according to their temporal
expression patterns in the wild-type and in the mutant strains. In the
first group are pooled the genes whose induction was almost completely
mediated by Cat8p. In the second group are pooled the genes for which
an induction was still observed in the mutant strain but to a lower
extent than in the presence of Cat8p. This indicates that in the
presence of glucose these genes are repressed and that the derepression
at the diauxic shift is Cat8p-independent or that another factor is
necessary for the full induction.
The comparison of the transcriptome and the proteome of a
wild-type strain and a CAT8-deleted strain led us to
characterize 34 genes whose expression at the diauxic shift is
Cat8p-dependent. These data present the first comprehensive
overview of the genes under the control of the transcriptional factor
Cat8p. For nine of these 34 genes, the control by Cat8p was already
known. The Cat8p dependence of the 25 remaining genes is reported here
for the first time. Eight of them are ORFs of unknown function. Whether the regulation of these 25 genes by Cat8p is direct cannot be inferred
from these expression data, but the fact that almost all of them have a
CSRE sequence in their promoter region strongly argues in favor of this hypothesis.
Cat8p Provides a Small Contribution to the Reprogramming of the
Yeast Genome Expression during the Diauxic Shift--
The data
reported here show that the control mediated by Cat8p concerns only a
small fraction of the yeast genome. Indeed, among the 3000 genes that
we have been able to consider by transcriptome analysis, only 1% were
found to display a Cat8p dependence (34 of 3000). This observation
indicates that this transcriptional activator plays a limited role in
the reprogramming of yeast genome expression during the diauxic shift.
DeRisi et al. (5) showed that 700 genes were induced by a
factor of >2 as the glucose was progressively depleted from the growth
medium. Accordingly, the Cat8p control would account for only
5% of the whole induction in gene expression observed at the diauxic shift.
Regulation of the Expression of the Cat8p-dependent
Genes--
Some of the genes identified as Cat8p-dependent
were completely repressed in a cat8
A previous investigation at the proteome level showed that the
expression of at least 39 genes at the diauxic shift could be
positively controlled by the transcription factors Msn2p and Msn4p (9).
None of them correspond to the Cat8p-dependent genes identified here. It seems thus excluded that Msn2p/Msn4p may trigger an
additional control in this case.
Also, among the genes subject to the Cat8p control, several are
significantly more expressed on oleate than on glucose. This is the
case for ACS1, CAT2, CRC1,
FBP1, ICL1, IDP2, JEN1,
MDH2, MLS1, PCK1, PUT4,
SFC1, and YCR010C (29, 30). This finding illustrates the relationships between different pathways involved in
the switch to alternative carbon sources.
Cat8p Plays an Important Role in the Reprogramming of the Yeast
Metabolism during the Diauxic Shift--
Because Cat8p is dispensable
to growth on fermentable carbon source and is essential to growth on
nonfermentable carbon source (ethanol, acetate, lactate, and glycerol),
it was expected that Cat8p essentially controls genes specifically
required for the use of nonfermentable carbon source. Considering the
function of the Cat8p-dependent genes characterized here,
we observed indeed that a large number of them encode proteins involved
in ethanol utilization. The metabolic pathways concerned are reported
in Fig. 5, and the activities under the
control of Cat8p are mapped on these pathways.
As can be seen, the Cat8p control begins with the early steps of
ethanol utilization, i.e. the conversion of ethanol into acetate via acetaldehyde and the subsequent activation of acetate into
acetyl-CoA. Cat8p is required for the induction of ADH2, ALD6, and ACS1, whose products catalyze each of
these steps. Once acetyl-CoA is produced, it can be used to fuel both
the glyoxylate cycle and the tricarboxylic acid cycle. For this
purpose, its acetyl moiety is associated to carnitine, and the
resulting acetyl-carnitine is transported across the peroxisomal and
the mitochondrial membranes. Cat8p also exerts a control on this step.
Two genes, CAT2 and YAT1, induced by Cat8p encode
carnitine acetyltransferase (26, 31), and CRC1, controlled
by Cat8p, encodes a mitochondrial carnitine carrier (32). Also, Cat8p
induces YER024W, which is homologous to YAT1.
The glyoxylate cycle also appears to be strongly dependent upon the
Cat8p control. This cycle allows carbon assimilation for biosyntheses
by converting two acetyl-CoA into oxaloacetate. The induction during
the diauxic shift of the enzymes catalyzing four of the five steps of
this conversion (Cit2p, Icl1p, Mls1p, and Mdh2p) is
Cat8p-dependent. Concerning CIT2, we showed that
in a CAT8-deleted strain the induction of CIT2 in
response to glucose exhaustion was not suppressed but only delayed. The
existence of a retrograde regulation of CIT2 in response to
changes in the functional state of mitochondria has been reported. It
involves the RTG genes (33). It is not known whether the
lack of Cat8p compromises the efficient functioning of the
tricarboxylic acid cycle. Thus, the delayed induction of
CIT2 in the mutant strain could be controlled by the
RTG gene products, as an indirect consequence of the
CAT8 deletion.
Each turn of the glyoxylate cycle generates one molecule of succinate.
This molecule is produced in the cytosol and can be transported into
the mitochondria for subsequent use. This transport is achieved by the
succinate-fumarate carrier encoded by SFC1. It exchanges
external succinate for internal fumarate across the mitochondrial inner
membrane (34). SFC1 is induced by Cat8p. In the
mitochondrial matrix, the succinate dehydrogenase complex catalyzes the
conversion of succinate to fumarate. The protein Sdh1p is one subunit
of this complex. Interestingly, we observed that Cat8p induced
YJL045W, homologous to SDH1.
Oxaloacetate produced from fumarate via malate can be fed into the
gluconeogenesis pathway to produce glucose 6-phosphate and its
derivatives. Cat8p controls the expression of PCK1 and FBP1, whose products are the key enzymes of the
gluconeogenesis pathway.
In summary, previous investigations led to the characterization of only
six genes regulated by Cat8p whose products are directly involved in
ethanol utilization. These genes are encoding an enzyme of one of the
first steps of the ethanol utilization (ACS1), two enzymes
of the glyoxylate cycle (MLS1 and ICL1), two
enzymes specific to the gluconeogenesis (FBP1 and
PCK1), and a mitochondrial carrier (SFC1). The
present study brings to 13 the number of genes involved in ethanol
utilization, which are under the control of Cat8p. Two additional
Cat8p-dependent genes (YER024W and
YJL045W), corresponding to genes of unknown function, are
also good candidates for participating in ethanol utilization, given
their homology to genes involved in carbon metabolism. Interestingly,
our study shows that the control of Cat8p is not limited to the
glyoxylate cycle and to the gluconeogenesis enzymes. It clearly extends
upstream of the glyoxylate cycle, over all of the first steps of
ethanol utilization, from ethanol to acetyl-CoA, and on the transport
of acetyl-CoA across the peroxisomal and mitochondrial membranes.
19 Genes Identified as Cat8p-dependent Encode Proteins
of Functions Not Directly Linked to the Utilization of
Ethanol--
Seven genes encode proteins linked to carbon metabolism,
ACH1, DLD1, IDP2, JEN1,
SIP4, STL1, and possibly REG2. Reg2p
has 48% similarity with Reg1p, which is required for the function of
GLC7-encoded PP1 in the glucose repression mechanism
(35).
Six genes encode proteins not involved in carbon metabolism,
CUP1A, CUP1B, CWP1, DAL81,
PUT4, and TIM17. Since CUP1A and
CUP1B share 100% nucleotide identity, because of
cross-hybridization it was impossible to know if both would be
controlled by Cat8p.
Finally, six genes are ORFs of unknown functions, YCR010C,
YFR039C, YGL128C, YGR067C,
YNR002C, and YPL156C. The YCR010C
product exhibits homology to GPR1 (glyoxylate pathway
regulator) from Yarrowia lipolytica, which may be involved
in a specific response of the cell to the toxic effects of acetic acid
and ethanol (36). Interestingly, the proteins encoded by
YCR010C and YNR002C have 78% identity, and the
deletion of CAT8 has opposite consequences on the expression
profiles of these two ORFs. Thus, Cat8p would positively regulate
YCR010C. In contrast, its homologous ORF YNR002C is more expressed in the absence of Cat8p. Putative CSRE sequences were
found only in the promoter region of YCR010C. These findings suggest that the increased expression of YNR002C in the
absence of Cat8p may be regarded as a compensation for the lack of
expression of YCR010C and may be an indirect consequence of
the CAT8 deletion.
In conclusion, although Cat8p participates moderately in the
change in gene expression occurring during the diauxic shift, it
controls many genes whose products are necessary, and sometime essential, to the growth on ethanol. It is interesting to note that the
products of Cat8p-dependent genes execute all of the steps
going from ethanol to acetyl-CoA and four steps of the glyoxylate cycle
out of five. The step not controlled by Cat8p, going from citrate to
isocitrate, could be executed in the mitochondria by Aco1p or Aco2p
(YJL200C). This hypothesis agrees with the fact that Cat8p does
not control any step of the tricarboxylic acid cycle or the oxidative
phosphorylation, both happening in the mitochondria. But the
understanding of the genetic control by Cat8p at the diauxic transition
cannot necessarily be restricted to a particular pathway,
because several genes identified as Cat8p-dependent encode
proteins with functions not linked directly to the utilization of ethanol.
strain during the diauxic shift. In this report, we demonstrate that,
in addition to the nine genes known as Cat8p-dependent,
there are 25 other genes or open reading frames whose expression at the
diauxic shift is altered in the absence of Cat8p. For all of the genes
characterized here, the Cat8p-dependent control results in
a parallel alteration in mRNA and protein synthesis. It appears
that the biochemical functions of the proteins encoded by
Cat8p-dependent genes are essentially related to the first
steps of ethanol utilization, the glyoxylate cycle, and
gluconeogenesis. Interestingly, no function involved in the
tricarboxylic cycle and the oxidative phosphorylation seems to be
controlled by Cat8p.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mutant, but overexpression of Sip4p compensates for the lack of Cat8p and restores
growth on ethanol. Although Sip4p and Cat8p seem to be closely related
in functions, transcription of SIP4 requires Cat8p (18). All
of the Cat8p-dependent genes are highly induced in response
to glucose exhaustion at the diauxic shift (5). These findings suggest
the importance of Cat8p in the regulation of gene expression at the
diauxic shift and in the establishment of aerobic metabolism.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, mal),
derived from S288C, was provided by F. Winston (Harvard Medical
School). The cat8
deletion was constructed in the
wild-type strain FY5 by replacing base pairs 36-4284 of the
CAT8 gene with the kanMX4 cassette (19). The single
chromosomal integration of kanMX4 at the CAT8 locus was
controlled by polymerase chain reaction and Southern blot analyses. The
transformed strains selected were named YP1, YP2, and YP3.
-33P]dCTP (Amersham Pharmacia Biotech; 10 mCi/ml, >2500 Ci/mmol), and 300 units of reverse transcriptase
(SuperScript Rnase H
RT; Life Technologies, Inc.) in a
reaction volume of 30 µl. The reaction was incubated for 3 h at
42 °C. The probe was purified by a passage through Micro Bio-Spin 30 Chromatography Column (Bio-Rad). The miniarray genomic filters
(Research Genetics) used in this work were hybridized with labeled
cDNA as recommended by the supplier. The filters were exposed to a
phosphor screen, which was scanned with a Molecular Dynamics
PhosphorImager. Detection and quantification of spots were performed
with ImageMaster software (Amersham Pharmacia Biotech). Two independent
experiments of comparison were performed with a single set of
membranes. Between hybridizations, the efficiency of stripping (95%)
was checked by scanning the filters. To allow comparison between
separate filter hybridizations, the intensity of each spot was
normalized to the intensity of the actin spot. Normalization to the
total hybridization signal gave similar results. A gene was considered
as potentially Cat8p-dependent when the spot displayed at
least a 2-fold difference between the mutant and the wild-type strains.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strain, this second growth phase does not occur.
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Fig. 1.
Expression profile of CAT8
during growth on glucose medium.
A, temporal profile of the cell density (open
circles) and glucose concentration (filled
circles) in the medium. Cell samples were withdrawn
at different time points, before and after glucose exhaustion,
indicated by the arrows. B, Northern blot
experiment with total RNA extracted from the cell samples and probed
with CAT8 sequence. mRNA quantification was normalized
to ACT1 mRNA.
strain are not affected before the onset of the
diauxic transition (data not shown). CAT8 starts to be
expressed less than 1 h before glucose is exhausted and is fully
induced 15 min after glucose exhaustion. To minimize the risk of
secondary effects due to the inability of the cat8
strain
to use ethanol, we decided to carry out our investigations 25 min after
glucose exhaustion.
Strain--
To identify all of the genes controlled by the
transcriptional activator Cat8p during the diauxic transition, we
compared the transcriptional patterns of a wild-type strain and of the isogenic cat8
strain. DNA miniarrays containing 6144 ORFs
of the yeast genome were used for this study. These miniarray filters were successively hybridized with 33P-labeled cDNA
probes synthesized from mRNA of the wild-type strain and of the
cat8
strain (Fig. 2).
Messenger RNA was obtained from cells grown on a synthetic medium
containing 2% glucose and harvested 25 min after glucose depletion.
About 3000 ORFs of the S. cerevisiae genome were expressed
at a level high enough to have their transcripts detectable by this
approach.
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Fig. 2.
Section of a yeast genome miniarray
filter. The filters were hybridized with 33P-labeled
cDNA probes synthesized from mRNA isolated from FY5
(wt) and from YP3 (cat8 ) cells. Cells were
harvested 25 min after glucose exhaustion. Circles indicate
spots corresponding to genes with transcript levels reduced in the
cat8
strain.
Cat8p-dependent genes identified by miniarray hybridization
mutant cells.
strain and thus can be considered as new Cat8p-dependent genes. Nine encode
proteins with unknown function. In most cases, the decrease in
transcription levels of the new Cat8p-dependent genes is
rather limited. The decrease factors range between 8 and 2. There are
two marked exceptions with YCR010C and YPL156C.
The transcription levels of these ORFs were drastically affected by the
cat8 inactivation such that their transcripts were
not detectable in the cat8
strain.
strain. Although YAT1 was not identified
as Cat8p target here, Northern analysis revealed a lower abundance of
YAT1 transcripts in the cat8
cells in
comparison with wild-type cells (see below and Fig. 4). Concerning this
special case, the result of miniarray hybridization remains unclear.
strain than in the wild-type strain.
Strain during the Diauxic Transition--
To analyze the
incidence of the deletion of CAT8 at the proteome level, we
compared the two-dimensional pattern of proteins synthesized during the
diauxic transition in cat8
cells and in wild-type cells
(Fig. 3). Cells grown on 2% glucose
medium were labeled for 10 min with [35S]methionine 15 min after glucose exhaustion. Thus, the cells were harvested 25 min
after glucose exhaustion as for the mRNA preparations. Proteins
were separated by two-dimensional gel electrophoresis. After exposure
to phosphor screen, individual spots were quantified and analyzed for
significant changes in the rate of protein synthesis.
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Fig. 3.
Comparison between proteins of FY5
(wt) and YP3 (cat8 )
synthesized at the diauxic transition by two-dimensional gel
electrophoresis. Cells were labeled 10 min with
[35S]methionine, 15 min after glucose exhaustion.
Variable spots not identified are indicated with a number
(from 1 to 17), and variable identified proteins
are indicated with the name of the corresponding genes.
strain. These spots are listed in Table
II. 26 of them were identified spots of
the yeast protein map (Ref. 25 and this study). They correspond to 12 different proteins. The discrepancy between the number of spots and the
number of corresponding proteins results from the fact that several
spots are isoforms of the same protein. Among the identified proteins,
six are the products of genes already known to be controlled by Cat8p
(Acs1p, Fbp1p, Icl1p, Idp2p, Mls1p, and Pck1p). The six other
identified proteins are Ach1p, Adh2p, Ald6p, Cit2p, Dld1p, and Sdh1p.
The demonstration that the synthesis of these proteins is
Cat8p-dependent suggests that their encoding genes could be
also controlled by Cat8p.
Cat8p-dependent proteins characterized by proteome analysis
mutant strain were also retained. One of these spots was only
detectable on the map of the mutant strain (spot named
KanR). Given its apparent pI and
Mr (5.1 and 27,700, respectively), it probably
corresponds to the product of the KanR gene used
for the CAT8 gene replacement (calculated pI and
Mr, 5.05 and 30,716, respectively). The other
spot, spot 17, has a relative intensity 3-fold higher in the mutant
strain. The migration of this spot on two-dimensional gel led us to
think that it could be the product of YNR002C, the only gene
found to be more expressed in the cat8
strain by our
transcriptome analysis. The apparent pI and
Mr of spot 17 were 4.62 and 26,700, respectively, whereas the calculated pI and
Mr of Ynr002cp were 4.98 and 30,700, respectively. However, identification of this protein will be necessary
to confirm this hypothesis.
strain are only
a consequence of a control of Cat8p at the transcriptional level, the
alterations in the levels of mRNA and in protein synthesis were
compared for each of these genes (Table II). A good correlation was
observed between these values. Hence, it appears that at the diauxic
transition, the alteration in synthesis of these
Cat8p-dependent proteins mainly reflects a change in
mRNA level.
strain (see below and Fig.
4).
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Fig. 4.
Northern blot assay of the expression of
Cat8p-dependent genes in FY5 (wt) and in
YP3 (cat8 ) during the diauxic
shift. RNA were extracted at different times during the diauxic
shift: 20 min before glucose exhaustion (1), 5 min before
(2), 10 min after (3), 25 min after
(4), and 40 min after (5). mRNA levels were
normalized to ACT1 mRNA at each time point. The genes
are classified in two groups, as defined under "Results." In
the second group, the genes are ordered according to the level of
induction in absence of Cat8p, from the more sensitive to the less
sensitive. The graphics represent examples of temporal pattern of gene
expression of each group.
strain and in the wild-type strain was performed by Northern blot.
Divergent sequences were used as probes for each of them to avoid
cross-hybridization. Surprisingly, the expression of SDH1
was not affected in the mutant strain, while the expression of the
homologous gene, YJL045W, was reduced in absence of Cat8p
(see below and Fig. 4). YJL045W may thus be considered as a
new Cat8p-dependent gene. According to the predicted
pI and Mr of Yjl045wp, its
comigration with Sdh1p cannot be ruled out. This could explain the
difference between the wild-type and the mutant strains concerning the
intensity of the spot identified as Sdh1p.
strain. When the
kinetic of expression of CIT2 was followed at the diauxic
transition using Northern blot experiment (see below and Fig. 4), the
gene was more expressed in the wild-type strain but only during a short lapse of time, in the first 30 min after glucose exhaustion. Since the
samplings for the proteome and the transcriptome analyses were realized
25 min after the glucose exhaustion, a 5-min difference between the
samples could explain the discrepancy in the results.
CSRE sites in the upstream region of the Cat8p-dependent
genes identified in this study
mutant strain (Fig. 4). For these assays, mRNA
were extracted from cells harvested at different times during the
diauxic transition. The sequences used as probes were chosen to prevent
cross-hybridization with homologous genes. 16 Cat8p-dependent genes characterized by our DNA filter
analysis were considered; we focused on genes or ORFs containing at
least one CSRE-related sequence. We also investigated the expression
profiles of ADH2, CIT2, and YJL045W, three genes
for which the existence of a control by Cat8p could be inferred from
our proteome analysis. As a control for these studies, we investigated
the temporal pattern of COR1. This gene is induced during
the diauxic shift, but its expression is Cat8p-independent. This
control allowed us to rule out the possibility of a general alteration
of genome expression in a cat8
strain related to the inability of this mutant to respond normally to glucose exhaustion (data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strain when glucose
was depleted, whereas other genes were partially derepressed. These
distinctions between the transcriptional responses to glucose
exhaustion in a cat8
strain suggest that, for a few
genes, several regulatory controls could participate to their optimal
expression, by additive effects or by switching from the Cat8p control
to another control. For example, requirement of the Hap2/3/4/5 complex
for DLD1 induction following a shift from fermentable to
nonfermentable carbon source has been demonstrated (27). Moreover, it
has been shown that the full expression of ADH2 in the
derepressing condition was dependent on Cat8p and Adr1p, another
positive regulator, in distinct activating pathways (28).
View larger version (24K):
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Fig. 5.
Scheme of metabolic pathways essential for
ethanol utilization in yeast. The genes controlled by Cat8p and
encoding proteins involved in this metabolic circuit are identified by
name in the boxes.
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ACKNOWLEDGEMENT |
---|
We thank Fred Winston for providing the strain FY5.
![]() |
FOOTNOTES |
---|
* 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 fellowship from the Conseil Régional d'Aquitaine.
To whom all correspondence should be addressed. Tel.:
33-5-5699-9021; Fax: 33-5-5699-9068; E-mail:
francis.sagliocco@ibgc.u- bordeaux2.fr.
Published, JBC Papers in Press, October 6, 2000, DOI 10.1074/jbc.M008752200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CSRE, carbon source-responsive element; ORF, open reading frame.
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REFERENCES |
---|
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---|
1. | Ronne, H. (1995) Trends Genet. 11, 12-17[CrossRef][Medline] [Order article via Infotrieve] |
2. | Entian, K. D., and Schüller, H. J. (1997) in Yeast Sugar Metabolism (Zimmermann, F. K. , and Entian, K. D., eds) , pp. 409-434, Technomic, Lancaster, PA |
3. | Carlson, M. (1998) Curr. Opin. Genet. Dev. 8, 560-564[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Gancedo, J. M.
(1998)
Microbiol. Mol. Biol. Rev.
62,
334-361 |
5. |
DeRisi, J. L.,
Iyer, V. R.,
and Brown, P. O.
(1997)
Science
278,
680-686 |
6. | Bataillé, N., Régnacq, M., and Boucherie, H. (1991) Yeast 7, 367-378[Medline] [Order article via Infotrieve] |
7. | Boy-Marcotte, E., Tadi, D., Perrot, M., Boucherie, H., and Jacquet, M. (1996) Microbiology 141, 459-467[Abstract] |
8. | De Winde, J. H., and Grivell, L. A. (1993) Prog. Nucleic Acids Res. Mol. Biol. 46, 51-91[Medline] [Order article via Infotrieve] |
9. |
Boy-Marcotte, E.,
Perrot, M.,
Bussereau, F.,
Boucherie, H.,
and Jacquet, M.
(1998)
J. Bacteriol.
180,
1044-1052 |
10. | Hedges, D., Proft, M., and Entian, K. D. (1995) Mol. Cell. Biol. 15, 1915-1922[Abstract] |
11. | Randez-Gil, F., Bojunga, N., Proft, M., and Entian, K. D. (1997) Mol. Cell. Biol. 17, 2502-2510[Abstract] |
12. | Rahner, A., Hiesinger, M., and Schüller, H. J. (1999) Mol. Microbiol. 34, 146-156[CrossRef][Medline] [Order article via Infotrieve] |
13. | Caspary, F., Hartig, A., and Schüller, H. J. (1997) Mol. Gen. Genet. 255, 619-627[CrossRef][Medline] [Order article via Infotrieve] |
14. | Kratzer, S., and Schüller, H. J. (1997) Mol. Microbiol. 26, 631-641[Medline] [Order article via Infotrieve] |
15. | Bojunga, N., Kotter, P., and Entian, K. D. (1998) Mol. Gen. Genet. 260, 453-461[CrossRef][Medline] [Order article via Infotrieve] |
16. | Bojunga, N., and Entian, K. D. (1999) Mol. Gen. Genet. 262, 869-875[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Casal, M.,
Paiva, S.,
Andrade, R. P.,
Gancedo, C.,
and Leao, C.
(1999)
J. Bacteriol.
181,
2620-2623 |
18. |
Vincent, O.,
and Gancedo, J. M.
(1998)
EMBO J.
17,
7002-7008 |
19. | Wach, A. (1996) Yeast 12, 259-265[CrossRef][Medline] [Order article via Infotrieve] |
20. | Miller, M. J., Xuong, N. H., and Geiduschek, E. P. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 5222-5225[Abstract] |
21. | Lindquist, S. (1981) Nature 293, 311-314[Medline] [Order article via Infotrieve] |
22. | Lehrach, H., Diamond, D., Wozney, J. M., and Boedtker, H. (1977) Biochemistry 16, 4743-4751[Medline] [Order article via Infotrieve] |
23. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
24. | Boucherie, H., Dujardin, G., Kermorgant, M., Monribot, C., Slonimsky, P., and Perrot, M. (1995) Yeast 11, 601-613[Medline] [Order article via Infotrieve] |
25. | Perrot, M., Sagliocco, F., Mini, T., Monribot, C., Schneider, U., Shevchenko, A., Mann, M., Jenö, P., and Boucherie, H. (1999) Electrophoresis 20, 2280-2298[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Schmalix, W.,
and Bandlow, W.
(1993)
J. Biol. Chem.
268,
27428-27439 |
27. | Lodi, T., Alberti, A., Guiard, B., and Ferrero, I. (1999) Mol. Gen. Genet. 262, 623-632[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Rahner, A.,
Scholer, A.,
Martens, E.,
Gollwitzer, B.,
and Schüller, H. J.
(1996)
Nucleic Acids Res.
24,
2331-2337 |
29. |
Karpichev, I. V.,
and Small, G. M.
(1998)
Mol. Cell. Biol.
18,
6560-6570 |
30. |
Kal, A. J.,
van Zonneveld, A. J.,
Benes, V.,
van den Berg, M.,
Koerkamp, M. G.,
Albermann, K.,
Strack, N.,
Ruijter, J. M.,
Richter, A.,
Dujon, B.,
Ansorge, W.,
and Tabak, H. F.
(1999)
Mol. Biol. Cell
10,
1859-1872 |
31. |
vanRoermund, C. W. T.,
Hettema, E. H.,
vandenBerg, M.,
Tabak, H. F.,
and Wanders, R. J. A.
(1999)
EMBO J.
18,
5843-5852 |
32. | Palmieri, L., Lasorsa, F. M., Iacobazzi, V., Runswick, M. J., Palmieri, F., and Walker, J. E. (1999) FEBS Lett. 462, 472-476[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Liu, Z.,
and Butow, R. A.
(1999)
Mol. Cell. Biol.
19,
6720-6728 |
34. | Fernandez, M., Fernandez, E., and Rodicio, R. (1994) Mol. Gen. Genet. 242, 727-735[Medline] [Order article via Infotrieve] |
35. | Tu, J., and Carlson, M. (1995) EMBO J. 14, 6789-6796 |
36. | Tzschoppe, K., Augstein, A., Bauer, R., Kohlwein, S. D., and Barth, G. (1999) Yeast 15, 1645-1656[CrossRef][Medline] [Order article via Infotrieve] |