The Transcriptional Activator Cat8p Provides a Major Contribution to the Reprogramming of Carbon Metabolism during the Diauxic Shift in Saccharomyces cerevisiae*

Valerie HaurieDagger §, Michel PerrotDagger , Thierry Mini, Paul Jenö, Francis SaglioccoDagger ||, and Helian BoucherieDagger

From the Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 cat8Delta 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

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 sip4Delta 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.

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.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Strains and Culture Conditions-- The wild-type strain S. cerevisiae FY5 (MATalpha , mal), derived from S288C, was provided by F. Winston (Harvard Medical School). The cat8Delta 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.

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 [alpha -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.

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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 cat8Delta 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.

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 cat8Delta 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 cat8Delta strain to use ethanol, we decided to carry out our investigations 25 min after glucose exhaustion.

Comparison of the Global Pattern of Gene Expression during the Diauxic Transition in a Wild-type Strain and in the Isogenic cat8Delta 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 cat8Delta 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 cat8Delta 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 (cat8Delta ) cells. Cells were harvested 25 min after glucose exhaustion. Circles indicate spots corresponding to genes with transcript levels reduced in the cat8Delta strain.

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.


                              
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Table I
Cat8p-dependent genes identified by miniarray hybridization
As expected, CAT8 mRNA was detected in wild-type cells by miniarray hybridization and was absent from cat8Delta mutant cells.

In addition, 22 other genes or ORFs were observed to have reduced transcription levels in the cat8Delta 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 cat8Delta strain.

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 cat8Delta strain. Although YAT1 was not identified as Cat8p target here, Northern analysis revealed a lower abundance of YAT1 transcripts in the cat8Delta cells in comparison with wild-type cells (see below and Fig. 4). Concerning this special case, the result of miniarray hybridization remains unclear.

Surprisingly, one ORF, YNR002C, was found to be more expressed in the cat8Delta strain than in the wild-type strain.

Characterization of Proteins Whose Synthesis Is Affected in the cat8Delta 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 cat8Delta 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 (cat8Delta ) 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.

42 spots were observed to have a significantly reduced intensity or were not detected on the two-dimensional pattern of the cat8Delta 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.


                              
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Table II
Cat8p-dependent proteins characterized by proteome analysis

Two spots more abundant on the protein map of the cat8Delta 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 cat8Delta 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.

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 cat8Delta 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.

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 cat8Delta 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 (cat8Delta ) 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.

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 cat8Delta 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.

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 cat8Delta 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.

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.


                              
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Table III
CSRE sites in the upstream region of the Cat8p-dependent genes identified in this study
Asterisks represent functional sites. (c), complementary strand. Lowercase letters indicate nucleotides that differ from the consensus sequence. Boldface letters indicate invariable nucleotides.

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 cat8Delta 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 cat8Delta strain related to the inability of this mutant to respond normally to glucose exhaustion (data not shown).

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 cat8Delta strain when glucose was depleted, whereas other genes were partially derepressed. These distinctions between the transcriptional responses to glucose exhaustion in a cat8Delta 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).

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.



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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.

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.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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