Dual Influence of the Yeast Catlp (Snflp) Protein Kinase on Carbon Source-Dependent Transcriptional Activation of Gluconeogenic Genes by the Regulatory Gene CAT8 (original) (raw)

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Antje Rahner, Anja Schöler, Erika Martens, Boris Gollwitzer, Hans-Joachim Schüller, Dual Influence of the Yeast Catlp (Snflp) Protein Kinase on Carbon Source-Dependent Transcriptional Activation of Gluconeogenic Genes by the Regulatory Gene CAT8, Nucleic Acids Research, Volume 24, Issue 12, 1 June 1996, Pages 2331–2337, https://doi.org/10.1093/nar/24.12.2331
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Abstract

The CSRE (carbon source-responsive element) is a sequence motif responsible for the transcriptional activation of gluconeogenic structural genes in Saccharomyces cerevisiae. We have isolated a regulatory gene, DILI (derepression of isocitrate lyase, = CAT8), which is specifically required for derepression of CSRE-dependent genes. Expression of CAT8 is carbon source regulated and requires a functional Cat1p (Snf1p) protein kinase. The derepression defect of CAT8 in a catl mutant could be suppressed by a mutant Mig1p repressor protein. Derepression of CAT8 also requires a functional HAP2 gene, suggesting a regulatory connection between respiratory and gluconeogenic genes. Carbon source-dependent protein-CSRE complexes detected in a gel retardation analysis with wild-type extracts were absent in cat8 mutant extracts. However, similar experiments with an epitope-tagged CAT8 gene product in the presence of tag-specific antibodies gave evidence against a direct binding of Cat8p to the CSRE. A constitutively expressed GAL4-CAT8 fusion gene revealed a carbon source-dependent transcriptional activation of a UASGAL-containing reporter gene. Activation mediated by Cat8p was no longer detectable in a catl mutant. Thus, biosynthetic control of CAT8 as well as transcriptional activation by Cat8p requires a functional Cat1p protein kinase. A model proposing _CAT8_as a specific activator of a transcription factor(s) binding to the CSRE is discussed.

Introduction

The utilization of non-fermentable carbon sources such as lactate, ethanol or acetate by the yeast Saccharomyces cerevisiae requires the coordinate biosynthesis of gluconeogenic enzymes. However, sugar phosphates required for protein glycosylation and generation of the cell wall can be more easily produced in the presence of fermentable carbon sources. Thus, genes encoding gluconeogenic enzymes are strictly regulated at the transcriptional level by the complex glucose repression network (reviewed in 1–4). This regulatory system also affects genes of alternative sugar metabolism (SUC, MAL or GAL genes), respiration and peroxisomal β-oxidation. The CAT1 gene (= SNF1, CCR1) encodes a serine/threonine-specific protein kinase which is essential for the derepression of most glucose-repressible genes (5–7). The CAT3 (= SNF4) gene product is physically associated with Catlp and may function as a stimulatory subunit of the protein kinase (8). Since cat1 and cat3 mutants fail to derepress glucose-repressible enzymes, pleiotropic growth defects on various carbon sources (raffinose, maltose, galactose or ethanol) can be observed. Some of these defects could be suppressed by the mig1 (= cat4, ssn1) mutation, leading to a glucose-insensitive expression of SUC, MAL and GAL genes (9–12). MIG1 encodes a zinc finger protein that binds to a GC-rich motif upstream of several glucose-repressible genes (13). Under conditions of glucose derepression, Catlp may lead to a deactivation of the Miglp repressor by a currently unknown mechanism.

Isocitrate lyase (encoded by the ICL1 gene), as a key enzyme of the glyoxylate cycle (considered as a subpathway of gluconeogenesis from C2 substrates), is regulated by transcriptional repression/derepression (14,15), phosphorylation (16) and proteolytic degradation (17). We have previously identified the CSRE (carbon source-responsive element), an upstream activation site (UAS) responsible for transcriptional derepression of the ICL1 gene (18). Similar sequence motifs were found upstream of the fructose-1,6-bisphosphatase gene FBP1 (18,19), the phosphoenolpyruvate carboxykinase gene PCK1 (20) and the acetyl-CoA synthetase gene ACS1 (21). Thus, the CSRE can be considered as a pathway-specific _cis_-element of genes involved in non-fermentative metabolism. Gene activation by a CSRE requires functional CAT1 and CAT3 genes (18). The carbon source-dependent binding of a protein factor, designated Ang1 (activator of non-fermentative growth) to the CSRE could no longer be observed in cat1 and cat3 mutants. We thus wished to identify _trans_-acting factors responsible for the signal transduction from pleiotropic regulators to the CSRE. In this paper we describe the characterization of the DIL1 (=CAT8) gene and identify its gene product as a carbon source-regulated, _CAT1_-dependent transcriptional activator.

Materials and Methods

Yeast strains and media

Strains of S.cerevisiae used in this work are listed in Table 1. Synthetic complete medium have been described elsewhere (7). Repressed cells were grown with 2% glucose until the mid log phase. For derepressing conditions, cells were directly grown with 0.2% glucose. Alternatively, repressed cells were harvested in the early log phase and subsequently transferred to derepression medium for 8 h.

Table 1

Strains of S.cerevisiae used in this work

Isolation of dil mutants

Cells of the wild-type strain JS92.32-11 containing an integrated ICLl-lacZ reporter gene were mutagenized with 1% ethylmethanesulfonate for 60 min (rate of survival ∼90%). Mutants unable to grow on synthetic complete medium with 3% ethanol (SCE) were further assayed on X-gal-containing plates, thereby screening for ICLl activation defects. In order to distinguish pleiotropic mutations affecting utilization of various carbon sources (such as catl/snfl or snf2;7,22) from those specific for CSRE-mediated gene expression, activation-deficient mutants were finally plated on medium containing raffinose, maltose or galactose. Mutants exhibiting the expected phenotype (no growth on SCE, reduced expression of the ICLl-lacZ reporter gene, normal growth with raffinose or galactose) were further investigated.

Gene isolation and plasmid constructions

For the isolation of the DILI gene by mutant complementation, strain JS94.9–9 was transformed with a plasmid library based on YEp24 (23). Plasmid YEp24-DIL1 was isolated from transformants showing restored growth with ethanol as the sole carbon source. Plasmid YEp13-DIL1 was isolated similarly.

Procedures for recombinant DNA followed established protocols (24). Episomal and integrative reporter plasmids containing ICLl-lacZ fusions (pJS310 and pJS330, respectively) have been described (14). For the synthetic promoter construct pAS74 (CSRE-_ICLl_-lacZ), the ICLl upstream region (−527 to −164) was deleted and replaced by the oligonucleotide ICLSM1 containing the CSRE of ICLl (5′-tcgaggatcCCATTCATCCG-ctagca-3′; authentic promoter sequence shown in capital letters). Reporter plasmids pJS151 (FBPl-lacZ; 25), pJS154 (ADH2-lacZ) and pJS334 (POTl-lacZ) contained at least 1 kb of the respective upstream regions. The CAT8-lacZ fusion construct pJS427 was obtained by ligation of a 1.4 kb _Eco_RI-Hin_dIII fragment containing 1.2 kb of the CAT8 upstream region into the lacZ fusion plasmid YEp356 (26). For the construction of a Δ_cat8 null mutation, a 2.5 kb _Eco_RI-_Kpn_I fragment from YEp24-DIL1 was subcloned into pGEM7 (Promega). Subsequently, a 1.45 kb _Pst_I-_Bgl_II fragment representing the CAT8 promoter and the N-terminus of its reading frame was replaced by the HIS3 gene, giving the cat8_-Δ_l::HIS3 construct pEM4 (cf. Fig. 1). For the heterologous expression of the zinc cluster domain of Cat8p, a 0.85 kb fragment (encoding amino acids 1–281) was amplified by PCR and subsequently ligated into the GST fusion plasmid pGEX-2TK (Pharmacia) to give pAR1. The GAL4-CAT8 fusion constructs pAR2-pAR5 were obtained by cloning the fragments shown in Figure 1 into plasmid pY1 (27). The CAT8 variant CAT8-FLAG was constructed by PCR-mediated addition of a sequence encoding the FLAG epitope (DYKDDDDK). The resulting plasmid pEM6 (2µm URA3 CAT8-FLAG) contains an extended CAT8 gene under native promoter control, epitopetagged at the C-terminus.

Gel retardation analysis

Labeling of promoter fragments and gel retardation experiments were carried out as described previously (18). Protein extracts were prepared from repressed or derepressed transformants of proteinase-deficient strains C13-ABY.S86 and ABYSΔdil1, respectively. The synthetic DNA fragment OAS12 containing the CSRE of the ICLl promoter has been described (18). The anti-FLAG antibody M2 was used for supershift studies.

Miscellaneous procedures

β-Galactosidase assays were performed with crude extracts of yeast transformants as described (18). Transformation of yeast strains followed established procedures (28). Isolation of plasmids and chromosomal DNA from yeast has been described (29). Verification of the cat8 null mutation was done by Southern blot hybridization (24). Synthetic oligonucleotides were purchased from MWG Biotech (Ebersberg, Germany). For the immunoblot analysis (30) of protein extracts from CAT8-FLAG transformants, the monoclonal anti-FLAG antibody M2 (Kodak/IBI) was used. The ECL chemiluminescence system (Amersham) was used for immunodetection.

Figure 1

Restriction map of the DIL1 (CAT8) gene and constructs derived thereof. The binuclear zinc cluster of Cat8p is shown as a hatched box. Sequences deleted for the construction of the _cat8-Δ_1::HIS3 null mutation are indicated by a gap. Thick lines represent parts of Cat8p fused to the DNA binding domain of the GAL4 gene (_GAL4_dbd). Specific β-galactosidase activities were measured in transformants of the strain YJOZ, grown under repressing (2% glucose, repr.) or derepressing (0.2% glucose, derepr.) conditions. B, _Bam_HI; Bg, _Bgl_II; E, _Eco_RI; H, _Hind_III; K, _Kpn_I; P, _Pst_I; S, _Sal_I; X, _Xba_I.

Results

Isolation of dill mutants

We have previously described the CSRE as an essential activating promoter element upstream of structural genes of gluconeogenesis (preliminary consensus sequence CCRTYCRTCCG; 18; modified). At least expression of the isocitrate lyase gene ICL1 depends almost completely on a functional CSRE. Thus, we considered an ICL1-lacZ fusion as a suitable reporter construct for the isolation of mutants specifically affected in CSRE-dependent gene expression. Following the procedure described above (see Materials and Methods), we isolated nine representatives of the dil1 complementation group (derepression of isocitrate lyase). In these mutants, ICL1-lacZ derepression was reduced to 1–14% of the wild-type level (not shown). Expression of reporter genes dependent on the FBP1 promoter or a CSRE-containing synthetic minimal promoter was similarly affected. On the other hand, enzymes of alternative sugar utilization, such as invertase, maltase or galactokinase, were normally derepressed in dil1 mutants (data not shown).

Isolation of the DILI gene and its identity with CAT8

The DIL1 wild-type gene was isolated by functional complementation of the growth defect of a dil1 mutant on ethanol medium. Two distinct plasmids recovered from the transformants contained inserts overlapping for 4.8 kb. Restriction mapping of this DNA segment (Fig. 1) revealed a fragment pattern similar to that of the recently isolated CAT8 gene necessary for efficient expression of the fructose bisphosphatase gene FBP1 (19). Indeed, partial DNA sequencing of the DIL1 gene proved its identity with CAT8. Thus, for reasons of uniformity, we shall use the designation CAT8 instead of DIL1 in the further text. The DNA sequence of CAT8 predicts a binuclear zinc cluster motif at the N-terminus of the respective 1433 amino acid protein (19; residues 69–99), reminiscent of the Gal4p protein family. To date, more than 50 members of this family have been described in fungal systems (not shown; a recent compilation of C6 zinc cluster proteins is available upon request). Thus, Cat8p may be considered as a DNA binding transcription factor directly or indirectly involved in activation of CSRE-dependent structural genes (see below).

A cat8 deletion mutation was constructed (cf. Fig. 1) and characterized for its influence on structural genes involved in C2 metabolism. As expected, cat8 null mutants failed to grow on non-fermentable carbon sources, such as lactate, ethanol or acetate, while maltose, raffinose or galactose were utilized normally. In the cat8 null mutant, expression of ICL1-lacZ or FBP1-lacZ fusion constructs under derepressing conditions was reduced to 1–2% of the wild-type level (Table 2). Expression of a reporter gene driven by a CSRE-containing minimal promotor was completely abolished in the cat8 mutant. A partial influence of the cat8 mutation was also found for the glucose-repressible alcohol dehydrogenase gene ADH2. This effect is independent of the ADH2 positive regulator Adr1p, since ADH2 expression is further decreased in a cat8 adr1 double mutant (data not shown). Obviously, the positive regulators CAT8 and ADR1 function in distinct activating pathways. A complete loss of derepression has been also reported for the phosphoenolpyruvate carboxykinase gene PCK1 (19). On the other hand, a functional CA T8 gene is not required for induction of the peroxisomal thiolase gene POT1 by oleic acid (Table 2). In summary, CAT8 is necessary for the efficient transcriptional derepression of all currently known CSRE-controlled structural genes, at least in part.

Table 2

Expression of carbon source-regulated structural genes in a cat8 null mutant

Table 3

Influence of increased CAT8 gene dosage on ICLl-lacZ expression in wild-type and pleiotropic regulatory mutants

Influence of CAT8 gene dosage on ICL1 expression

In order to investigate the position of CAT8 in the regulatory hierarchy of CSRE-dependent gene activation, a yeast strain containing an integrated ICLl-lacZ reporter gene was transformed with the episomal plasmid YEp24-DIL1 containing the entire CAT8 gene. In addition, isogenic catl, migl and catl migl derivatives of this strain were treated similarly. An increased CAT8 gene dosage did not influence ICLl-lacZ expression in the wild-type, the catl mutant or the migl mutant, neither under repressing nor under derepressing conditions (Table 3). However, a substantial increase in reporter gene expression was found in CAT8 multi-copy transformants of the catl migl strain under derepressing conditions (28% of the wild-type level), but not under repressing conditions. This CAT8 dosage-dependent suppression of the derepression defect caused by the catl mutation allowed a slow growth of the transformants even on synthetic complete medium containing ethanol as the sole carbon source. For this suppression to occur, the MIGl gene must be defective. Thus, the Mig1p repressor must fulfil some negative function even under conditions of non-fermentative growth. In contrast to previous CAT8 gene dosage studies (19), no suppression of growth defects of the catl single mutant by episomal CAT8 could be observed. This discrepancy may be due to the rich medium used by these authors for growth assays. In conclusion, our data provide evidence for a function of CAT8 downstream of the pleiotropic Cat1p protein kinase system. Since we could not detect CAT8-dependent gene dosage effects in a wild-type strain, positive regulators in addition to Cat8p presumably affect CSRE-dependent structural genes.

Expression of CAT8 in wild-type and regulatory mutants

Several transcription factors involved in various regulatory networks have been shown to be regulated at the biosynthetic level (e.g. Gal4p and Gcn4p; 11,31). We thus investigated the expression of a CAT8-lacZ reporter gene in wild-type and regulatory mutant strains under repressing and derepressing conditions. In agreement with recent data (19), we found CAT8 expression to increase ∼45-fold in derepression medium (Table 4). Repression of CAT8 was significantly alleviated in the migl mutant, while CAT8 derepression required a functional CATl gene. However, migl turned out to be epistatic to catl, since the CAT8 derepression defect observed in the catl mutant could be suppressed by a migl mutation. This result can be easily explained by the Mig1p repressor binding site identified upstream of CAT8 (19). Thus, the Cat1p protein kinase system appears to be required for deactivation of the Mig1p repressor.

Table 4

Carbon source-regulated expression of CAT8 in wild-type and regulatory mutants

The HXK2 structural gene encoding hexokinase PII has been shown as essential for glucose repression of SUC and MAL genes (32). However, a hxk2 null mutation did not cause a significantly increased CAT8 expression under repressing conditions (Table 4). Interestingly, derepression of CAT8 was severely affected in a hap2 mutant, indicating a regulatory connection between respiration and gluconeogenesis. The sequence motif TGATTGGT upstream of CAT8 (—199 to —192) agrees completely with the consensus binding site of the heteromeric Hap2/3/4/5p complex (33), which may mediate a respiration-coupled activation of CAT8. The CAT8 gene appears to be negatively autoregulated, since expression of the CAT8-lacZ reporter gene in a cat8 mutant increases >3-fold under either growth conditions when compared with the isogenic wild-type. Presently, the _cis_-acting element responsible for this control and its possible regulatory significance are unknown.

Importance of CAT8 for protein-CSRE interactions

In a previous study, we described two protein-CSRE complexes specifically detectable with extracts from derepressed wild-type cells (comprising Ang1 and Ang2; 18). The presence of a binuclear zinc cluster motif in the N-terminus of Cat8p indicates its binding to DNA. The importance of the terminal CCG nucleotides within the recognition site has been shown for some members of this family (e.g. Gal4p, ArgRIIp, Pprlp and Put3p; 34,35). Since the CSRE indeed contains a CCG motif, Cat8p may be considered as a CSRE binding factor. Thus, we expressed the N-terminus of Cat8p (amino acids 1–281) as a GST fusion construct in E.coli (plasmid pAR1; cf. Fig. 1) and used the affinity-purified protein for gel retardation studies with a CSRE probe. However, we could not observe any GST-Cat8p-CSRE interaction in this assay (not shown). This result would be consistent with a yeast-specific modification of Cat8p being required for CSRE binding or the importance of a possible Cat8p binding partner. Alternatively, protein factors distinct from Cat8p could bind to the CSRE. In order to investigate a possible Cat8p-CSRE interaction in the homologous yeast system, we constructed an epitope-tagged variant of Cat8p (encoded by the CAT8-FLAG gene in plasmid pEM6). In an immunoblot analysis of extracts prepared from CAT8-FLAG transformants, a carbon source-dependent signal at an apparent size of ∼160 kDa was detected (Fig. 2, lane 6). No signal was obtained with extracts from the wild-type or the cat8 mutant (lanes 1–4). Protein extracts of the respective transformants were subsequently used for a supershift analysis in the presence of a FLAG-specific monoclonal antibody. As demonstrated by the gel retardation experiment shown in Figure 3, the carbon source-dependent protein-CSRE complexes CI and CII (comprising Ang1 and Ang2, respectively) observed in derepressed wild-type extracts (lane 3) were absent in a cat8 null mutant (lane 5). Introduction of the CAT8-FLAG gene into the cat8 mutant restored the band shift pattern typical of the wild-type (lane 6). However, no alteration of the electrophoretic mobilities of complexes CI or CII was observed in the presence of the FLAG-specific antibody. We thus conclude that DNA binding proteins distinct from Cat8p bind to the CSRE. The absence of protein-CSRE complexes CI and CII in cat8 null mutants suggests that CAT8 may encode a positive regulator of the respective genes.

Figure 2

Immunological detection of a FLAG-tagged Cat8p variant. Protein extracts (50 µg) from the wild-type C13-ABY.S86 (lanes 1 and 2), the cat8 null mutant ABYS.Δdill (lanes 3 and 4) and pEM6 transformants (CAT8-FLAG) were separated by SDS-PAGE and subsequently blotted to nitrocellulose. As a primary antibody, the monoclonal antibody M2 (Kodak/IBI) was used. Secondary antibody was detected by a chemiluminescence reaction. Strains were grown under repressing (lanes 1, 3 and 5) or derepressing (lanes 2, 4 and 6) conditions.

Figure 3

Gel retardation analysis of protein binding to the CSRE probe OAS12. Binding reactions contained 30 µg cellular protein. The following protein extracts were added: lane 1, no extract added; lane 2, repressed wild-type cells; lanes 3 and 4, derepressed wild-type cells; lane 5, derepressed cat8 mutant; lanes 6–9, derepressed cat8 mutant, transformed with pEM6 (CAT8-FLAG). For competition studies, a 100-fold molar excess of unlabeled oligonucleotide OAS12 was used (lanes 4, 7 and 9). In lanes 8 and 9, anti-FLAG antibody M2 (1 µg) was added. CI-CIV, protein-CSRE complexes.

Cat8p functions as a C427-dependent transcriptional activator

A large number of transcription factors can be functionally separated into DNA binding and transcriptional activation domains. We thus fused various parts of CAT8 to the DNA binding domain of GAL4 (controlled by the ADH1 promoter) and assayed the expression of a _GAL4_-dependent reporter gene. As shown in Figure 1, none of the tested CAT8 subfragments could mediate transcriptional activation. On the other hand, a strong activation was observed with the entire CAT8 gene (plasmid pAR4). Interestingly, this activation was completely carbon source dependent. In contrast, a GAL4_DBD_-INO2 fusion (36), used as a control, showed a constant activation under repressing and derepressing conditions (Table 5). Gene activation mediated by Cat8p was no longer observed in a cat1 null mutant. This defect was not suppressed in a cat1 mig1 double mutant. Interestingly, _INO2_-dependent activation was also significantly reduced, although not completely abolished, in the cat1 mutant. This result may explain the inositol auxotrophy caused by a cat1 mutation in some strain backgrounds (37; Table 5).

Table 5

Transcriptional activation of a GAL1-lacZ reporter gene by a _GAL4_DBD-CAT8 fusion construct in the wild-type and pleiotropic regulatory mutants

These data prove (direct or indirect) transcriptional activation mediated by the positive regulatory protein Cat8p. The loss of this function in the cat1 mutant suggests a possible signal transduction pathway from the pleiotropically acting Cat1p protein kinase system to the pathway-specific regulator Cat8p affecting CSRE-dependent structural genes.

Discussion

The complex pattern of carbon source-dependent transcription in the yeast S.cerevisiae involves several pleiotropic as well as pathway-specific regulators. Previously, we identified the CSRE as a UAS element upstream of the isocitrate lyase gene ICL1 and other co-regulated genes of non-fermentative metabolism (18,21). In this work, we describe the DIL1 gene as a transcriptional regulator specifically affecting CSRE-containing structural genes. DIL1 turned out to be identical to the recently isolated CAT8 gene, which is required for derepression of the gluconeogenic genes FBP1 and PCK1 (19). This identity was not surprising, since the promoters of ICL1, FBP1 and PCK1 all contain at least a single copy of a functional CSRE (18–20). Cat8p also functions as a positive regulator of the glucose-repressible ADH2 gene. Although we did not find anADH2 promoter sequence element convincingly similar to the CSRE, the previously described UAS2 region immediately upstream of the Adr1p binding site (38) may be considered as a putative target of Cat8p. On the other hand, CAT8 is clearly not involved in the oleate induction pathway (mediated by the oleate-responsive element ORE; 39,40), responsible for the efficient proliferation of peroxisomal structures.

Previous gel retardation experiments led to the identification of two CSRE binding protein factors (Ang1 and Ang2) exclusively detectable in extracts from derepressed yeast cells (18). The presence of a binuclear C6 zinc cluster motif in the N-terminus of Cat8p strongly argues for its binding to DNA. Thus, Cat8p may be considered as a CSRE binding factor possibly related to Ang1 and/or Ang2. This idea would be consistent with the binding of several members of the Gal4p family to sites containing a CCG sequence (34,35), which is also present within the CSRE. The carbon source-dependent binding pattern of Ang factors to the CSRE would be easily explained by the regulation of CAT8 at the biosynthetic level shown here and elsewhere (19). Although Ang-CSRE interactions could no longer be observed in a cat8 null mutant, we were unable to demonstrate CSRE binding of a GST-Cat8p fusion protein produced in E.coli. Thus, Cat8p is necessary but not sufficient for protein binding to the CSRE. Even extracts from derepressed yeast cells containing a full-length, epitope-tagged protein variant gave no evidence for Cat8p being at least a part of an Ang factor. Although a failure of the antibody used in the supershift experiment to detect the tag under native conditions cannot completely be ruled out, we favor a model with CAT8 being an indirect activator of CSRE-containing genes. Two additional findings support this view: first, overexpression of CAT8 by introduction of a multi-copy plasmid into a wild-type strain does not cause any gene dosage effects, neither under repressing nor under derepressing conditions (Table3). Dosage effects on structural genes have been frequently observed when directly activating transcription factors are overexpressed (e.g. in the case of GAL4;41). Second, both UAS elements of the carbon source-regulated FBP1 promoter require a functional CAT8 gene (19; this work), although distinct protein factors bind to UAS1 and UAS2 (25,42). Such a result would not be expected when assuming the sole and direct binding of Cat8p to both UAS elements. In conclusion, these data can be easily explained by considering Cat8p as a transcriptional regulator of activating proteins (Ang factors) finally binding to the CSRE and other UAS elements of gluconeogenic genes. Thus, it will be necessary in the future to identify the genes encoding Ang factors. According to our hypothesis (cf. Fig. 4), their control regions may contain sequence elements recognized by the putative DNA binding domain of Cat8p.

Figure 4

Hypothesis on the function of CAT8 for the derepression of gluconeogenic structural genes. The dual function of the Cat1p-Cat3p protein kinase complex for deactivation of the Mig1p repressor and generation of Cat8p-dependent transcriptional activation should be emphasized. Cat8p may lead to derepression of the ANG gene(s) encoding CSRE binding factors. A co-regulation of respiratory and gluconeogenic genes is suggested by the Hap2/3/4/5p binding site upstream of CAT8.

By fusing its entire reading frame to the DNA binding domain of Gal4p, Cat8p could be identified as a transcriptional activator. Interestingly, activation turned out to be regulated by the carbon source. Thus, besides the biosynthetic control of CAT8 expression, a second level of carbon source control affects Cat8p function. In both cases, a functional Cat1p protein kinase system is required, suggesting a dual role of this pleiotropic factor of glucose derepression for Cat8p function. However, both levels of Cat1p influence on Cat8p are unequally affected by the Mig1p repressor. While the biosynthetic defect of CAT8 in a cat1 mutant was suppressed by a mig1 mutation, no suppression could be observed for the activation defect. Thus, expression of CAT8 is under negative control by MIG1, while activation by Cat8p is not. This result provides a simple explanation for the failure of cat1 mig1 double mutants to derepress gluconeogenic genes and, subsequently, to utilize non-fermentable carbon sources. Cat8p must be able to mediate some basal transcriptional activation even in the absense of a functional CAT1 gene. This follows from the partial derepression of the ICL1 gene in a CAT8 multi-copy transformant of a cat1 mig1 double mutant, thereby allowing slow growth even on ethanol-containing medium.

Although the C-terminal 229 amino acids of Cat8p were necessary for activation, no effect was observed when this domain was fused separately to Gal4p. This result was surprising, since protein structures comprising <30 amino acids can activate efficiently (43). Possibly, an inhibitory domain also present within the C-terminus of Cat8p may account for this. Inhibitory glucose response domains were previously identified within the central part of Gal4p (44). A more detailed analysis is necessary to localize the transcriptional activation domain of Cat8p precisely. No obvious similarities to previously classified transcriptional activation motifs were found within Cat8p. At present, we cannot rule out indirect activation by a distinct protein tethered to Cat8p by protein-protein interactions.

Based on these considerations of Cat8p function, we propose the following model for the derepression of gluconeogenic structural genes (Fig. 4). Expression of CAT8 is positively controlled by the Hap2/3/4/5p complex, arguing for a genetic co-regulation of respiration and gluconeogenesis. A connection of both pathways is also suggested by the recent characterization of the CAT5 gene involved in ubiquinone biosynthesis and glucose derepression (45). Under derepression conditions, the Cat1p protein kinase may deactivate the Mig1p repressor (46), thereby allowing CAT8 transcription. At present, the nature of the derepression signal is unknown, although a regulation of Cat1p (Snf1p) by phosphorylation has been suggested (47). Subsequently, Cat1p leads to efficient transcriptional activation of downstream regulatory genes (ANG genes) by Cat8p. It remains to be shown whether direct phosphorylation of Cat8p is required for this process. ANG gene products then bind to the CSRE (and possibly to functionally related sequence variants) and mediate derepression of the respective structural genes.

Acknowledgements

This paper is dedicated to Prof. Dr E.Schweizer on the occasion of his 60th birthday. The work described was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. We wish to thank C.Denis, L.Guarente, S.Hohmann, S.Johnston, K.Melcher, H.Ronne, I.Sadowski and D.Wolf for providing strains or plasmids used in this work. We also thank E.Schweizer for kind support and B.Hoffmann for excellent technical assistance.

References

1

,

The Molecular and Cellular Biology of the Yeast Saccharomyces

,

1992

, vol.

2

Cold Spring Harbor, NY

Cold Spring Harbor Laboratory Press

(pg.

193

-

281

)

2

,

Eur. J. Biochem

,

1992

, vol.

206

(pg.

297

-

313

)

3

,

Trends Biochem. Sci.

,

1992

, vol.

17

(pg.

506

-

510

)

4

,

Trends Genet.

,

1995

, vol.

11

(pg.

12

-

17

)

5

,

Mol. Gen. Genet.

,

1977

, vol.

151

(pg.

95

-

103

)

6

,

Science

,

1986

, vol.

233

(pg.

1175

-

1180

)

7

,

Mol. Gen. Genet.

,

1987

, vol.

209

(pg.

366

-

373

)

8

,

Mol. Cell. Biol

,

1989

, vol.

9

(pg.

5045

-

5054

)

9

,

J. Bacteriol.

,

1991

, vol.

173

(pg.

2045

-

2052

)

10

,

EMBO J.

,

1990

, vol.

9

(pg.

2891

-

2898

)

11

,

EMBO J.

,

1991

, vol.

10

(pg.

3373

-

3377

)

12

,

Genetics

,

1994

, vol.

137

(pg.

49

-

54

)

13

,

Mol. Cell. Biol

,

1994

, vol.

14

(pg.

1979

-

1985

)

14

,

Curr. Genet.

,

1993

, vol.

23

(pg.

375

-

381

)

15

,

FEBS Lett.

,

1993

, vol.

333

(pg.

238

-

242

)

16

,

J. Gen. Microbiol.

,

1988

, vol.

134

(pg.

2499

-

2505

)

17

,

FEBS Lett.

,

1995

, vol.

367

(pg.

219

-

222

)

18

,

Mol. Cell. Biol

,

1994

, vol.

14

(pg.

3613

-

3622

)

19

,

Mol. Cell. Biol

,

1995

, vol.

15

(pg.

1915

-

1922

)

20

,

Mol. Gen. Genet

,

1995

, vol.

246

(pg.

367

-

373

)

21

,

Gene

,

1995

, vol.

161

(pg.

75

-

79

)

22

,

Genetics

,

1984

, vol.

108

(pg.

845

-

858

)

23

,

Cell

,

1982

, vol.

28

(pg.

145

-

154

)

24

,

Molecular Cloning: A Laboratory Manual

,

1989

2nd Edn

Cold Spring Harbor, NY

Cold Spring Harbor Laboratory Press

25

,

Curr. Genet.

,

1992

, vol.

22

(pg.

363

-

370

)

26

,

Gene

,

1987

, vol.

45

(pg.

299

-

310

)

27

,

Gene

,

1992

, vol.

118

(pg.

137

-

141

)

28

,

Curr. Genet.

,

1993

, vol.

24

(pg.

455

-

459

)

29

,

Gene

,

1987

, vol.

57

(pg.

267

-

272

)

30

,

Antibodies: A Laboratory Manual

,

1988

Cold Spring Harbor, NY

Cold Spring Harbor Laboratory Press

31

,

Proc. Natl. Acad. Sci. USA

,

1984

, vol.

81

(pg.

6442

-

6446

)

32

,

Mol. Gen. Genet.

,

1980

, vol.

178

(pg.

633

-

637

)

33

,

Mol. Cell. Biol

,

1988

, vol.

8

(pg.

647

-

654

)

34

,

Mol. Cell. Biol

,

1992

, vol.

12

(pg.

68

-

81

)

35

,

Science

,

1993

, vol.

261

(pg.

909

-

911

)

36

,

Nucleic Acids Res.

,

1995

, vol.

23

(pg.

230

-

237

)

37

,

Genes Dev.

,

1992

, vol.

6

(pg.

2288

-

2298

)

38

,

Mol. Cell. Biol.

,

1989

, vol.

9

(pg.

34

-

42

)

39

,

Eur. J. Biochem.

,

1993

, vol.

214

(pg.

323

-

331

)

40

,

Gene

,

1993

, vol.

132

(pg.

49

-

55

)

41

,

Proc. Natl. Acad. Sci. USA

,

1982

, vol.

79

(pg.

6971

-

6975

)

42

,

J. Biol. Chem

,

1995

, vol.

270

(pg.

12832

-

12838

)

43

,

Hoppe-Seyler Z. Biol. Chem.

,

1994

, vol.

375

(pg.

463

-

470

)

44

,

EMBO J.

,

1993

, vol.

12

(pg.

1375

-

1385

)

45

,

EMBO J

,

1995

, vol.

14

(pg.

6116

-

6126

)

46

,

Mol. Cell. Biol.

,

1996

, vol.

16

(pg.

753

-

761

)

47

,

J. Biol. Chem.

,

1994

, vol.

269

(pg.

19509

-

19515

)

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