INTRODUCTION

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Saccharomyces cerevisiae and many other yeasts may thrive on a variety of carbon sources, but glucose and fructose are the preferred ones. When one of these sugars is present, the enzymes required for the utilization of alternative carbon sources are synthesized at low rates or not at all. This phenomenon is known as carbon catabolite repression, or simply catabolite repression, and since no "catabolite" derived from glucose and involved in the repression has been yet identified, the term "glucose repression" has also been proposed. In this review, I still use the term "catabolite repression" as well as glucose repression, to stress that other sugars, such as galactose or maltose, are able to affect the synthesis of enzymes repressed by glucose (Table 1).

A comprehensive picture of the mechanism(s) of catabolite repression is not yet available, in spite of the accumulation of information on the subject (for earlier reviews, see references 95, 96a, 96b, 124, 163, 289, and 346). Although the solution of the puzzle has progressed, important pieces are still missing and it has been found that other pieces, originally thought to belong, do not really pertain to the basic frame. The last few years have seen important advances, which are reviewed and discussed in this article. I also propose some models for catabolite repression of different genes and discuss some perspectives for future research. Although the review deals mainly with S. cerevisiae, reference to other yeast species is made, as far as information is available.

For easy reference, Table 2 provides an overview of the alternative names given to genes related to catabolite repression, since these genes have been repeatedly isolated by different groups and given different names.

Control of the mRNA translation rate is not common in yeast; however, in the case of the transcriptional activator Adr1, glucose appears to act at this step. While the concentration of Adr1 is at least 10-fold higher in ethanol-grown yeast than in glucose-grown yeast, there is only a 2-fold difference in the levels of the corresponding mRNAs (354). Since the half-life of Adr1 itself is not longer in ethanol-grown cells than in glucose-grown cells, it has been concluded that the observed decrease in the level of Adr1 is due mainly to a reduction in the rate of Adr1 synthesis brought about by glucose. The molecular mechanism by which glucose acts remains unclear, but it has been shown that the translational control does not depend on the long untranslated 5' leader sequence of ADR1 mRNA (354). Removal of the gene sequence corresponding to the 681 C-terminal residues of Adr1 (more than half the length of the protein) did not disrupt the translational control, but the ADR1 coding sequence between amino acids 262 and 642 is required for the control of ADR1 translation by glucose.

While translational control by glucose is rare, glucose triggers inactivation and/or proteolysis of a number of proteins. By analogy to catabolite repression, this phenomenon has been called catabolite inactivation (151); it affects a variety of proteins, from gluconeogenic enzymes to transport molecules, but it is not yet known whether the same mechanism underlies the inactivation in the different cases (125a). Inactivation of fructose-1,6-bisphosphatase (FbPase) by glucose (121) has been most extensively studied, and it has been shown that glucose causes a very rapid phosphorylation of FbPase and a proteolytic degradation of the enzyme (119, 224, 225, 236). Two alternative mechanisms for the proteolysis have been described: (i) transfer of FbPase to the vacuole and degradation by vacuolar proteases (56), and (ii) ubiquitination of FbPase (310) followed by degradation by the proteasome (309). The relative contribution of each pathway could depend on the physiological state of the yeast (306). Proteolysis of FbPase triggered by glucose can occur even in the absence of phosphorylation (291), but it has not been established whether phosphorylation may be a requirement or a facilitator of at least one of the degradation systems. The mechanism by which glucose triggers the proteolysis is not known, but glucose appears to induce the synthesis of proteins required for the degradation process. Some of these proteins would be transported to the vacuole, via the secretory pathway, and facilitate the uptake of FbPase by the vacuole (56). Other proteins would be required for ubiquitination of FbPase and its degradation in the proteasome (310). Recently, a series of mutants have been isolated in which inactivation and degradation of FbPase are uncoupled and the inactive protein accumulates in the cytosol and in small vesicles in the cytoplasm (149). Although the proteins involved in the process have not been identified yet, there is some evidence that in wild-type cells, FbPase could be imported in cytoplasmic vesicles before being degraded in the vacuole (155).

The most general system of catabolite repression involves a parallel decrease in mRNA and protein levels. Glucose has been reported to destabilize the corresponding mRNAs in a few systems: the functional half-life of CYC1 mRNA was shown to decrease from 12 min in derepressed cells to about 2 min when glucose was present (400, 401); for MAL6S (MAL62) mRNA the decrease was from 25 to 6 min (107); glucose also had a very strong effect on the mRNAs encoding subunits of succinate dehydrogenase, decreasing the half-life from more than 60 min to less than 10 min (198); for PCK1 mRNA, glucose accelerated the degradation rate only twofold (230). In no case, however, was information available about a possible mechanism for this effect of glucose.

More recently, it has been shown that the 5' untranslated end of the mRNA encoding the iron protein (Ip) subunit of succinate dehydrogenase is able to promote rapid degradation of a fusion mRNA upon glucose addition (48). The 5' exonuclease Xrn1 seems to play an important role in the mRNA degradation, and it has been suggested that the rate of degradation of mRNA could be set by a competition between initiation of translation and nuclease action (48). In this case, glucose could promote mRNA degradation by blocking mRNA translation. Phosphorylation of glucose or fructose was required to trigger Ip mRNA turnover, but any of the hexose kinases would be effective. Although further metabolism of the hexose phosphate formed does not seem to be required (both glucose and fructose increased turnover in a pgi mutant), addition of 2-deoxyglucose to a derepressed yeast culture did not decrease Ip mRNA stability (49). A possible interpretation for this could be that 2-deoxyglucose would decrease ATP levels and that the triggering of mRNA degradation would be energy dependent. Most factors required for glucose repression of genes such as SUC2 or the GAL genes (Hxk2, Grr1, Tup1, and Cyc8) did not markedly affect mRNA turnover in the presence of glucose, and the protein Ume5 (Srb10), which has been reported to destabilize SPO13 mRNA in a glucose-containing medium (331), was not needed for differential turnover of Ip mRNA (49). On the other hand, the regulatory protein Reg1 was required for increased degradation of Ip mRNA upon glucose addition (49).

The mechanism(s) regulating mRNA turnover in response to the carbon source remain to be worked out, but it is clearly established that for a subset of genes regulated by glucose, control is operating on mRNA stability instead of (or in addition to) on transcription rates. However, in the rest of this review, catabolite repression is considered in the narrow sense of the repression of transcription caused by glucose, since, as mentioned above, this is the major control mechanism.

Glucose and other repressing sugars can affect the rate of transcription by two basic mechanisms: they interfere with activators of transcription, or they facilitate the action of proteins with a negative effect on transcription. In bacteria, one or the other of these control mechanisms regulates catabolite repression in different species (297). Our present picture is that in yeast, the different sugars do not act directly on DNA-binding proteins but produce signals that are transmitted through a series of proteins to the promoters of the corresponding genes. To unravel the mechanisms of catabolite repression, it is therefore necessary to identify the signals produced by the sugar, the proteins which respond to them, and their substrates, down to the proteins binding to the promoter of the regulated genes.

To find the different elements that participate in the cascade of reactions between glucose and the final target, mutants affected in the process have been invaluable. Two kinds of mutants were sought, mutants for which glucose was no longer repressing and mutants in which derepression did not occur even when the glucose in the medium had been used up. To obtain these mutants, a variety of strategies were used (see reference 125 for a review), and the number of different genes isolated has increased in a bewildering manner. However, in the last few years it has become apparent that a number of these genes are not specifically related to the control by glucose but play a more general role in the control of transcription.

In the following sections, we consider in detail the different elements of the system: proteins which act as specific transcriptional repressors or activators, intermediary regulatory factors, and elements involved in glucose signalling. A number of proteins which somehow affect catabolite repression but are not directly involved in the response to glucose are also discussed.

The Hap2/3/4/5 complex. A large number of genes are regulated by a complex containing the proteins Hap2, Hap3, Hap4, and Hap5 (see reference 79 for a compilation of such genes). The complex, which activates transcription when yeast grows on a nonfermentable carbon source, binds DNA and makes contacts with a consensus ACCAA(T/C)NA sequence called the CCAAT box (255). In a search for yeasts unable to activate a CCAAT box-containing fusion gene, hap2, hap3, and hap4 mutants were isolated (114, 134, 136). Hap5 was identified by the two-hybrid assay with the core region of Hap2 as a bait (227). Hap2, Hap3, and Hap5 are absolutely required for CCAAT-binding activity, as shown in band shift experiments, and the three subunits are also sufficient for DNA binding, since a mixture of the three purified recombinant proteins allows binding to a CCAAT box in vitro (227). This is in contrast to initial reports that Hap4 was required for binding (114). It appears now that Hap4 is mainly responsible for the activation of transcription produced by the complex (257). The stoichiometry of the complex is not yet clear, but it has been reported to contain a single Hap2 molecule (384).

Gal4. The protein Gal4 (184) activates the transcription of a family of genes, GAL1, GAL2, GAL7, GAL10, and MEL1, involved in the catabolism of galactose and melibiose (for a review, see references 161 and 228). These genes contain one to four copies of a regulatory element, UAS_GAL, with the palindromic consensus binding site CGGA(G/C)GACAGTC(C/G)TCCG (129), to which Gal4 can bind.

Mal63. In different S. cerevisiae strains, the genes required for maltose utilization may be found at different loci called MAL1, MAL2, MAL3, MAL4, or MAL6. The most extensively studied gene complex is MAL6; in this complex, MAL61 (MAL6T) encodes maltose permease, MAL62 (MAL6S) encodes maltase, and MAL63 (MAL6R) encodes a protein which activates the expression of the genes MAL61 and MAL62 and probably that of the MAL63 gene itself (51, 240).

Adr1. Adr1 is a zinc finger protein belonging to the C₂H₂ family (138), identified as a positive effector of the expression of ADH2, a gene which is repressed by glucose and which encodes alcohol dehydrogenase II (57). Adr1 has been localized in the nucleus, and its zinc fingers are essential for DNA binding (14). Adr1 binds a 22-bp palindromic sequence in the promoter of ADH2 (94) and may also bind sequences in the promoters of genes encoding peroxisomal proteins or proteins involved in glycerol utilization (266, 320). A consensus sequence for Adr1 binding, C(T/C)CC(A/G)N_6-38(T/C)GG(A/G)G, has been proposed (53).

Other activators. There are a number of proteins related to catabolite repression which have features of a transcriptional activator but for which no targets have been identified. The characteristics of Sip3 and Sip4 which have been isolated by their capacity to interact with the protein kinase Snf1 in the two-hybrid assay (186, 187) are described in the section on the Snf1 complex (below).

The Mig1 complex. The MIG1 gene, an important element in glucose repression, was identified in a search for genes which would turn off the GAL1 promoter of S. cerevisiae (243). Mutations called cat4 or ssn1, which turned out to be allelic to MIG1, were also isolated as extragenic suppressors of snf1 and snf4 mutations (311, 356). Mig1 is a C₂H₂ zinc finger protein that is able to bind to the promoters of a variety of genes repressed by glucose. Binding requires a GC box with the consensus sequence (G/C)(C/T)GGGG, but it also requires an AT-rich region 5' to the GC box (200). It has been suggested that finger 1 from Mig1 recognizes a G(G/A)G triplet and finger 2 recognizes a (G/C)(C/T)G triplet. The specific residues involved in the contacts would be an arginine at position 21, a histidine at position 18, and an arginine at position 15 for the first finger and an arginine, a glutamic acid, and an arginine at the same positions for the second finger. The AT-rich region would be required to stabilize the interaction, since it would allow bending of the DNA and facilitate further protein-DNA contacts (200).

View larger version (10K): [in this window] [in a new window]   FIG. 1.   Sequences of the Mig1 proteins from different yeasts which may be substrates of the protein kinase Snf1; the serine which may be phosphorylated is underlined. Amino acids which were found to be important in a study with artificial peptides as substrates for Snf1 (68) are shown in boldface type; amino acids in parentheses are suboptimal. Equivalent regions in the different proteins are aligned.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Schematic view of the mode of action of Mig1 and its regulation. In the presence of glucose, Mig1 is found in the nucleus, where it represses the transcription of genes encoding activators such as GAL4 and MAL63 and of genes whose products are implicated in the metabolism of alternative carbon sources. Glucose removal causes both phosphorylation of Mig1, depending on the Snf1 complex, and its translocation to the cytoplasm. For details, see the text.

Other repressors. There is not much evidence about proteins different from the Mig1 family that are able to bind to the promoters of genes subject to catabolite repression and to inhibit the transcription of these genes. The SKO1 gene, which was isolated by the same procedure as MIG1, encodes a protein that is able to bind to the promoter of SUC2, but its role in mediating repression by glucose is doubtful (242). Although transcription of SUC2 is increased up to twofold in a sko1 strain, the effect of the SKO1 disruption is more marked during growth on raffinose than in the presence of glucose. Moreover, the lack of Sko1 has no effect on the expression of GAL genes or on growth on different carbon sources (242).

The Snf1 complex. The SNF1 gene (also called CAT1 or CCR1) is absolutely required for the derepression of genes repressed by glucose (40, 58, 399). SNF1 encodes a Ser/Thr protein kinase (44), the first member of a growing family of protein kinases to be identified (9). In particular, it has a mammalian homolog, which is the catalytic  subunit of the AMP-activated protein kinase (AMPK) (36, 234, 380).

sip1 sip2 gal83

sip1 sip2 gal83

View larger version (29K): [in this window] [in a new window]   FIG. 3.   Model for the regulation of the Snf1 complex by glucose. The bridging protein (Brp) between Snf1 and Snf4 can be Gal83, Sip1, Sip2, or some other, as yet unidentified, protein. Glucose affects the interaction between the catalytic domain (KD) and the regulatory domain (RD) of Snf1, presumably by inhibiting the (auto)phosphorylation of Snf1 and/or activating its dephosphorylation. Glucose may act at the level of the corresponding kinase and phosphatase but may also alter the conformation of Snf1 or even Brp, making Snf1 a worse or better substrate for the corresponding enzyme. Hxk2 and Grr1 are required for transmitting the glucose signal. Redrawn from reference 159.

sip4

sip1

sip2

sip3

The Glc7 complex. GLC7 (DIS2S1) is an essential gene, encoding a protein phosphatase type 1, which controls a variety of processes including glycogen accumulation (108, 254). A mutation called cid1, which partially relieved the repression of invertase by glucose (245), turned out to be a glc7-T152K mutation (348). This mutation did not impair glycogen accumulation (348), while a different mutation, glc7-1, which interfered with glycogen synthesis (34, 267), did not affect the repression of invertase by glucose (348). Since the mutant protein Glc7-1 was shown to be defective in its interaction with Gac1, a protein that appears to be a glycogen-specific regulatory subunit of the protein phosphatase type 1 (330), it was suggested that Glc7-T152K could be impaired in its interaction with a different regulatory protein, which would direct the phosphatase to some substrate(s) specifically related to the control of catabolite repression (348).

Glucose signaling has a variety of effects. It causes the repression and the inactivation of many enzymes but also the induction and the activation of other enzymes (122). It is possible that some signals cause different kinds of effects while others are more specific for particular systems. For genes encoding glycolytic enzymes and requiring glucose for full expression, induction by glucose depends on the accumulation of intermediary metabolites (16, 18, 238). For some genes, an increase in the level of hexose-6-phosphates is required, while for others, induction is triggered by glycolytic three-carbon metabolites.

There is strong evidence for specific glucose sensors in the yeast membrane. Among the large number of hexose transporter (HXT) genes identified in S. cerevisiae (17, 175), two genes, SNF3 and RGT2, are expressed at very low levels compared to most HXT genes and play a specific regulatory role (247, 259). While SNF3 is repressed by a high concentration of glucose, RGT2 is expressed constitutively. Since Snf3 is required for the induction by low levels of glucose of some hexose transporter genes such as HXT2, it is likely to function as a sensor for low glucose levels. Rgt2, which is required for the induction of HXT1 by high glucose levels, would be a sensor for high glucose levels (259). Since both Snf3 and Rgt2, unlike other glucose transporters, have a long cytoplasmic domain at the C terminus, it has been speculated that the binding of glucose to these proteins causes a conformational change affecting a putative C-terminal signaling domain (47, 259). Since the whole yeast genome has been now sequenced, it can be ascertained that no further protein with a structure similar to that of Snf3 and Rgt2 remains to be discovered (175).

A snf3 mutation does not relieve the repression of SUC2, GAL10, or ADH2 by high glucose levels (192, 247), but it abolishes the ability of raffinose to repress ADH2 (192) and the induction of SUC2 by low glucose levels (264). While an rgt2 mutation does not affect the repression of SUC2 or GAL1 by high glucose levels (259), an snf3 rgt2 double mutant grows very poorly in glucose, and in this mutant neither GAL1 nor SUC2 are repressed by 4% glucose (258b).

It is important to note that signaling by glucose does not have the same requirements for different systems. In a yeast strain with a mutant protein Rgt2-1, a gene such as HXT2 is induced even in the absence of glucose in the medium, suggesting that the signaling domain of the mutant protein is permanently activated. However, in an RGT2-1 strain, the SUC2 gene is still normally derepressed in a medium with low glucose (214).

Could other glucose transporters be involved in the signaling pathway leading to catabolite repression? To address this question, a yeast mutant strain, unable to transport glucose, has been constructed where all the genes from HXT1 to HXT7 are disrupted and a series of derived strains expressing one or several of the genes HXT1 to HXT7 have been studied with regard to their capacity to repress the MAL2, SUC2, or GAL1 genes (283). The results indicate that glucose repression does not depend on any specific transporter but that in the different strains, the extent of repression is correlated with the glucose uptake capacity, suggesting that the rate of glucose utilization determines the strength of the relevant glucose signal. Similarly, it has been reported that in a strain which retains only the HXT6/7 gene and has a low glucose transport capacity, SUC2 is no longer repressed by glucose (368). In contrast to these results, repression of SUC2 and ADH2 was observed in a yeast strain lacking the transport genes HXT1 through HXT4 and HXT6/7, after overnight growth in a medium containing glycerol, ethanol, and 5% glucose (192). It is possible, however, that under these particular conditions other transport genes are expressed at sufficient levels to allow glucose uptake and catabolite repression.

Other reports have shown a correlation between transport capacity and catabolite repression. A yeast strain with the dominant mutation DGT1-1 expresses glucose transporters at low levels, and in this strain glucose repression of a variety of enzymes, including the gluconeogenic ones, is totally or partially relieved while repression by galactose is not affected (120). Another dominant mutation, HTR1-23, has similar but less strong effects (260). HTR1 is allelic to MTH1 (312), and it has now been found that although HTR1-23 and DGT1-1 differ in some of their effects, they are alleles of the same gene (179). MTH1 appears to be involved in the expression of the HXT genes, and although its exact role has not been yet elucidated, another protein, Std1/Msn3, structurally and functionally related to Mth1, has been reported to interact directly with the TATA binding protein (341).

Another mutation, grr1/cat80, isolated as conferring resistance to catabolite repression (3, 97), has also been found to affect the regulation of glucose transporters. Induction of the HXT1 to HXT4 genes is defective in grr1 mutants (261). There is now evidence that Grr1, a large protein with multiple leucine-rich repeats and tightly associated with a particulate fraction (112), is a component of a ubiquitin-conjugating enzyme complex which would regulate, perhaps indirectly, Rgt1 (189). Rgt1 is a DNA-binding protein that is able to repress HXT genes in the absence of glucose and to activate some genes such as HXT1 at high concentrations of glucose (263). Grr1 would be required both at low glucose concentrations to inactivate the Rgt1 repressor function and at high glucose concentrations to turn Rgt1 into an activator. The glucose signal would be transmitted to Grr1 via Snf3 or Rgt2, depending on the concentration of external glucose. In the absence of Grr1, Rgt1 permanently acts as a repressor, the glucose transport capacity of the cell is low, and glucose repression of a number of genes is relieved. In fact, the lack of Rgt1 restores both glucose-induced expression of the HXT1 gene and glucose repression in a grr1 mutant (102, 261, 356a).

In K. lactis, sensitivity to glucose repression is correlated with the hexose transporter genes present in the yeast genome. Strains containing two transporter genes, KHT1 and KHT2, in tandem are very sensitive to glucose. Natural isolates in which most of KHT2 has been lost by a recombination event between KHT1 and KHT2 which generated the gene RAG1 are moderately repressed by glucose. Mutant rag1 strains, which do not synthesize the low-affinity glucose transporter, are nearly insensitive to glucose repression (372).

Among the earliest mutants isolated as defective in catabolite repression were mutants lacking hexokinase II (398); the mutation was first called hex1 (97) and was renamed hxk2 once the affected gene had been identified. Since there are three hexose kinases in S. cerevisiae, hexokinase I, hexokinase II, and glucokinase (195), and the hxk2 mutants can still phosphorylate glucose efficiently, it was first assumed that hexokinase II had a specific regulatory domain required for glucose repression (96). However, the study of a large number of strains with different mutations in the HXK2 gene showed a parallelism between glucose repression and the residual phosphorylating capacity of the mutated hexokinase (205). Later, strains containing either Hxk1 or Hxk2 or hybrid hexokinases between Hxk1 and Hxk2 were constructed, and a strong correlation was found between the capacity of these strains to phosphorylate glucose or fructose and the repression of maltase and invertase by glucose or fructose (290). None of these results supported the hypothesis of a separate regulatory domain in hexokinase II. Hxk2 is phosphorylated in vivo and can be autophosphorylated in vitro (143, 366). Although Hxk2 is more highly phosphorylated when the glucose concentration in the medium is low (366), this phosphorylation does not seem connected with catabolite repression: while the main residue phosphorylated in vivo is Ser-15 (174), a deletion of the N-terminal 15 amino acids of hexokinase II does not affect glucose repression (204).

Kinetic studies, monitoring SUC2 mRNA levels after the addition of glucose to derepressed yeast cells, have shown that glucose has short-term and long-term effects on SUC2 expression (80, 303). Although it is difficult to compare the results of the two groups, since there are wide differences in the experimental conditions, the sugar kinase requirements appear to be different for the two processes. When glucose is added to fully derepressed cells, the early repression response occurs when any of the hexose kinases is present whereas the late response requires Hxk2 (303). On the other hand, while SUC2 expression is not repressed in an hxk2 mutant grown on glucose, growth on fructose has still a strong repressing effect in such a mutant (80).

At this stage, we can conclude that for a sugar to exert catabolite repression, it should be phosphorylated. The extent to which the structure of the phosphorylating protein plays a role in the repression process is still uncertain. However, the recent observation that Hxk2 can be found in the yeast nucleus suggests that this protein plays a regulatory role distinct from its capacity to phosphorylate sugars (279a).

The situation in other yeasts is not uniform. While it has been reported that mutants of Schwanniomyces occidentalis or Pachysolen tannophilus lacking a hexokinase isoenzyme have a defect in catabolite repression (226, 371), in Candida utilis a strong decrease in hexokinase activity does not suppress glucose repression of -glucosidase (103), and in Aspergillus nidulans, too, hexokinase is not involved in glucose repression (295).

The next question is how extensively glucose should be metabolized to be able to repress transcription. By using strains with a range of reduced amounts of phosphoglucose isomerase, it has been observed that even when the level of phosphoglucose isomerase is less than 2% of that found in a wild-type yeast, both glucose and fructose strongly repress maltase and invertase (290). It has then been concluded that for catabolite repression, glucose signaling does not require any metabolic step in the glycolytic pathway beyond phosphorylation.

Glucose is known to trigger an immediate, transient increase in the intracellular concentration of cAMP in derepressed cells of S. cerevisiae (225, 357, 373); it is less widely appreciated that glucose also has a long-term effect on cAMP levels. The fact that in different yeasts the intracellular levels of cAMP are higher in the presence of glucose and other sugars than under derepressed conditions (100) could suggest a role for cAMP in catabolite repression.

In S. pombe, where adenylate cyclase is dispensable for growth (207), the gluconeogenic enzyme FbPase is no longer repressed by glucose in strains lacking this enzyme or other elements of the glucose-induced adenylate cyclase activating pathway (28, 147). It is not yet established, however, how general this effect of cAMP is in S. pombe. A very recent report indicates that in a strain where the cyr