In S. cerevisiae
Genes repressed by glucose
An important signal for glucose repression is an increase in intracellular cAMP, which allows the activation of PKA. This is shown by the large number of genes for which the transcription rate decreases on activation of Ras2 (Wang et al., 2004). Under artificial conditions, this downregulation of transcription can take place in the absence of glucose, but is dependent on the presence of a protein kinase subject to activation by cAMP. This shows that, for a number of genes, the activation of PKA is sufficient to trigger transcriptional repression. An analysis of the data of Wang et al. (2004) shows, however, that not every gene repressed by glucose responds in the same way to the activation of Ras2 and PKA, as illustrated by some selected cases shown in Table 1. For some genes, cAMP is the main signal and glucose repression in a tpkw bcy1 background is impaired or absent. Representative examples are genes related to the metabolism of trehalose or glycogen (TPS1, TPS2, GPH1), glucose utilization (HXK1, GLK1, TKL2, GPD1), or ubiquitination (COQ3, YJR036). In contrast, other genes do not respond to the activation of Ras2, among them SUC2, one of the model genes used to study repression by glucose. It should be noted that genes involved in the same or related metabolic pathways do not always behave in parallel. For instance, considering genes encoding enzymes from the gluconeogenic pathway or the glyoxylate cycle, it can be observed that activation of Ras2 blocks transcription of FBP1, while its effect on the transcription of PCK1, ICL1 or MLS1 is weak or absent. This is consistent with results showing that external cAMP has a stronger inhibitory effect on derepression of Fbp1 than on derepression of Pck1 or Icl1 (Zaragoza et al., 1999). Among genes involved in respiratory metabolism, CYB2 and FOX2 are strongly repressed when Ras2 is activated, while the response of COX6 is weak and CYC1 shows no response. For most glucose-repressed genes signalling through cAMP is redundant with another glucose-signalling pathway, as shown by the fact that glucose repression takes place normally in a tpkw bcy1 strain. The nature of the corresponding signal has not been yet elucidated, but it does not involve the plasma membrane glucose sensors Snf3 or Rgt2, and depends on glucose metabolism (Belinchón & Gancedo, 2007b). In fact, in mutants where glycolytic flux is strongly reduced, glucose repression is partially relieved (Gamo et al., 1994; Elbing et al., 2004).
Table 1. Elements affecting different glucose repressed genes
|cAMP||Galactose*||GAL10-RAS2Val19 gal1||FBP1, GDH2, HXK1, COQ3, CYB2, TPS2, FOX2||COX6, ADH2, MLS1||ICL1, PCK1, CYC1, SUC2|
|cAMP||External cAMP† (no glucose present)||pde2||FBP1||PCK1, ICL1, GDH2||SUC2|
|cAMP-dependent protein kinase||2% Glucose*||tpkw bcy1||FBP1, ICL1, PCK1, FOX2||TPS1, HXK1, GLK1, TKL2||TPS2, GPH1, GPD1, COQ3, YJR036|
|Snf3, Rgt2, Gpr1||2% Glucose‡||snf3 rgt2 gpr1||FBP1, GDH2, SUC2||–||–|
|Hxk2||2% Glucose‡,§||hxk2||FBP1, ICL1, ADH2, FOX1||GDH2, GLK1, CYC1, CYB2||SUC2, HXK1, GAL1|
|Hxk1, Hxk2||2% Glucose‡,§||hxk1 hxk2||FBP1, ICL1, PCK1||GDH2||GLK1|
|Hxk1, Hxk2, Glk1||2% Glucose‡||hxk1 hxk2 glk1||–||–||FBP1, ICL1, GDH2|
For the repression of some genes such as SUC2, HXK1 or the GAL genes, the glucose-phosphorylating enzyme Hxk2 is specifically required (Zimmermann & Scheel, 1977; Entian, 1980; Rodríguez et al., 2001) (see section on Hxk2). On the other hand, repression of ADH2 or FOX1 is not relieved in an hxk2 mutant (Dombek et al., 1993; Stanway et al., 1995) and relief is only partial for genes such as CYC1, CYB2, GLK1 or GDH2 (Ma & Botstein, 1986; Brown et al., 1995; Rodríguez et al., 2001; Belinchón & Gancedo, 2007b). Some genes such as FBP1, PCK1 or ICL1 are completely repressed by glucose, even in a double mutant hxk1 hxk2 (Yin et al., 1996; Belinchón & Gancedo, 2007b) and, while long-term repression of SUC2 requires Hxk2, SUC2 mRNA levels show a strong, transient, decrease on addition of glucose to an hxk1hxk2 strain (Sanz et al., 1996). A further element modulating repression by glucose is the protein kinase Pho85, because in its absence genes involved in gluconeogenesis and the glyoxylate cycle can be expressed at low concentrations of glucose (Nishizawa et al., 2004).
Genes induced by glucose
A genome-wide screening has shown that, for most of the genes induced by high glucose, the increase in transcript levels triggered by glucose is modest, between 1.5- and 4-fold. This increase is usually rapid, <20 min, but it is sometimes transient, mRNA levels decreasing markedly at 60 min. There are exceptions, as for some genes the degree of induction is higher, about fivefold for RPA12 or RPC53, 20-fold for HXT3, and even 100-fold for HXT1, while for genes such as TRP3, ARG3 or MET14, the kinetics of induction are slower (Wang et al., 2004). The role that different sensing elements play in the induction of different genes is examined in the next paragraphs.
Binding of glucose to the plasma membrane sensors Snf3/Rgt2 triggers the induction of a limited number of genes through a cascade of reactions discussed previously. Most of these genes encode glucose transporters (Özcan & Jonhston, 1999), but a few others have been identified (Kaniak et al., 2004), and among them MIG2 is of special interest, as increased levels of the DNA-binding protein Mig2 contribute to glucose repression (Lutfiyya et al., 1998). The other plasma membrane sensor, Gpr1 (see the corresponding section), is often required for a maximal response to glucose; in its absence, the degree of induction decreases by a factor of two in many cases (Wang et al., 2004). Gpr1 is not needed, however, for the complete induction of HXT1 or pyruvate decarboxylase (Belinchón & Gancedo, 2007a) (Table 2).
Table 2. Elements affecting different genes induced by glucose
|cAMP||Galactose||GAL10-RAS2Val19gal1||THR4, RPC53, RPO31||ARG3, MET14, HXT3||HXT1, HXK2, ENO2|
|cAMP-dependent protein kinase||Glucose*||tpkwbcy1||ENO2, ARG3, THR4, GCN3, RPA43, RPC53||HXT1, HXT3, RPC82||MET14, RPO31|
|Snf3, Rgt2||Glucose*||snf3 rgt2||PDC1||SUC2†||HXT1, HXT3|
|Gpr1||Glucose*||gpr1||PDC1, HXT1||SUC2†|| |
|Hxk2||Glucose*||hxk2||PDC1, HXT3||HXT1, HXT2†, HXT3†||–|
|Hxk1, Hxk2||Glucose*||hxk1 hxk2||SUC2†, PDC1†||PDC1||–|
|Hxk1, Hxk2, Glk1||Glucose*||hxk1 hxk2 glk1||SUC2||HXT1||PDC1|
|Hxk1, Hxk2, Glk1, Gpr1||Glucose*||hxk1 hxk2 glk1 gpr1||–||HXT1||SUC2|
Artificial activation of Ras2, in the absence of glucose (see section on ‘Signalling by intracellular metabolites’), can cause an increase in mRNA levels for many genes; for some genes the extent of induction is similar to that triggered by glucose, while for others induction is low, or even absent for genes such as HXK2 or ENO2 (Table 2). Ras2 activation experiments performed in a strain with a weak PKA insensitive to cAMP (tpkw bcy1 background) have led to the conclusion that Ras signalling takes place exclusively through a cAMP-regulated PKA (Wang et al., 2004). It should be noted, however, that for HXT1 and HXT3 there was at least a 10-fold induction by activated Ras2 in this background (supplementary information in Wang et al., 2004). Although these results have not yet been confirmed by northern analysis or quantitative reverse transcriptase (RT)-PCR, it would be interesting to follow this lead and investigate a potential alternative pathway for Ras signalling.
To investigate whether a cAMP-regulated PKA is not only sufficient, but also necessary, for glucose induction of transcription, glucose was added to a tpkw bcy1 mutant strain growing on glycerol (Wang et al., 2004), because in such a strain PKA is no longer sensitive to the changes in cAMP intracellular concentration triggered by glucose. In a large number of cases there was a strong induction of transcription by glucose (at least 10-fold), thus showing that metabolism of glucose can provide for most genes an induction mechanism independent of an increase in PKA activity. It may be observed that for many genes, mRNA levels were much lower (5–20-fold) in the mutant strain than in the wild type, during growth in glycerol. This suggests that in a wild-type yeast grown in glycerol, the PKA activity is greater than in the mutant and sufficient to cause a partial induction of the corresponding genes. Surprisingly, while galactose does not act as an inducer in the wild-type strain, for some genes it can cause large increases in mRNA levels in the tpkw bcy1 mutant, although the strain lacks Gal1 and is therefore unable to metabolize galactose (Wang et al., 2004). This is a result that remains difficult to interpret.
The role of the glucose phosphorylating enzymes in the induction of transcription by high glucose is not the same for different genes (Table 2). In mutant strains lacking Hxk2, there is a strong decrease in the induction of HXT1 and HXK2, but HXT3 and pyruvate decarboxylase are fully induced (Özcan & Johnston, 1995; Rodríguez et al., 2001; Belinchón & Gancedo, 2007a). While induction of pyruvate decarboxylase is decreased by 50% in a double mutant hxk1 hxk2 and blocked in the triple hxk1 hxk2 glk1 mutant, in this last mutant there is a partial induction of HXT1 and SUC2 is highly induced. The need for glucose phosphorylation to induce pyruvate decarboxylase is in agreement with the observation that this induction is dependent on products of glucose metabolism such as glucose-6P and three-carbon metabolites (Boles & Zimmermann, 1993). It should be noted that, in the absence of glucose phosphorylation, Gpr1 is absolutely required for the induction of SUC2 but is dispensable for that of HXT1 (Belinchón & Gancedo, 2007a).
A low concentration of glucose, <0.1%, is able to induce a number of genes, and in this case also, there are different signalling pathways controlling the induction process (Özcan & Johnston, 1995; Belinchón & Gancedo, 2007a). The induction of HXT2, HXT3 and HXT4, encoding glucose transporters, requires the glucose sensor Snf3 and the expression of these genes decreases two- to fivefold in the absence of Hxk2 (Özcan & Johnston, 1995). The induction of SUC2 has only a partial requirement for Snf3/Rgt2 and does not depend on Hxk2, or Hxk1, but it is slightly impaired in the absence of Gpr1 (Belinchón & Gancedo, 2007a) and considerably decreased in mutants lacking PKA (J.M. Gancedo, unpublished results). Induction of pyruvate decarboxylase by low glucose is little affected by the lack of both Hxk1 and Hxk2 (Belinchón & Gancedo, 2007a).
Proteins activated by glucose
When glucose becomes available to glucose-starved yeast, the activity of a number of enzymes increases. Prominent among them, because it initiates an important signalling pathway, is adenylate cyclase. Activation of adenylate cyclase requires the Ras1/Ras2 proteins, located in the plasma membrane and able to interact directly with the adenylate cyclase complex (Shima et al., 2000). The exact mechanism for activation of the Ras proteins is not known, but it has been established that glucose causes a modest increase in Ras1/2 GTP loading and that this increase requires glucose phosphorylation; the increase is impaired in a strain lacking Cdc25, the GTP/GDP exchange factor for the Ras proteins. Inhibition of the Ira1/Ira2 proteins, which increase the GTPase activity of Ras1/2, contributes to the activation of the Ras proteins triggered by glucose (Colombo et al., 2004). The increased GTP loading of the Ras proteins does not depend on the glucose receptor Gpr1 or on its G-binding protein Gpa2 (Colombo et al., 2004); however, the Gpr1/Gpa2 system is required for full activation of adenylate cyclase (Rolland et al., 2000). Only the GTP-bound, active form of Gpa2 is able to bind adenylate cyclase (Peeters et al., 2006), but this binding is not enough to trigger a full response, because in strains unable to phosphorylate glucose the activation of adenylate cyclase observed, which is dependent on Gpr1 and on a high concentration of glucose, is very weak (Kraakman et al., 1999a). The activation of adenylate cyclase causes an increase in the intracellular concentration of cAMP, which in turn activates PKA. Activated PKA phosphorylates different enzymes such as the neutral trehalase Nth1 (Ortiz et al., 1983; Uno et al., 1983; François & Parrou, 2001) or the 6-phosphofructo-2-kinase Pfk26 (François et al., 1984; Dihazi et al., 2003) and thereby increases their activity.
The activation by glucose of the plasma membrane H+-ATPase is also related to phosphorylation of the protein at different positions, but PKA is not involved in the process, at least directly (Portillo et al., 1991). While phosphorylation of Thr912 from the H+-ATPase appears to mediate an increase in the Vmax of the enzyme (Portillo et al., 1991), phosphorylation of Ser899 would cause a decrease in the Km for ATP (Eraso & Portillo, 1994). Phosphorylation of Ser899 is performed by the protein kinase Ptk2 (Eraso et al., 2006), but the protein kinase responsible for Thr912 phosphorylation has not been identified, in spite of a systematic search using protein kinase mutants (Goossens et al., 2000). The activity of Ptk2 is the same in membranes isolated from cells incubated in the presence or in the absence of glucose and is not affected by glucose itself (Eraso et al., 2006), but it could be modulated by some glucose-derived metabolite as indicated by the fact that activation of the H+-ATPase is strongly reduced in an hxk1 hxk2 mutant and extremely low in a mutant unable to phosphorylate glucose (Belinchón & Gancedo, 2007b). On the other hand, the activation of the H+-ATPase is independent of the glucose sensors Snf3/Rgt2 and Gpr1 (Belinchón & Gancedo, 2007b).
Proteins inactivated by glucose
Glucose also triggers the inactivation of different proteins, a phenomenon called catabolite inactivation (Holzer, 1976). Phosphorylation of FbPase by PKA causes a partial inactivation of the enzyme (Gancedo et al., 1983; Rittenhouse et al., 1987); this process is impaired in a mutant unable to phosphorylate glucose and the impairment is stronger when both the glucose phosphorylating enzymes and the glucose sensor Gpr1 are absent (Belinchón & Gancedo, 2007b). This is consistent with the observation that phosphorylation of FbPase by PKA is strongly activated by fructose-2,6-bisphosphate, a regulatory metabolite that is formed when glucose is metabolized (Gancedo et al., 1983). While partial inactivation of isocitrate lyase by glucose is also dependent on phosphorylation by PKA (Ordiz et al., 1996), there is no information on the protein kinase(s) performing the phosphorylation of the malate dehydrogenase isoenzyme Mdh2 that takes place on glucose addition and causes inactivation of the enzyme (Minard & McAlister-Henn, 1994).
Glucose not only causes the inactivation of metabolic enzymes, but also that of the protein kinase Snf1, which plays a major role in allowing the transcription of glucose-repressed genes. In this case inactivation is due to dephosphorylation of Snf1 by the Glc7-Reg1 complex (Sanz et al., 2000). Glucose may shift the equilibrium between the phosphorylated and nonphosphorylated forms of Snf1, by activating Glc7-Reg1 and/or by inhibiting the protein kinases Sak1, Tos3 and Elm1 that phosphorylate Snf1 (Hong et al., 2003; Nath et al., 2003; Sutherland et al., 2003). However, the mechanism by which glucose modifies Snf1 activity has not yet been unravelled (Hedbacker & Carlson, 2008; Rubenstein et al., 2008).
Proteins degraded by glucose
Glucose also triggers the degradation of a number of enzymes, a process that has been most thoroughly studied for FbPase and Mdh2. Two conflicting views on the mechanism of FbPase degradation have been offered. One of them contemplates a regulated transfer of FbPase to the vacuole and its degradation by vacuolar proteases (Chiang & Schekman, 1991; Shieh et al., 2001); the other one proposes that FbPase is first subject to ubiquitination (Schork et al., 1995) and then degraded by the proteasome (Schork et al., 1994). These views may be reconciled by the observation that, depending on the physiological conditions of the yeast cells, degradation of FbPase, and also of Mdh2, can take place in the vacuole or in the proteasome (Hung et al., 2004). Degradation in the vacuole requires the phosphorylation of glucose but may proceed in the absence of Hxk1 and Hxk2 (Hung et al., 2004; Belinchón & Gancedo, 2007b), while degradation by the proteasome is strictly dependent on Hxk2 (Horak et al., 2002). Reg1 and Grr1 play a role in the degradation of FbPase in the proteasome (Horak et al., 2002), perhaps related to their possible involvement in the ubiquitination process. Reg1 is also required for the transport of FbPase from intermediate vesicles to the vacuoles (Cui et al., 2004). On the other hand, the cAMP-signalling pathway is required for the vacuolar-dependent degradation of FbPase (Hung et al., 2004; Belinchón & Gancedo, 2007b) but not for degradation by the proteasome (Horak et al., 2002; Hung et al., 2004). The transcriptional repressor Rgt1 is required for FbPase degradation in some conditions (Belinchón & Gancedo, 2007b) but not in others (Horak et al., 2002); its specific role, however, has not been elucidated. Degradation of phosphoenolpyruvate carboxykinase appears to proceed by a mechanism similar to that described for FbPase and Mdh2 (Müller et al., 1981; Hämmerle et al., 1998).
Mth1, a regulatory protein involved in the transcription control of the HXT genes, is also degraded on glucose addition as described in the section on Snf3 and Rgt2. This degradation is considerably reduced in a reg1 mutant, and this seems to be related to the fact that the Glc7-Reg1 complex is required for glucose activation of the kinases Yck1/2 (Gadura et al., 2006), as activation of Yck1/2 is an early step in the pathway leading to Mth1 ubiquitination and degradation (Moriya & Johnston, 2004). This role of Reg1 could explain the observation that HXT1 induction is strongly impaired in a reg1 mutant but appears in contradiction with the fact that the HXT2, HXT3 or HXT4 genes are normally induced in this mutant (Özcan & Johnston, 1995).
On glucose addition different kinds of transporters are internalized and degraded, among them the maltose/H+ symporter Mal61 (Riballo et al., 1995), the galactose permease Gal2 (Horak & Wolf, 1997), the monocarboxylate/H+ symporter Jen1 (Paiva et al., 2002) and the glycerol/H+ symporter Stl1 (Ferreira et al., 2005). Because the first step in the internalization of the transporters is their ubiquitination (Horak & Wolf, 1997; Lucero & Lagunas, 1997; Paiva et al., 2002), the corresponding signal is likely to be the binding of glucose to Snf3/Rgt2 and the subsequent activation of Yck1/2 as occurs for Mth1 (Gadura et al., 2006). The fact that Gal2 inactivation is not prevented in an snf3 or in an rgt2 mutant (Horak et al., 2002) may be due to the partial functional redundancy of Snf3 and Rgt2. Degradation of the ubiquitinated transporters is independent of the proteasome and takes place within the vacuole. Although Grr1, the F-box protein of one of the SCF ubiquitin ligase complexes, is required for the degradation of the Gal2 transporter, it does not seem to be required for the ubiquitination of Gal2 itself (Horak & Wolf, 2005). Degradation of the maltose permease Mal61 by glucose requires Rgt2 and Grr1 but it is not impaired in an rgt1 mutant (Jiang et al., 1997).
Other processes controlled by glucose
In addition to the processes already considered, the presence of glucose affects yeast physiology in many other ways. Glucose increases the turnover of a number of mRNA species (Lombardo et al., 1992; Mercado et al., 1994), while it causes a transient stabilization of ribosomal protein mRNAs (Yin et al., 2003). The increased turnover of SDH2 mRNA, encoding the iron–sulfur protein subunit of succinate dehydrogenase, takes place only at high levels of glucose; in contrast, the degradation rates of the FBP1 and PCK1 mRNAs, encoding gluconeogenic enzymes, are already increased by the addition of 0.02% glucose (Yin et al., 2000). Glucose phosphorylation is required to enhance the turnover of SDH2, FBP1, PCK1 or SUC2 mRNAs, but Hxk2 is not specifically needed, except for SDH2 (Cereghino & Scheffler, 1996; Yin et al., 2000). In all cases the effect of glucose is blocked in an reg1 mutant, where the activity of the protein kinase Snf1 is less sensitive to glucose (Sanz, 2007). Although external cAMP (in a pde2 background) can trigger degradation of PCK1 and SDH2 mRNAs, the increased degradation of PCK1 mRNA observed in the presence of 0.02% glucose also occurs in the absence of a Ras-cAMP pathway (Yin et al., 2000). It appears therefore that glucose controls mRNA turnover through alternative, partially redundant signalling pathways. The same applies to the stabilization by glucose of different ribosomal protein encoding mRNAs (Yin et al., 2003). Specifically, the effect of glucose on RPL3 mRNA stability, but not on that of RPS6 mRNA, is retained in the absence of glucose phosphorylation, while the Ras-cAMP pathway is required for stabilization of RPL3 and RPL24 mRNAs but not for that of RPS6 mRNA.
Glucose also affects the translation rate of mRNA. On transfer of glucose-growing yeast to a medium lacking glucose, there is a very rapid (1–2 min) inhibition of translation (Ashe et al., 2000). This effect of carbon source removal is specific for glucose (or fructose), as it does not occur in yeast grown in sucrose, maltose or galactose. Inhibition of translation upon glucose withdrawal does not involve the pathways implicated in the inhibition of translation caused by amino acid starvation (Ashe et al., 2000). Translation is restored rapidly (5 min) by addition of glucose (Ashe et al., 2000) and slowly (hours) by incubation of the yeast cells under aerobic conditions in the absence of a carbon source (Uesono et al., 2004). Although several proteins such as Pat1, Ddh1 or Sbp1 have been identified as destabilizing translation initiation complexes upon glucose removal (Coller & Parker, 2005; Segal et al., 2006), there is no information on the mechanism(s) through which glucose may control their activity. Inhibition of translation after glucose removal is prevented if yeast cells are in a metabolic condition that mimicks to some extent the absence of glucose. This occurs in mutants with a very low PKA activity or in reg1 or hxk2 mutants, provided Snf1 is present (Ashe et al., 2000). Translation is also strongly inhibited when yeast cells are transferred from a medium with glucose to a medium with ethanol, and this inhibition requires the Gat1 protein, involved in signalling the quality of the carbon source through the Tor pathway (Kuruvilla et al., 2001).
The yeast vacuolar H+ATPase (V-ATPase) is a multisubunit complex responsible for the acidification of the organelle. The assembly of this V-ATPase is regulated by glucose, 0.1% glucose increasing the level of assembly from 10 to 25%, while in the presence of 2% glucose, 60% of the subunits are assembled in complexes (Parra & Kane, 1998). Glucose metabolism beyond glucose-6-phosphate is required for triggering V-ATPase assembly but a cAMP signal is neither sufficient nor necessary for the process to take place (Parra & Kane, 1998). Because aldolase is specifically needed for assembly, it has been suggested that aldolase may act in this process as a sensor for the presence of glucose (Lu et al., 2004). V-ATPase assembly is also dependent on the RAVE complex, formed by the Rav1, Rav2 and Skp1 proteins (Seol et al., 2001), but it is not known whether glucose directly controls RAVE.
The growth rate of S. cerevisiae and its cell size depend on the carbon source in the medium and both are highest in glucose-grown cells (Johnston et al., 1979). The effect of glucose on cell size is mediated by the Gpr1-Gpa2 system (Tamaki et al., 2005; Vanoni et al., 2005) and requires Cdc25, the Ras GTP–GDP exchange protein (Belotti et al., 2006). Glucose increases the growth rate by inducing a diverse set of genes: glycolytic and ribosomal protein genes, as well as genes related to the cell division cycle. An important signal for this process is the activation of PKA by cAMP, which increases the transcription rate of Rap1 target genes (Klein & Struhl, 1994) and causes an increase in the level of the cyclin Cln3 (Hall et al., 1998). The increase in Cln3 concentration triggered by glucose is due both to a PKA-dependent stimulation of its synthesis (Hall et al., 1998) and to a PKA-independent activation of CLN3 transcription, involving the transcription factor Azf1 (Newcomb et al., 2002). It has been shown that the induction by glucose of genes such as CLN3, BCK2 and CDC28, which promote progress through the start phase of the cell cycle, does not require signalling through Snf3/Rgt2 or Hxk2 (Newcomb et al., 2003). On the other hand, it does not occur in pfk1 or pfk2 mutants or in the presence of iodoacetate, an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, thus suggesting that some product of glucose metabolism plays a role in the control of growth rate (Newcomb et al., 2003). The transcriptional activator Gcr1 also affects the response to glucose, as it is required to stimulate cellular metabolism and transcription of CLN genes (Willis et al., 2003; Barbara et al., 2007). Although the effects of Gcr1 may be indirect, it appears more likely that Gcr1 acts in concert with the transcription factor Rap1 (Santangelo, 2006).
Glucose blocks the sporulation developmental pathway, initiated in diploid cells by the phosphorylation of the transcriptional activator Ime1. Glucose regulates the process by inhibiting Rim11, a homologue of glycogen synthase kinase 3-β, able to phosphorylate a tyrosine residue, and possibly a serine residue, in Ime1 (Rubin-Bejerano et al., 2004). The glucose signal is transmitted through the cAMP/PKA pathway that mediates, directly or indirectly, the phosphorylation of Ser5, Ser8 and/or Ser12 in Rim11, and the inhibition of its kinase activity (Rubin-Bejerano et al., 2004). An additional element involved in sporulation is the protein kinase Ime2, able to phosphorylate Sic1, an inhibitor of meiotic DNA replication, thus allowing its degradation. Ime2 also stimulates a transcription factor, Ndt80, involved in the G2-M transition (Benjamin et al., 2003). In the presence of glucose Ime2 is destabilized, a process dependent on ubiquitination by the SCFGrr1 ubiquitin ligase (Purnapatre et al., 2005). The mode of action of glucose has not yet been established, but by analogy with the mechanism of regulation of Mth1 (Moriya & Johnston, 2004) it could involve the binding of glucose to the Snf3/Rgt2 receptors.
In some strains of S. cerevisiae, a further developmental pathway may take place, pseudohyphal growth caused by nitrogen starvation (Gimeno et al., 1992; Gancedo, 2001). Under such conditions, glucose (or sucrose) and the Gpr1-Gpa2 system are required to trigger the formation of pseudohyphae (Lorenz et al., 2000). It has been reported recently that pseudohyphal growth can be triggered by maltose or maltotriose through a signalling pathway independent of Gpr1 (Van de Velde & Thevelein, 2008).
In other yeasts
In other yeasts, there is only scattered information on the elements controlling repression by glucose. In S. pombe, repression of fbp1, encoding FbPase, is dependent on the phosphorylation of the transcription factor Rst2 by a cAMP-dependent protein kinase (Higuchi et al., 2002). In mutants lacking this protein kinase, or unable to activate it, fbp1 is no longer sensitive to repression by glucose (Byrne & Hoffman, 1993; Hoffman, 2005b). In contrast, glucose repression of inv1, encoding invertase, is maintained in the absence of the cAMP-signalling pathway (Tanaka et al., 1998). While the lack of Scr1, a repressor protein orthologous of Mig1, relieves repression of inv1, it has no effect on the repression of fbp1 (Tanaka et al., 1998). Nevertheless, in the presence of glucose Scr1 binds to the UAS2 site of the fbp1 promoter, and when glucose is removed Scr1 is exported to the cytoplasm, in a process not regulated by the cAMP pathway, and replaced by the activator Rst2 (Hirota et al., 2006). In K. lactis, glucose repression of different genes is impaired in strains with a decreased rate of glucose transport (Weirich et al., 1997; Milkowski et al., 2001). The fact that, in K. lactis, the strong repression of invertase by glucose is independent of Mig1 (Georis et al., 1999) may be related to the observation that expression of invertase in this yeast does not require Fog2, the orthologue of Snf1 (Goffrini et al., 1996). In P. angusta, glucose fails to repress peroxisomal enzymes in a mutant lacking the orthologue of Snf3, Gcr1 (Stasyk et al., 2004), while repression of alcohol oxidase is only partially relieved in a mig1 mig2 double mutant (Stasyk et al., 2007).
Regarding the signals controlling induction of transcription by glucose in other yeasts, there are few data available. In K. lactis, induction of pyruvate decarboxylase by glucose is abolished in a mutant where the genes KHT1 and KHT2, encoding for glucose transporters, have been deleted (Milkowski et al., 2001). Induction of RAG1, a gene encoding a glucose transporter, requires the RAG5 gene, encoding hexokinase; although the mode of action of Rag5 is not known, it should be noted that induction of RAG1 by galactose is also dependent on Rag5, suggesting that this protein plays a regulatory role independent of its metabolic function (Prior et al., 1993). Induction by glucose of RAG1 and of several glycolytic genes is only partial in the absence of the transcriptional activator Sck1 (Lemaire et al., 2002). Because expression of both RAG1 and SCK1 is blocked by the repression factor Rgt1 (Rolland et al., 2006; Neil et al., 2007), the Rag4/Rag8 pathway that inactivates Rgt1 (Rolland et al., 2006)[cf. section above on Snf3/Rgt2] is essential for the induction by glucose of RAG1 and of other glycolytic genes. In C. albicans, induction by glucose of genes encoding hexose transporters requires Hgt4, an orthologue of the Snf3 glucose sensor (Brown et al., 2006).
In yeast genera different from Saccharomyces, there is little evidence for activation or inactivation of proteins by glucose. An exception is the activation of adenylate cyclase by glucose in S. pombe (Hoffman, 2005a, b). As discussed in the section on Gpr1, it depends on the binding of glucose to Git3, a plasma membrane protein that plays the same role as ScGpr1, and on the trimeric G-protein formed by the Gpa2, Git5 and Git11 subunits. In contrast with the situation in S. cerevisiae, the SpRas1 protein from S. pombe plays no role in glucose/cAMP signalling (Fukui et al., 1986) but the direct activation of adenylate cyclase by Gpa2 requires the Git1 protein (Kao et al., 2006). Git1 lacks sequence homologues in other fungi and its mechanism of action remains to be determined. Regarding catabolite inactivation, only a few systems have been studied and it was found that it does not affect FbPase in S. pombe (Vassarotti et al., 1982) or isocitrate lyase in K. lactis (López et al., 2004). In methylotrophic yeasts, peroxisomal enzymes can be degraded after peroxisomes have been engulfed within the vacuole (Dunn et al., 2005), but this process of pexophagy is induced both by ethanol and by glucose and is therefore not equivalent to the classical catabolite inactivation. There is, nevertheless, a case where degradation is specifically triggered by glucose; this is the glucose-induced microautophagy in Pichia pastoris, which requires the α-subunit of phosphofructokinase (Yuan et al., 1997). The role of phosphofructokinase has not yet been established, but a catalytically inactive phosphofructokinase is still functional for this process. Interestingly, the glucose-induced degradation of peroxisomal enzymes in P. pastoris does not require the Gpr1/Gpa2 proteins, while in S. cerevisiae lack of Gpr1 or Gpa2 suppresses the degradation of the peroxisomal thiolase induced by glucose (Nazarko et al., 2007, 2008).
As observed in S. cerevisiae for pseudohyphal growth, hyphae formation in C. albicans, in a glucose-containing medium, is also dependent on both Gpr1 and Gpa2 (Miwa et al., 2004).