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The yeast S senses glucose through Snf3 and Rgt2, transmembrane proteins that generate an intracellular signal in response to glucose that leads to inhibition of the Rgt1 transcriptional repressor and consequently to derepression of . cerevisiae genes encoding glucose transporters. Snf3 and Rgt2 are thought to be glucose receptors because they are similar to glucose transporters. In contrast to glucose transporters, they have unusually long C-terminal tails that bind to Mth1 and Std1, paralogous proteins that regulate function of the Rgt1 transcription factor. We show that the C-terminal tail of Rgt2 is not responsible for its inability to transport glucose. To gain insight into how the glucose sensors generate an intracellular signal, we identified HXT mutations that cause constitutive signal generation. Most of the mutations alter evolutionarily-conserved amino acids in the transmembrane spanning regions of Rgt2 that are predicted to be involved in maintaining an outward-facing conformation or to be in the substrate binding site. Our analysis of these mutations suggests they cause Rgt2 to adopt inward-facing or occluded conformations that generate the glucose signal. These results support the idea that Rgt2 and Snf3 are glucose receptors that signal in response to binding of extracellular glucose and inform the basis of their signaling.RGT2 mutations that cause constitutive signal generation. Most of the mutations alter evolutionarily-conserved amino acids in the transmembrane spanning regions of Rgt2 that are predicted to be involved in maintaining an outward-facing conformation or to be in the substrate binding site. Our analysis of these mutations suggests they cause Rgt2 to adopt inward-facing or occluded conformations that generate the glucose signal. These results support the idea that Rgt2 and Snf3 are glucose receptors that signal in response to binding of extracellular glucose and inform the basis of their signaling., (Copyright © 2018 Scharff-Poulsen et al.)

The ability to respond to available nutrients is critical for all living cells. The AMP-activated protein kinase (SNF1 in yeast) is a central regulator of metabolism that is activated when energy is depleted. We found that SNF1 activity in the nucleus is regulated by controlled relocalization of the SNF1 activator Std1 into puncta. This process is regulated by glucose through the activity of the previously uncharacterized protein kinase Vhs1 and its substrate Sip5, a protein of hitherto unknown function. Phosphorylation of Sip5 prevents its association with Std1 and triggers Std1 accretion. Reversible Std1 puncta formation occurs under non-stressful, ambient conditions, creating non-amyloid inclusion bodies at the nuclear-vacuolar junction, and it utilizes cellular chaperones similarly to the aggregation of toxic or misfolded proteins such as those associated with Parkinson's, Alzheimer's, and CJD diseases. Our results reveal a controlled, non-pathological, physiological role of protein aggregation in the regulation of a major metabolic cellular pathway., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The ability to design and engineer organisms demands the ability to predict kinetic responses of novel regulatory networks built from well-characterized biological components. Surprisingly, few validated kinetic models of complex regulatory networks have been derived by combining models of the network components. A major bottleneck in producing such models is the difficulty of measuring in vivo rate constants for components of complex networks. We demonstrate that a simple, genetic approach to measuring rate constants in vivo produces an accurate kinetic model of the complex network that Saccharomyces cerevisiae employs to regulate the expression of genes encoding glucose transporters. The model predicts a transient pulse of transcription of HXT4 (but not HXT2 or HXT3) in response to addition of a small amount of glucose to cells, an outcome we observed experimentally. Our model also provides a mechanistic explanation for this result: HXT2-4 are governed by a type 2, incoherent feed forward regulatory loop involving the Rgt1 and Mig2 transcriptional repressors. The efficiency with which Rgt1 and Mig2 repress expression of each HXT gene determines which of them have a pulse of transcription in response to glucose. Finally, the model correctly predicts how lesions in the feed forward loop change the kinetics of induction of HXT4 expression.

Efficient uptake of glucose is especially critical to Saccharomyces cerevisiae because its preference to ferment this carbon source demands high flux through glycolysis. Glucose induces expression of HXT genes encoding hexose transporters through a signal generated by the Snf3 and Rgt2 glucose sensors that leads to depletion of the transcriptional regulators Mth1 and Std1. These paralogous proteins bind to Rgt1 and enable it to repress expression of HXT genes. Here we show that Mth1 and Std1 can substitute for one another and provide nearly normal regulation of their targets. However, their roles in the glucose signal transduction cascade have diverged significantly. Mth1 is the prominent effector of Rgt1 function because it is the more abundant of the two paralogs under conditions in which both are active (in the absence of glucose). Moreover, the cellular level of Mth1 is quite sensitive to the amount of available glucose. The abundance of Std1 protein, on the other hand, remains essentially constant over a similar range of glucose concentrations. The signal generated by low levels of glucose is amplified by rapid depletion of Mth1; the velocity of this depletion is dependent on both its rate of degradation and swift repression of MTH1 transcription by the Snf1-Mig1 glucose repression pathway. Quantitation of the contributions of Mth1 and Std1 to regulation of HXT expression reveals the unique roles played by each paralog in integrating nutrient availability with metabolic capacity: Mth1 is the primary regulator; Std1 serves to buffer the response to glucose.

S. cerevisiae senses glucose and galactose differently. Glucose is detected through sensors that reside in the cellular plasma membrane. When activated, the sensors initiate a signal-transduction cascade that ultimately inactivates the Rgt1 transcriptional repressor by causing degradation of its corepressors Mth1 and Std1. This results in the expression of many HXT genes encoding glucose transporters. The ensuing flood of glucose into the cell activates Mig1, a transcriptional repressor that mediates "glucose repression" of many genes, including the GAL genes; hence, glucose sensing hinders galactose utilization. Galactose is sensed in the cytoplasm via Gal3. Upon binding galactose (and ATP), Gal3 sequesters the Gal80 protein, thereby emancipating the Gal4 transcriptional activator of the GAL genes. Gal4 also activates expression of MTH1, encoding a corepressor critical for Rgt1 function. Thus, galactose inhibits glucose assimilation by encouraging repression of HXT genes. C. albicans senses glucose similarly to S. cerevisiae but does not sense galactose through Gal3-Gal80-Gal4. Its genome harbors no GAL80 ortholog, and the severely truncated CaGal4 does not regulate CaGAL genes. We present evidence that C. albicans senses galactose with its Hgt4 glucose sensor, a capability that is enabled by transcriptional "rewiring" of its sugar-sensing signal-transduction pathways. We suggest that galactose sensing through Hgt4 is ancestral in fungi.

The Hgt4 protein of Candida albicans (orf19.5962) is orthologous to the Snf3 and Rgt2 glucose sensors of Saccharomyces cerevisiae that govern sugar acquisition by regulating the expression of genes encoding hexose transporters. We found that HGT4 is required for glucose induction of the expression of HGT12, HXT10, and HGT7, which encode apparent hexose transporters in C. albicans. An hgt4Delta mutant is defective for growth on fermentable sugars, which is consistent with the idea that Hgt4 is a sensor of glucose and similar sugars. Hgt4 appears to be sensitive to glucose levels similar to those in human serum ( approximately 5 mM). HGT4 expression is repressed by high levels of glucose, which is consistent with the idea that it encodes a high-affinity sugar sensor. Glucose sensing through Hgt4 affects the yeast-to-hyphal morphological switch of C. albicans cells: hgt4Delta mutants are hypofilamented, and a constitutively signaling form of Hgt4 confers hyperfilamentation of cells. The hgt4Delta mutant is less virulent than wild-type cells in a mouse model of disseminated candidiasis. These results suggest that Hgt4 is a high-affinity glucose sensor that contributes to the virulence of C. albicans.

The yeast Saccharomyces cerevisiae deploys two different types of glucose sensors on its cell surface that operate in distinct glucose signaling pathways: the glucose transporter-like Snf3 and Rgt2 proteins and the Gpr1 receptor that is coupled to Gpa2, a G-protein alpha subunit. The ultimate target of the Snf3/Rgt2 pathway is Rgt1, a transcription factor that regulates expression of HXT genes encoding glucose transporters. We have found that the cAMP-dependent protein kinase A (PKA), which is activated by the Gpr1/Gpa2 glucose-sensing pathway and by a glucose-sensing pathway that works through Ras1 and Ras2, catalyzes phosphorylation of Rgt1 and regulates its function. Rgt1 is phosphorylated in vitro by all three isoforms of PKA, and this requires several serine residues located in PKA consensus sequences within Rgt1. PKA and the consensus serine residues of Rgt1 are required for glucose-induced removal of Rgt1 from the HXT promoters and for induction of HXT expression. Conversely, overexpression of the TPK genes led to constitutive expression of the HXT genes. The PKA consensus phosphorylation sites of Rgt1 are required for an intramolecular interaction that is thought to regulate its DNA binding activity. Thus, two different glucose signal transduction pathways converge on Rgt1 to regulate expression of glucose transporters.

Expression of the HXT genes encoding glucose transporters in the budding yeast Saccharomyces cerevisiae is regulated by two interconnected glucose-signaling pathways: the Snf3/Rgt2-Rgt1 glucose induction pathway and the Snf1-Mig1 glucose repression pathway. The Snf3 and Rgt2 glucose sensors in the membrane generate a signal in the presence of glucose that inhibits the functions of Std1 and Mth1, paralogous proteins that regulate the function of the Rgt1 transcription factor, which binds to the HXT promoters. It is well established that glucose induces degradation of Mth1, but the fate of its paralogue Std1 has been less clear. We present evidence that glucose-induced degradation of Std1 via the SCF(Grr1) ubiquitin-protein ligase and the 26S proteasome is obscured by feedback regulation of STD1 expression. Disappearance of Std1 in response to glucose is accelerated when glucose induction of STD1 expression due to feedback regulation by Rgt1 is prevented. The consequence of relieving feedback regulation of STD1 expression is that reestablishment of repression of HXT1 expression upon removal of glucose is delayed. In contrast, degradation of Mth1 is reinforced by glucose repression of MTH1 expression: disappearance of Mth1 is slowed when glucose repression of MTH1 expression is prevented, and this results in a delay in induction of HXT3 expression in response to glucose. Thus, the cellular levels of Std1 and Mth1, and, as a consequence, the kinetics of induction and repression of HXT gene expression, are closely regulated by interwoven transcriptional and posttranslational controls mediated by two different glucose-sensing pathways.

Because glucose is the principal carbon and energy source for most cells, most organisms have evolved numerous and sophisticated mechanisms for sensing glucose and responding to it appropriately. This is especially apparent in the yeast Saccharomyces cerevisiae, where these regulatory mechanisms determine the distinctive fermentative metabolism of yeast, a lifestyle it shares with many kinds of tumour cells. Because energy generation by fermentation of glucose is inefficient, yeast cells must vigorously metabolize glucose. They do this, in part, by carefully regulating the first, rate-limiting step of glucose utilization: its transport. Yeast cells have learned how to sense the amount of glucose that is available and respond by expressing the most appropriate of its 17 glucose transporters. They do this through a signal transduction pathway that begins at the cell surface with the Snf3 and Rgt2 glucose sensors and ends in the nucleus with the Rgt1 transcription factor that regulates expression of genes encoding glucose transporters. We explain this glucose signal transduction pathway, and describe how it fits into a highly interconnected regulatory network of glucose sensing pathways that probably evolved to ensure rapid and sensitive response of the cell to changing levels of glucose.

Rgt1 is a transcription factor that regulates expression of HXT genes encoding glucose transporters in the yeast Saccharomyces cerevisiae. Rgt1 represses HXT gene expression in the absence of glucose; high levels of glucose cause Rgt1 to activate expression of HXT1. We identified four functional domains of Rgt1. A domain required for transcriptional repression (amino acids 210-250) is required for interaction of Rgt1 with the Ssn6 corepressor. Another region of Rgt1 (320-380) is required for normal transcriptional activation, and sequences flanking this region (310-320 and 400-410) regulate this function. A central region (520-830) and a short sequence adjacent to the zinc cluster DNA-binding domain (80-90) inhibit transcriptional repression when glucose is present. We found that this middle region of Rgt1 physically interacts with the N-terminal portion of the protein that includes the DNA-binding domain. This interaction is inhibited by the Rgt1 regulator Mth1, which binds to Rgt1. Our results suggest that Mth1 promotes transcriptional repression by Rgt1 by binding to it and preventing the intramolecular interaction, probably by preventing phosphorylation of Rgt1, thereby enabling Rgt1 to bind to DNA. Glucose induces HXT1 gene expression by causing Mth1 degradation, allowing Rgt1 phosphorylation, and leading to the intramolecular interaction that inhibits DNA binding of Rgt1.

The yeast Saccharomyces cerevisiae senses glucose through two transmembrane glucose sensors, Snf3 and Rgt2. Extracellular glucose causes these sensors to generate an intracellular signal that induces expression of HXT genes encoding glucose transporters by inhibiting the function of Rgt1, a transcriptional repressor of HXT genes. We present the following evidence that suggests that the glucose sensors are coupled to the membrane-associated protein kinase casein kinase I (Yck1). (i) Overexpression of Yck1 leads to constitutive HXT1 expression; (ii) Yck1 (or its paralogue Yck2) is required for glucose induction of HXT1 expression; (iii) Yck1 interacts with the Rgt2 glucose sensor; and (iv) attaching the C-terminal cytoplasmic tail of Rgt2 to Yck1 results in a constitutive glucose signal. The likely targets of Yck1 in this signal transduction pathway are Mth1 and Std1, which bind to and regulate function of the Rgt1 transcription factor and bind to the C-terminal cytoplasmic domain of glucose sensors. Potential casein kinase I phosphorylation sites in Mth1 and Std1 are required for normal glucose regulation of HXT1 expression, and Yck1 catalyzes phosphorylation of Mth1 and Std1 in vitro. These results support a model of glucose signaling in which glucose binding to the glucose sensors causes them to activate Yck1 in the cell membrane, which then phosphorylates Mth1 and Std1 bound to the cytoplasmic face of the glucose sensors, triggering their degradation and leading to the derepression of HXT gene expression. Our results add nutrient sensing to the growing list of processes in which casein kinase I is involved.

The yeast Saccharomyces cerevisiae senses glucose, its preferred carbon source, through multiple signal transduction pathways. In one pathway, glucose represses the expression of many genes through the Mig1 transcriptional repressor, which is regulated by the Snf1 protein kinase. In another pathway, glucose induces the expression of HXT genes encoding glucose transporters through two glucose sensors on the cell surface that generate an intracellular signal that affects function of the Rgt1 transcription factor. We profiled the yeast transcriptome to determine the range of genes targeted by this second pathway. Candidate target genes were verified by testing for Rgt1 binding to their promoters by chromatin immunoprecipitation and by measuring the regulation of the expression of promoter lacZ fusions. Relatively few genes could be validated as targets of this pathway, suggesting that this pathway is primarily dedicated to regulating the expression of HXT genes. Among the genes regulated by this glucose signaling pathway are several genes involved in the glucose induction and glucose repression pathways. The Snf3/Rgt2-Rgt1 glucose induction pathway contributes to glucose repression by inducing the transcription of MIG2, which encodes a repressor of glucose-repressed genes, and regulates itself by inducing the expression of STD1, which encodes a regulator of the Rgt1 transcription factor. The Snf1-Mig1 glucose repression pathway contributes to glucose induction by repressing the expression of SNF3 and MTH1, which encodes another regulator of Rgt1, and also regulates itself by repressing the transcription of MIG1. Thus, these two glucose signaling pathways are intertwined in a regulatory network that serves to integrate the different glucose signals operating in these two pathways.

Binding of the MIG1 repressor to the glucose-repressible GAL1 and GAL4 promoters was analyzed in vivo by cyclobutane dimer footprinting in two yeast strains that show different glucose repression responses. Mig1p binding to the two promoters in both strains was glucose-induced. In cells subject to rapid and stringent glucose repression (S288c), long-term Mig1p binding in glucose-grown cells was inhibited by the formation of a competing chromatin structure. Under conditions where glucose repression was only partially effective (gal80 - or low glucose), the chromatin structure did not form and long-term Mig1p binding was observed The same long-term binding of Mig1p was seen in cells of a different strain (W303A) that shows only partial glucose repression of the GAL1 promoter. We conclude from these experiments that Mig1p binding to glucose-repressed promoters is glucose-dependent but transient. We suggest that Mig1p functions at an early step in repression, but is not required to maintain the repressed state.

Glucose is the primary fuel for most cells. Because the amount of available glucose can fluctuate wildly, organisms must sense the amount available to them and respond appropriately. Altering gene expression is one of the major effects glucose has on cells. Two different glucose sensing and signal transduction pathways in the yeast S. cerevisiae--one for repression, and one for induction of gene expression--have recently come into focus. What we have learned about these glucose sensing and signaling mechanisms might shed light on how other cells sense and respond to glucose.

Mig1 and Mig2 are proteins with similar zinc fingers that are required for glucose repression of SUC2 expression. Mig1, but not Mig2, is required for repression of some other glucose-repressed genes, including the GAL genes. A second homolog of Mig1, Yer028, appears to be a glucose-dependent transcriptional repressor that binds to the Mig1-binding sites in the SUC2 promoter, but is not involved in glucose repression of SUC2 expression. Despite their functional redundancy, we found several significant differences between Mig1 and Mig2: (1) in the absence of glucose, Mig1, but not Mig2, is inactivated by the Snf1 protein kinase; (2) nuclear localization of Mig1, but not Mig2, is regulated by glucose; (3) expression of MIG1, but not MIG2, is repressed by glucose; and (4) Mig1 and Mig2 bind to similar sites but with different relative affinities. By two approaches, we have identified many genes regulated by Mig1 and Mig2, and confirmed a role for Mig1 and Mig2 in repression of several of them. We found no genes repressed by Yer028. Also, we identified no genes repressed by only Mig1 or Mig2. Thus, Mig1 and Mig2 are redundant glucose repressors of many genes.

How eukaryotic cells sense availability of glucose, their preferred carbon and energy source, is an important, unsolved problem. Bakers' yeast (Saccharomyces cerevisiae) uses two glucose transporter homologs, Snf3 and Rgt2, as glucose sensors that generate a signal for induction of expression of genes encoding hexose transporters (HXT genes). We present evidence that these proteins generate an intracellular glucose signal without transporting glucose. The Snf3 and Rgt2 glucose sensors contain unusually long C-terminal tails that are predicted to be in the cytoplasm. These tails appear to be the signaling domains of Snf3 and Rgt2 because they are necessary for glucose signaling by Snf3 and Rgt2, and transplantation of the C-terminal tail of Snf3 onto the Hxt1 and Hxt2 glucose transporters converts them into glucose sensors that can generate a signal for glucose-induced HXT gene expression. These results support the idea that yeast senses glucose using two modified glucose transporters that serve as glucose receptors.

Glucose represses the transcription of many genes in bakers yeast (Saccharomyces cerevisiae). Mig1 is a Cys2-His2 zinc finger protein that mediates glucose repression of several genes by binding to their promoters and recruiting the general repression complex Ssn6-Tup1. We have found that the subcellular localization of Mig1 is regulated by glucose. Mig1 is imported into the nucleus within minutes after the addition of glucose and is just as rapidly transported back to the cytoplasm when glucose is removed. This regulated nuclear localization requires components of the glucose repression signal transduction pathway. An internal region of the protein separate from the DNA binding and repression domains is necessary and sufficient for glucose-regulated nuclear import and export. Changes in the phosphorylation status of Mig1 are coincident with the changes in its localization, suggesting a possible regulatory role for phosphorylation. Our results suggest that a glucose-regulated nuclear import and/or export mechanism controls the activity of Mig1.

High levels of glucose repress expression of the SUC2 gene in the yeast Saccharomyces cerevisiae. We have discovered that low levels of glucose are required for maximal transcription of SUC2: SUC2 expression is induced about five- to ten-fold in cells growing on low levels of glucose (0.1%) compared to cells growing on galactose or glycerol. Two pieces of evidence suggest that this low-glucose-induced expression is mediated by a repression mechanism that involves an upstream repression site in the SUC2 promoter (URS(SUC2)). First, deletion of the URS(SUC2) results in expression of the SUC2 gene in the absence of glucose, and second the URS(SUC2) mediates a six-fold repression of a reporter gene when inserted into a heterologous promoter. However, this URS(SUC2) mediated repression occurs on all tested carbon sources, suggesting that this URS element acts in concert with all other promoter elements to respond to low concentrations of glucose. This repression requires the general repressor SSn6p. SNF3, which encodes a glucose transporter that appears to be a sensor of low levels of glucose, is also required for low-glucose-induced expression of SUC2.

Transcription of the yeast HXT2 and HXT4 genes, which encode glucose transporters, is induced only by low levels of glucose. This low-glucose-induced expression is mediated by two independent repression mechanisms: in the absence of glucose, transcription of both genes is prevented by Rgt1p, a C6 zinc cluster protein; at high levels of glucose, expression of HXT2 and HXT4 is repressed by Mig1p. Only at low glucose concentrations are both repressors inactive, leading to a 10- to 20-fold induction of gene expression. Mig1p and Rgt1p act directly on HXT2 and HXT4 by binding to their promoters. This transcriptional regulation is physiologically very important to the yeast cell because it causes these glucose transporters to be expressed only in low-glucose media, in which they are required for growth.

Expression of the SUC2 gene in Saccharomyces cerevisiae, which encodes invertase, is repressed about 200-fold by high levels of glucose. Mig1p is a Cys2His2 zinc-finger-containing protein required for glucose repression of SUC2 and several other genes. However, SUC2 expression is still about 13-fold repressed by glucose in a mig1 mutant. We have identified a second repressor, Mig2p, containing zinc fingers very similar to those of Mig1p that is responsible for this remaining glucose repression of SUC2 expression. Overexpression of MIG2 represses SUC2 under nonrepressing conditions, and a LexA-Mig2p fusion represses transcription of a lexO-containing promoter in a glucose-dependent manner, supporting the idea that Mig2p is a glucose-activated repressor. We have shown that Mig2p binds to the Miglp-binding sites in the SUC2 promoter. Even though Mig1p and Mig2p bind to similar sites and share almost identical zinc fingers, they differ in their relative affinities for various Mig1p-binding sites. This could explain our observation that MIG2 appears to have little role in glucose repression of other promoters with MIG1-binding sites.

The HXT genes (HXT1 to HXT4) of the yeast Saccharomyces cerevisiae encode hexose transporters. We found that transcription of these genes is induced 10- to 300-fold by glucose. Analysis of glucose induction of HXT gene expression revealed three types of regulation: (i) induction by glucose independent of sugar concentration (HXT3); (ii) induction by low levels of glucose and repression at high glucose concentrations (HXT2 and HXT4); and (iii) induction only at high glucose concentrations (HXT1). The lack of expression of all four HXT genes in the absence of glucose is due to a repression mechanism that requires Rgt1p and Ssn6p. GRR1 seems to encode a positive regulator of HXT expression, since grr1 mutants are defective in glucose induction of all four HXT genes. Mutations in RGT1 suppress the defect in HXT expression caused by grr1 mutations, leading us to propose that glucose induces HXT expression by activating Grr1p, which inhibits the function of the Rgt1p repressor. HXT1 expression is also induced by high glucose levels through another regulatory mechanism: rgt1 mutants still require high levels of glucose for maximal induction of HXT1 expression. The lack of induction of HXT2 and HXT4 expression on high levels of glucose is due to glucose repression: these genes become induced at high glucose concentrations in glucose repression mutants (hxk2, reg1, ssn6, tup1, or mig1). Components of the glucose repression pathway (Hxk2p and Reg1p) are also required for generation of the high-level glucose induction signal for expression of the HXT1 gene. Thus, the glucose repression and glucose induction mechanisms share some of the same components and may share the same primary signal generated from glucose.

Expression of the GAL genes of Saccharomyces cerevisiae is induced during growth on galactose by a well-characterized regulatory mechanism that relieves Gal80p inhibition of the Gal4p transcriptional activator. Growth on glucose overrides induction by galactose. Glucose repression acts at three levels to reduce GAL1 expression: (i) it reduces the level of functional inducer in the cell; (ii) it lowers cellular levels of Gal4p by repressing GAL4 transcription; and (iii) it inhibits Gal4p function through a repression element in the GAL1 promoter. We quantified the amount of repression provided by each mechanism by assaying strains with none, one, two, or all three of the repression mechanisms intact. In a strain lacking all three repression mechanisms, there was almost no glucose repression of GAL1 expression, suggesting that these are the major, possibly the only, mechanisms of glucose repression acting upon the GAL genes. The mechanism of repression that acts to reduce Gal4p levels in the cell is established slowly (hours after glucose addition), probably because Gal4p is stable. By contrast, the repression acting through the upstream repression sequence element in the GAL1 promoter is established rapidly (within minutes of glucose addition). Thus, these three mechanisms of repression collaborate to repress GAL1 expression rapidly and stringently. The Mig1p repressor is responsible for most (possibly all) of these repression mechanisms. We show that for GAL1 expression, mig1 mutations are epistatic to snf1 mutations, indicating that Mig1p acts after the Snf1p protein kinase in the glucose repression pathway, which suggests that Snf1p is an inhibitor of Mig1p.

We selected and analyzed extragenic suppressors of mutations in four genes--GRR1, REG1, GAL82 and GAL83-required for glucose repression of the GAL genes in the yeast Saccharomyces cerevisiae. The suppressors restore normal or nearly normal glucose repression of GAL1 expression in these glucose repression mutants. Tests of the ability of each suppressor to cross-suppress mutations in the other glucose repression genes revealed two groups of mutually cross-suppressed genes: (1) REG1, GAL82 and GAL83 and (2) GRR1. Mutations of a single gene, SRG1, were found as suppressors of reg1, GAL83-2000 and GAL82-1, suggesting that these three gene products act at a similar point in the glucose repression pathway. Mutations in SRG1 do not cross-suppress grr1 or hxk2 mutations. Conversely, suppressors of grr1 (rgt1) do not cross-suppress any other glucose repression mutation tested. These results, together with what was previously known about these genes, lead us to propose a model for glucose repression in which Grr1p acts early in the glucose repression pathway, perhaps affecting the generation of the signal for glucose repression. We suggest that Reg1p, Gal82p and Gal83p act after the step(s) executed by Grr1p, possibly transmitting the signal for repression to the Snf1p protein kinase.

Glucose (catabolite) repression is mediated by multiple mechanisms that combine to regulate transcription of the GAL genes over at least a thousandfold range. We have determined that this is due predominantly to modest glucose repression (4- to 7-fold) of expression of GAL4, the gene encoding the transcriptional activator of the GAL genes. GAL4 regulation is affected by mutations in several genes previously implicated in the glucose repression pathway; it is not dependent on GAL4 or GAL80 protein function. GAL4 promoter sequences that mediate glucose repression were found to lie downstream of positively acting elements that may be "TATA boxes." Two nearly identical sequences (10/12 base pairs) in this region that may be binding sites for the MIG1 protein were identified as functional glucose-control elements. A 4-base-pair insertion in one of these sites causes constitutive GAL4 synthesis and leads to substantial relief (50-fold) of glucose repression of GAL1 expression. Furthermore, promoter deletions that modestly reduce GAL4 expression, and therefore presumably the amount of GAL4 protein synthesized, cause much greater reductions in GAL1 expression. These results suggest that GAL4 works synergistically to activate GAL1 expression. Thus, glucose repression of GAL1 expression is due largely to a relatively small reduction of GAL4 protein levels caused by reduced GAL4 transcription. This illustrates how modest regulation of a weakly expressed regulatory gene can act as a sensitive genetic switch to produce greatly amplified responses to environmental changes.

Expression of the GAL1 gene in Saccharomyces cerevisiae is strongly repressed by growth on glucose. We show that two sites within the GAL1 promoter mediate glucose repression. First, glucose inhibits transcription activation by GAL4 protein through UASG. Second, a promoter element, termed URSG, confers glucose repression independently of GAL4. We have localized the URSG sequences responsible for glucose repression to an 87-base-pair fragment located between UASG and the TATA box. Promoters deleted for small (20-base-pair) segments that span this sequence are still subject to glucose repression, suggesting that there are multiple sequences within this region that confer repression. Extended deletions across this region confirm that it contains at least two and possibly three URSG elements. To identify the gene products that confer repression upon UASG and URSG, we have analyzed glucose repression mutants and found that the GAL83, REG1, GRR1, and SSN6 genes are required for repression mediated by both UASG and URSG. In contrast, GAL82 and HXK2 are required only for UASG repression. A mutation designated urr1-1 (URSG repression resistant) was identified that specifically relieves URSG repression without affecting UASG repression. In addition, we observed that the SNF1-encoded protein kinase is essential for derepression of both UASG and URSG. We propose that repression of UASG and URSG is mediated by two independent pathways that respond to a common signal generated by growth on glucose.

The promoters of the highly expressed and stringently regulated GAL genes of Saccharomyces cerevisiae, are useful for expressing proteins in this organism. However, two problems complicate their use. First, because growth on glucose causes prolonged repression of GAL expression, cells are most rapidly induced after growth on nonfermentable carbon sources, conditions which usually support poor growth. Second, because the inducer of the GAL genes (galactose) also serves as a carbon source, the level of inducer is continually diminishing during growth of a Gal+ strain, which may lead to reduced GAL expression. To solve the first problem, we have employed strains that carry the reg1-501 mutation, which eliminates glucose repression of GAL expression. This gene has been shown to be located on the right arm of chromosome IV, distal but tightly linked to the TRP1 gene. We demonstrate that expression from GAL promoters is efficiently and rapidly induced in these reg1 strains by the addition of galactose to a culture growing in glucose medium. Levels of galactose as low as 0.02% can be used to obtain a 1500-fold induction of gene expression from GAL promoters in this strain. To surmount the second problem, we have used a gal1 mutant, deficient in the enzyme that catalyzes the first step of galactose utilization. We show that high levels of expression from GAL promoters are achieved rapidly in these mutants, for which galactose is a gratuitous inducer.(ABSTRACT TRUNCATED AT 250 WORDS)