Glucose Repression In Yeast Research Articles

Reverse Evolution of a Classic Gene Network in Yeast Offers a Competitive Advantage

Glucose repression is a central regulatory system in yeast that ensures the utilization of carbon sources in a highly economical manner. The galactose (GAL) metabolism network is stringently regulated by glucose repression in yeast and has been a classic system for studying gene regulation. We show here that a Saccharomyces cerevisiae (S.cerevisiae) lineage in spontaneously fermented milk has swapped all its structural GAL genes (GAL2 and the GAL7-10-1 cluster) with early diverged versions through introgression. The rewired GAL network has abolished glucose repression and conversed from a strictly inducible to a constitutive system through polygenic changes in the regulatory components of the network, including a thymine (T) to cytosine (C) and a guanine (G) to adenine (A) transition in the upstream repressing sequence (URS) sites of GAL1 and GAL4, respectively, which impair Mig1p-mediated repression, loss of function of the repressor Gal80p through a T146I substitution in the protein, and subsequent futility of GAL3. Furthermore, the milk lineage of S.cerevisiae has achieved galactose-utilization rate elevation and galactose-over-glucose preference switch through the duplication of the introgressed GAL2 and the loss of function of the main glucose transporter genes HXT6 and HXT7. In addition, we demonstrate that GAL2 requires GAL7 or GAL10 for its expression, and Gal2p likely requires Gal1p for its transportation function in the milk lineage of S.cerevisiae. We show a clear case of reverse evolution of a classic gene network for ecological adaptation and provide new insights into the regulatory model of the canonical GAL network.

The Hsp70 homolog Ssb and the 14-3-3 protein Bmh1 jointly regulate transcription of glucose repressed genes in Saccharomyces cerevisiae.

Chaperones of the Hsp70 family interact with a multitude of newly synthesized polypeptides and prevent their aggregation. Saccharomyces cerevisiae cells lacking the Hsp70 homolog Ssb suffer from pleiotropic defects, among others a defect in glucose-repression. The highly conserved heterotrimeric kinase SNF1/AMPK (AMP-activated protein kinase) is required for the release from glucose-repression in yeast and is a key regulator of energy balance also in mammalian cells. When glucose is available the phosphatase Glc7 keeps SNF1 in its inactive, dephosphorylated state. Dephosphorylation depends on Reg1, which mediates targeting of Glc7 to its substrate SNF1. Here we show that the defect in glucose-repression in the absence of Ssb is due to the ability of the chaperone to bridge between the SNF1 and Glc7 complexes. Ssb performs this post-translational function in concert with the 14-3-3 protein Bmh, to which Ssb binds via its very C-terminus. Raising the intracellular concentration of Ssb or Bmh enabled Glc7 to dephosphorylate SNF1 even in the absence of Reg1. By that Ssb and Bmh efficiently suppressed transcriptional deregulation of Δreg1 cells. The findings reveal that Ssb and Bmh comprise a new chaperone module, which is involved in the fine tuning of a phosphorylation-dependent switch between respiration and fermentation.

Overexpression of HAP4 in glucose-derepressed yeast cells reveals respiratory control of glucose-regulated genes.

A link between control of respiration and glucose repression in yeast is reported. The HAP4 gene was overexpressed in a Delta mig1 deletion background, generating a mutant in which respiratory function is stimulated and glucose repression is diminished. Although this combination does not result in derepression of genes encoding proteins involved in respiratory function, it nevertheless generates resistance against 2-deoxyglucose and hence contributes to more derepressed growth characteristics. Unexpectedly, overexpression of HAP4 in the Delta mig1 deletion strain causes strong repression of several target genes of the Mig1p repressor. Repression is not restricted to glucose growth conditions and does not require the glucose repressors Mig2p or Hxk2p. It was observed that expression of the SUC2 gene is transiently repressed after glucose is added to respiratory-growing Delta mig1 cells. Additional overexpression of HAP4 prevents release from this novel repressed state. The data presented show that respiratory function controls transcription of genes required for the metabolism of alternative sugars. This respiratory feedback control is suggested to regulate the feed into glycolysis in derepressed conditions.

The Snf1 kinase controls glucose repression in yeast by modulating interactions between the Mig1 repressor and the Cyc8-Tup1 co-repressor.

Among lower eukaryotes, glucose repression is a conserved, widely spread mechanism regulating carbon catabolism. The yeast Snf1 kinase, the Mig1 DNA-binding repressor and the Mig1-interacting co-repressor complex Cyc8(Ssn6)-Tup1 are central components of this pathway. Previous experiments suggested that cytoplasmic translocation of Mig1, upon its phosphorylation by Snf1 in the nucleus, is the key regulatory step for releasing glucose repression. In this report we re-evaluate this model. We establish the coordinated repressive action of Mig1 and Cyc8-Tup1 on GAL1 transcription, but we find that Cyc8-Tup1 is not tethered by Mig1 to the promoter DNA. We demonstrate that both negative regulators occupy GAL1 continuously under either repression or activation conditions, although the majority of the Mig1 is redistributed to the cytoplasm upon activation. We show that Snf1-dependent phosphorylation of Mig1 abolishes interaction with Cyc8-Tup1, and we propose that regulation of this interaction, not the Mig1 cytoplasmic localization, is the molecular switch that controls transcriptional repression/de-repression.

Glucose repression in yeast

The Snf1 protein kinase is a central component of the signaling pathway for glucose repression in yeast. Recent studies have addressed the regulation of Snf1 kinase activity and elucidated mechanisms by which Snf1 controls repression and activation of glucose-repressed genes. Important advances include evidence that Snf1 regulates the localization of the Mig1 repressor and that Snf1 functions at multiple points to control Cat8 and Sip4, the activators of gluconeogenic genes.

Four hydrophobic amino acid residues in the C-terminal effector domain of the yeast Mig1p repressor are important for its in vivo activity.

The Mig1 repressor is a zinc finger protein that mediates glucose repression in yeast. Previous work in Saccharomyces cerevisiae has shown that two domains in Miglp are required for repression: the N-terminal zinc finger region and a C-terminal effector domain. Both domains are also conserved in Miglp homologs from the distantly related yeasts Kluyveromyces lactis and K. marxianus, and these Mig1 proteins can fully replace the endogenous Mig1p in S. cerevisiae. We have now made a detailed analysis of the conserved C-terminal effector domain in Mig1p from K. marxianus, using expression in S. cerevisiae to monitor its function. First, a series of small deletions were made within the effector domain. Second, an alanine scan mutagenesis was carried out across the effector domain. Third, double, triple and quadruple mutants were made that affect certain residues within the effector domain. Our results show that four conserved residues within the effector domain, three leucines and one isoleucine, are particularly important for its function in vivo. The analysis further revealed that while the C-terminal effector domain of KmMig1p mediates a seven- to nine-fold repression of the reporter gene, a five- to sixfold residual effect also exists that is independent of the C-terminal effector domain. Similar results were obtained when the corresponding mutations were made in ScMig1p. Moreover, we found that mutations in these residues affect the interaction between Mig1p and the general corepressor subunit Cyc8p (Ssn6p). Modeling of the C-terminal effector domain using a protein of known structure suggests that it may be folded into an alpha-helix.

Multiple regulatory proteins mediate repression and activation by interaction with the yeast Mig1 binding site.

A major mediator of glucose repression in yeast is Mig1, a zinc finger protein that binds to a GC-rich recognition sequence found upstream of many glucose-repressible genes. Because these Mig1 sites are found upstream of genes under different modes of regulation, we studied regulation of transcription mediated by an isolated Mig1 site placed upstream of a reporter gene under control of UAS(CYC1). The Mig1 site responded appropriately to glucose control and regulatory mutations, including snf1, reg1, cyc8, and tup1, mimicking the behavior of the SUC2 gene. Deletion of the MIG1-coding gene reduced but did not eliminate glucose repression mediated by the Mig1 site. Complete loss of repression was seen in a mig1 mig2 double mutant. When the UAS(CYC1) was replaced by UAS(ADH1) in the reporter plasmid, the Mig1 site activated transcription under most conditions. Mutations of the two Mig1 binding sites in the SUC2 promoter resulted in loss of activation of SUC2 expression. These results suggest the presence of an unknown activator or activators that binds to the Mig1 site. The activator is not any of the proteins previously proposed to bind to this site, including Mig1, Mig2, Msn2, or Msn4. Band shift assays showed that Mig1 is the major protein in yeast cell extracts that binds to the Mig1 site in vitro. This binding is not regulated by glucose or mutations in CYC8 or TUP1.

Molecular cloning of a rac family protein kinase and identification of a serine/threonine protein kinase gene family of Entamoeba histolytica

Eleven Entamoeba histolytica protein-serine/threonine-kinase gene segments were identified using the polymerase chain reaction (PCR) and degenerate oligonucleotide primers to conserved amino acids in subdomains VI and VIII of the catalytic domain of protein-serine/threonine kinases. These ameba gene segments were homologous to myosin light chain kinases, protein kinase C, phosphorylase b kinase, and kinases that regulate glucose repression in yeast and cell growth in mammalian cells. One of these PCR products, which was homologous to the Dictyostelium discoideum protein kinase 2, was used to identify a full-length protein-serine/threonine-kinase gene ( Eh racl) from an E. histolytica genomic library. The open reading frame of Eh racl was 409 amino acids long (encoding a 47-kDa protein) and included an amino terminal segment containing 87 mostly charged and polar amino acids and a 322-amino acid carboxyl terminal segment containing the catalytic domain. The catalytic domain of Eh racl was homologous to the rac family of protein-serine/threonine-kinases, which are related to cAMP-dependent protein kinases and protein kinase Cs. Southern blots of ameba DNA showed that the Eh racl gene was present as a single copy in all strains tested, however pathogenic amebae expressed four times more Eh racl mRNAs than did nonpathogenic amebae. These studies suggest that E. histolytica, a primitive unicellular eukaryote, has a complex protein kinase family.

Identification of a negative regulatory function for steroid receptors.

This report describes the identification of a negative regulator of estrogen and progesterone receptor function. Using a reconstituted estrogen-responsive transcription system in Saccharomyces cerevisiae, we have identified a "repressor function," which when mutated, increases the transcriptional activity of the estrogen and progesterone receptors. In the case of the estrogen receptor this mutation increases the sensitivity of estrogen-mediated activation by at least four orders of magnitude. Analysis of derivatives of the estrogen receptor indicated that this repressor specifically affects the transcription activity of the TAF1 activation domain of the estrogen receptor. The repressor was cloned by complementation and identified as SSN6, a previously described mediator of glucose repression in yeast. Our results indicate that SSN6 is likely to be involved also in the repression of other cellular activators. Interestingly, deletion of the SSN6 protein allows the antiestrogens ICI 164384 and nafoxidine to behave as more potent agonists of estrogen receptor function, while RU486 also becomes a more potent activator of progesterone receptor function. These data suggest that in wild-type cells the role of hormone is twofold: it promotes DNA binding of the receptor and it also induces a conformational change in the receptor which overcomes the effects of this repressor function.

Genetic analysis of serine biosynthesis and glucose repression in yeast

Serine and glycine biosynthesis in yeast proceed by two pathways: a "glycolytic" pathway, using 3-phosphoglycerate, and a "gluconeogenic" pathway, using glyoxylate. We used a mutation in the cat1 gene to abolish the glucose-repressible "gluconeogenic" pathway and re-isolated two mutants, ser1 and ser2, in the "glycolytic" pathway. The ser1 mutation corresponded to phosphoserine transaminase and ser2 to that of phosphoserine phosphatase. Mutagenesis of a ser1 ser2 cat1 triple mutant facilitated the isolation of a mutation in a new gene, SER10. SER10 appears to be part of a pathway which, under normal growth conditions, is less important in serine biosynthesis. The ser1 ser2 ser10 triple mutants were totally serine auxotrophic on glucose media but serine prototrophic during growth on non-fermentable carbon sources. This phenotype was used to select for possible regulatory mutants that synthesize serine by the gluconeogenic pathway even in the presence of glucose, e.g., with a non-glucose repressible glyoxylate cycle. In an alternative approach to isolate such mutants URA3 and TRP1 expression were placed under the control of the glucose-repressible FBP1 (fructose-1,6-bisphosphatase) promoter. Although both systems resulted in strong selection pressure we could not isolate constitutively derepressed mutants. These results indicate that transcription of glucose-repressible gluconeogenic enzymes is mainly dependent on positive regulatory elements.

Glucose repression in the yeast Saccharomyces cerevisiae.

Understanding the mechanism of glucose repression in yeast has proved to be a difficult and challenging problem. A multitude of genes in different pathways are repressed by glucose at the level of transcription. The SUC2 gene, which encodes invertase, is an excellent reporter gene for glucose repression, since its expression is controlled exclusively by this pathway. Genetic analysis has identified numerous regulatory mutations which can either prevent derepression of SUC2 or render its expression insensitive to glucose repression. These mutations allow us to sketch the outlines of a pathway for general glucose repression, which has several key elements: hexokinase PII, encoded by HXK2, which seems to play a role in the sensing of glucose levels; the protein kinase encoded by SNF1, whose activity is required for derepression of many glucose-repressible genes; and the MIG1 repressor protein, which binds to the upstream regions of SUC2 and other glucose-repressible genes. Repression by MIG1 requires the activity of the CYC8 and TUP1 proteins. Glucose repression of other sets of genes seems to be controlled by the general glucose repression pathway acting in concert with other mechanisms. In the cases of the GAL genes and possibly CYC1, regulation is mediated by a cascade in which the general pathway represses expression of a positive transcriptional activator.

Characterization of Hex2 protein, a negative regulatory element necessary for glucose repression in yeast

The regulatory HEX2 gene plays an important role in glucose repression in the yeast Saccharomyces cerevisiae. The hex2 mutants have pleiotropic defects in the regulation of glucose-repressible enzymes, hexokinase PII synthesis and maltose uptake [Entian, K.-D. & Zimmermann, F.K. (1980) Mol. Gen. Genet. 177, 345-350]. The HEX2 gene encodes a protein of 114137 Da, deduced from its DNA sequence. There were no strong similarities to previously known genes. HEX2-lacZ fusions revealed a largely constitutive expression when repressing and non-repressing growth conditions were compared. Cellular fractionation studies indicated a nuclear localization of the Hex2 protein. The hex2 mutation was shown to be allelic to reg1, which releases galactose pathway enzymes from glucose repression [Matsumoto, K., Yoshimatsu, T. & Oshima, Y. (1983) J. Bacteriol. 153, 1405-1414]. Overexpression of HEX2 resulted in a 70% reduction of GAL1 expression under induced growth conditions. Our studies support the view that protein Hex2 is a negative regulatory element in glucose repression which may directly influence transcription, possibly by interaction with transcriptional factors. Deletion experiments identified a central core of Hex2, spanning only 492 out of 1026 amino acid residues, as mainly important for glucose repression. There are two strongly acidic regions within this part of the protein, their possible importance is discussed.

Cloning and characterization of the CYC8 gene mediating glucose repression in yeast

Mutations m the CYC8( = SSN6) gene of Saccharomyces cerevisiae alleviate glucose repression of many glucose-repressible genes. The gene was isolated by screening for complementation of a cyc8 effect on colony morphology. Subclones containing a 5.3-kb SalI- XbaI fragment provided complete complementation. The gene was further localized to 3.5 kb by mapping of the CYC8 mRNA and insertional mutagenesis. Insertion and deletion mutations are viable and produce the same array of phenotypes as point mutations. CYC8 disruptions also had effects on the mating ability and morphology of MATα cells similar to that of tup1 mutations. The nucleotide sequence of a 4866-bp fragment, including CYC8, was determined. One long open reading frame of 966 amino acids predicts a protein of molecular weight 107215. The predicted protein is extremely glutamine-rich, with blocks of 16 and 31 glutamines in tandem at the N and C regions, respectively. The CYC8 gene product lacks consensus sequences for DNA-binding domains, suggesting that its function may be different from classical represser proteins.

Isolation and characterization of the regulatory HEX2 gene necessary for glucose repression in yeast.

The HEX2 gene which is necessary for glucose repression and is involved in the regulation of hexokinase PII synthesis and maltose uptake, has been cloned by complementation of a hex2 mutant, and selection for restored growth on maltose. Glucose repression in the transformants was like that in the wild type. The HEX2 gene was localized within a 2.15 kb fragment. The restriction map was confirmed by Southern hybridization of genomic DNA. Based on 30 tetrads, the linkage between HEX2 and TRP1 was determined as 10 cM. Plasmid integration directed to the genomic site of the cloned gene also gave a similar linkage distance between the amino acid auxotroph plasmid marker and genomic TRP1. Gene disruption of HEX2 yielded nonrepressible transformants with elevated hexokinase PII activity showing inhibition by maltose; this provides clear evidence that the HEX2 gene has been isolated.

Glucose repression and hexokinase isoenzymes in yeast. Isolation and characterization of a modified hexokinase PII isoenzyme.

Hexokinase PII, but not isoenzyme PI, has a unique role in glucose repression in yeasts [Entian, K.-D. (1980) Mol. Gen. Genet. 178, 633-637; Entian, K.-D. and Mecke, D. (1982) J. Biol. Chem. 257, 870-874; Entian, K.-D. and Fröhlich, K.-U. (1984) J. Bacteriol. 158, 29-35]. The number of hexokinase isoenzymes in crude extracts was re-examined by chromatofocusing. In addition to the known isoenzymes PI and PII, a third isoenzyme, PIIM, was detected. The activity of this enzyme was only about 5-10% of that of hexokinase PII and was independent of growth conditions. Experiments with hexokinase transformants and purified hexokinase isoenzymes clearly indicated that the PIIM form is also present in vivo. Fingerprint mapping of purified hexokinases showed that hexokinase PIIM is closely related to PII. Hybridization experiments between totally restricted yeast DNA and the previously isolated PII gene clearly indicated that PIIM is also coded by one of the two known hexokinase genes. No mRNA specific for hexokinase PIIM was detected after hybridization experiments with the previously cloned hexokinase PII gene [Fröhlich et al. (1984) Mol. Gen. Genet. 194, 144-148]. Hexokinase PIIM appears to be derived from hexokinase PII by a posttranslational event. The Km values of each of the purified isoenzymes, PII and PIIM, were identical for glucose, fructose and ATP. Both isoenzymes were strongly inhibited by high physiological concentrations for ATP; such inhibition has not been described previously. The possible role of hexokinase PIIM in glucose repression is discussed.

Mitochondrial DNA and glucose repression in yeast

The amount of mitochondrial DNA, oxygen uptake, cytochrome content and number of mitochondria were examined in Saccharomyces carlsbergensis subjected to various degrees of glucose repression in chemostate cultures. The proportion of mitochondrial DNA varied from 11.5% of nuclear DNA in nonrepressed to 3.3% in repressed cells. Even at 0.2% glucose in the medium a decline in all parameters is observed. From calculations of the number of mitochondrial genomes per mitochondrion a constant number of 3 – 4 appears. Our results contradict recent reports which claim the proportion of yeast mitochondrial DNA to be constant under all degrees of glucose repression.