ABSTRACT
We have characterized the monocarboxylate permease family of Saccharomyces cerevisiae comprising five proteins. We could not find any evidence that the monocarboxylate transporter-homologous (Mch) proteins of S. cerevisiae are involved in the uptake or secretion of monocarboxylates such as lactate, pyruvate or acetate across the plasma membrane. A yeast mutant strain deleted for all five MCH genes exhibited no growth defects on monocarboxylic acids as the sole carbon and energy sources. Moreover, the uptake and secretion rates of monocarboxylic acids were indistinguishable from the wild-type strain. Additional deletion of the JEN1 lactate transporter gene completely blocked uptake of lactate and pyruvate. However, uptake of acetate was not even affected after the additional deletion of the gene YHL008c, which had been proposed to code for an acetate transporter. The mch1-5 mutant strain showed strongly reduced biomass yields in aerobic glucose-limited chemostat cultures, pointing to the involvement of Mch transporters in mitochondrial metabolism. Indeed, intracellular localization studies indicated that at least some of the Mch proteins reside in intracellular membranes. However, pyruvate uptake into isolated mitochondria was not affected in the mch1-5 mutant strain. It is concluded that the yeast monocarboxylate transporter-homologous proteins perform other functions than do their mammalian counterparts.
Subject(s)
Carboxylic Acids/metabolism , Fungal Proteins/metabolism , Membrane Transport Proteins/metabolism , Monocarboxylic Acid Transporters/metabolism , Saccharomyces cerevisiae/metabolism , Biomass , Cell Membrane/enzymology , Cell Membrane/metabolism , Fungal Proteins/genetics , Gene Expression Regulation, Fungal/physiology , Genes, Fungal , Glucose/metabolism , Kinetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Membrane Transport Proteins/genetics , Mitochondria/metabolism , Mitochondria/physiology , Monocarboxylic Acid Transporters/genetics , Mutagenesis , Open Reading Frames , Oxygen Consumption , Pyruvic Acid/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , beta-Galactosidase/analysisABSTRACT
The yeast Snf3 protein has been described to function as a sensor for low concentrations of extracellular glucose. We have found that Snf3 is able to transduce a signal in the complete absence of extracellular glucose. High basal activity of the HXT7 promoter during growth on ethanol required Snf3 as well as other components of the signalling pathway activated by Snf3. Moreover, the C-terminal domain of Snf3 was sufficient to complement the role of Snf3 in this regulation. As the C-terminal tail of Snf3 interacted with other components at the plasma membrane independent of the carbon source, our data suggest that Snf3 is involved in signalling complexes which can be activated by other signals than extracellular glucose.
Subject(s)
Glucose/metabolism , Membrane Proteins/metabolism , Monosaccharide Transport Proteins/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Signal Transduction , Base Sequence , Biological Transport , DNA Primers , Transcription, GeneticABSTRACT
In the respirofermentative yeast Kluyveromyces lactis, only a single genetic locus encodes glucose transporters that can support fermentative growth. This locus is polymorphic in wild-type isolates carrying either KHT1 and KHT2, two tandemly arranged HXT-like genes, or RAG1, a low-affinity transporter gene that arose by recombination between KHT1 and KHT2. Here we show that KHT1 is a glucose-induced gene encoding a low-affinity transporter very similar to Rag1p. Kht2p has a lower K(m) (3.7 mM) and a more complex regulation. Transcription is high in the absence of glucose, further induced by low glucose concentrations, and repressed at higher glucose concentrations. The response of KHT1 and KHT2 gene regulation to high but not to low concentrations of glucose depends on glucose transport. The function of either Kht1p or Kht2p is sufficient to mediate the characteristic response to high glucose, which is impaired in a kht1 kht2 deletion mutant. Thus, the KHT genes are subject to mutual feedback regulation. Moreover, glucose repression of the endogenous beta-galactosidase (LAC4) promoter and glucose induction of pyruvate decarboxylase were abolished in the kht1 kht2 mutant. These phenotypes could be partially restored by HXT gene family members from Saccharomyces cerevisiae. The results indicate that the specific responses to high but not to low glucose concentrations require a high rate of glucose uptake.
Subject(s)
Fungal Proteins , Gene Expression Regulation, Bacterial , Glucose/metabolism , Kluyveromyces/metabolism , Monosaccharide Transport Proteins/genetics , Transcription, Genetic , Culture Media , Kinetics , Kluyveromyces/genetics , Kluyveromyces/growth & development , Monosaccharide Transport Proteins/metabolism , MutationABSTRACT
Glucose-induced cAMP signalling in Saccharomyces cerevisiae requires extracellular glucose detection via the Gpr1-Gpa2 G-protein coupled receptor system and intracellular glucose-sensing that depends on glucose uptake and phosphorylation. The glucose uptake requirement can be fulfilled by any glucose carrier including the Gal2 permease or by intracellular hydrolysis of maltose. Hence, the glucose carriers do not seem to play a regulatory role in cAMP signalling. Also the glucose carrier homologues, Snf3 and Rgt2, are not required for glucose-induced cAMP synthesis. Although no further metabolism beyond glucose phosphorylation is required, neither Glu6P nor ATP appears to act as metabolic trigger for cAMP signalling. This indicates that a regulatory function may be associated with the hexose kinases. Consistently, intracellular acidification, another known trigger of cAMP synthesis, can bypass the glucose uptake requirement but not the absence of a functional hexose kinase. This may indicate that intracellular acidification can boost a downstream effect that amplifies the residual signal transmitted via the hexose kinases when glucose uptake is too low.
Subject(s)
Cyclic AMP/metabolism , GTP-Binding Protein alpha Subunits , Gene Expression Regulation, Fungal , Glucose/metabolism , Receptors, G-Protein-Coupled , Saccharomyces cerevisiae/metabolism , Signal Transduction , Biological Transport , Fungal Proteins/metabolism , Heterotrimeric GTP-Binding Proteins/metabolism , Hexokinase/metabolism , Hydrogen-Ion Concentration , Phosphorylation , Receptors, Cell Surface/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
In Saccharomyces cerevisiae, glucose activation of cAMP synthesis requires both the presence of the G-protein-coupled receptor (GPCR) system, Gpr1-Gpa2, and uptake and phosphorylation of the sugar. In a hxt-null strain that lacks all physiologically important glucose carriers, glucose transport as well as glucose-induced cAMP signalling can be restored by constitutive expression of the galactose permease. Hence, the glucose transporters do not seem to have a regulatory function but are only required for glucose uptake. We established a system in which the GPCR-dependent glucose-sensing process is separated from the glucose phosphorylation process. It is based on the specific transport and hydrolysis of maltose providing intracellular glucose in the absence of glucose transport. Preaddition of a low concentration (0.7 mM) of maltose to derepressed hxt-null cells and subsequent addition of glucose restored the glucose-induced cAMP signalling, although there was no glucose uptake. Addition of a low concentration of maltose itself does not increase the cAMP level but enhances Glu6P and apparently fulfils the intracellular glucose phosphorylation requirement for activation of the cAMP pathway by extracellular glucose. This system enabled us to analyse the affinity and specificity of the GPCR system for fermentable sugars. Gpr1 displayed a very low affinity for glucose (apparent Ka = 75 mM) and responded specifically to extracellular alpha and beta D-glucose and sucrose, but not to fructose, mannose or any glucose analogues tested. The presence of the constitutively active Gpa2val132 allele in a wild-type strain bypassed the requirement for Gpr1 and increased the low cAMP signal induced by fructose and by low glucose up to the same intensity as the high glucose signal. Therefore, the low cAMP increases observed with fructose and low glucose in wild-type cells result only from the low sensitivity of the Gpr1-Gpa2 system and not from the intracellular sugar kinase-dependent process. In conclusion, we have shown that the two essential requirements for glucose-induced activation of cAMP synthesis can be fulfilled separately: an extracellular glucose detection process dependent on Gpr1 and an intracellular sugar-sensing process requiring the hexose kinases.
Subject(s)
Cyclic AMP/metabolism , Fungal Proteins/metabolism , GTP-Binding Protein alpha Subunits , Glucose/metabolism , Heterotrimeric GTP-Binding Proteins/metabolism , Hexokinase/metabolism , Receptors, Cell Surface/metabolism , Receptors, G-Protein-Coupled , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Signal Transduction , Amino Acid Substitution , Biological Transport , Carbohydrate Metabolism , Extracellular Space , Fructose/metabolism , Fructose/pharmacology , Glucose/pharmacology , Glucose-6-Phosphate/metabolism , Hexoses/metabolism , Monosaccharide Transport Proteins/metabolism , Phosphorylation , Saccharomyces cerevisiae/drug effectsABSTRACT
Anaerobic xylulose fermentation was compared in strains of Zygosaccharomyces and Saccharomyces cerevisiae, mutants and wild-type strains to identify host-strain background and genetic modifications beneficial to xylose fermentation. Overexpression of the gene (XKS1) for the pentose phosphate pathway (PPP) enzyme xylulokinase (XK) increased the ethanol yield by almost 85% and resulted in ethanol yields [0.61 C-mmol (C-mmol consumed xylulose)(-1)] that were close to the theoretical yield [0.67 C-mmol (C-mmol consumed xylulose)(-1)]. Likewise, deletion of gluconate 6-phosphate dehydrogenase (gnd1delta) in the PPP and deletion of trehalose 6-phosphate synthase (tps1delta) together with trehalose 6-phosphate phosphatase (tps2delta) increased the ethanol yield by 30% and 20%, respectively. Strains deleted in the promoter of the phosphoglucose isomerase gene (PGI1) - resulting in reduced enzyme activities - increased the ethanol yield by 15%. Deletion of ribulose 5-phosphate (rpe1delta) in the PPP abolished ethanol formation completely. Among non-transformed and parental strains S. cerevisiae ENY. WA-1A exhibited the highest ethanol yield, 0.47 C-mmol (C-mmol consumed xylulose)(-1). Other non-transformed strains produced mainly arabinitol or xylitol from xylulose under anaerobic conditions. Contrary to previous reports S. cerevisiae T23D and CBS 8066 were not isogenic with respect to pentose metabolism. Whereas, CBS 8066 has been reported to have a high ethanol yield on xylulose, 0.46 C-mmol (C-mmol consumed xylulose)(-1) (Yu et al. 1995), T23D only formed ethanol with a yield of 0.24 C-mmol (C-mmol consumed xylulose)(-1). Strains producing arabinitol did not produce xylitol and vice versa. However, overexpression of XKS1 shifted polyol formation from xylitol to arabinitol.
Subject(s)
Saccharomyces cerevisiae/metabolism , Xylulose/metabolism , Zygosaccharomyces/metabolism , Anaerobiosis , Fermentation , Glucose-6-Phosphate Isomerase/genetics , Glucose-6-Phosphate Isomerase/metabolism , Glucosyltransferases/genetics , Glucosyltransferases/metabolism , Glycolysis/genetics , Mutation , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Trehalose/metabolism , Zygosaccharomyces/enzymology , Zygosaccharomyces/geneticsABSTRACT
Saccharomyces cerevisiae HTR1 mutants are severely impaired in the uptake of glucose. We have cloned dominant HTR1 mutant alleles and show that they encode mutant forms of the Mth1 protein. Mth1 is shown to be involved in carbon source-dependent regulation of its own, invertase and hexose transporter gene expression. The mutant forms block the transduction of the Snf3- and Rgt2-mediated glucose signals upstream of the Rgt1 transcriptional regulator.
Subject(s)
Cell Cycle Proteins , Fungal Proteins/genetics , Glucose/physiology , Membrane Proteins/physiology , Monosaccharide Transport Proteins/physiology , Repressor Proteins , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Signal Transduction/genetics , Adaptor Proteins, Signal Transducing , Alleles , Cloning, Molecular , Gene Expression Regulation, Fungal , Glycoside Hydrolases/metabolism , Monosaccharide Transport Proteins/genetics , Mutation , RNA-Binding Proteins , Saccharomyces cerevisiae/physiology , beta-FructofuranosidaseABSTRACT
We have identified and characterized three genes, SUT1, SUT2 and SUT3, that encode glucose transporters of the yeast Pichia stipitis. When expressed in a Saccharomyces cerevisiae hxt null mutant strain that is unable to take up monosaccharides, all three proteins restored growth on glucose. Sequencing of the genes revealed open reading frames coding for 553 amino acids in the case of SUT1, and for 550 amino acids in the case of SUT2 and of SUT3. The derived protein sequences are closely related to one another, and show distinct sequence similarities to the S. cerevisiae hexose transporter family and to monosaccharide transporters of other organisms. The Sut2 and Sut3 proteins are nearly identical and differ only in one amino acid. Determination of substrate specificities and kinetic parameters of the individual Sut proteins expressed in a S. cerevisiae hxt1-7 mutant revealed Sut1, Sut2 and Sut3 as glucose transporters with K(m) values in the millimolar range. The proteins were also able to transport xylose and other monosaccharides, but with a considerably lower affinity. In P. stipitis, transcription of SUT1 was strongly induced by glucose and was independent of the oxygen supply. In contrast, SUT2 and SUT3 were only expressed under aerobic conditions, but independent of the carbon source. Cells disrupted for the SUT1 gene did not show any obvious growth phenotype, however low-affinity glucose uptake was lost. Further investigations suggest that the Sut proteins constitute a subfamily of glucose transporters in P. stipitis, and that other and probably unrelated proteins exist additionally mediating high-affinity glucose and xylose uptake.
Subject(s)
Fungal Proteins , Genes, Fungal , Monosaccharide Transport Proteins/genetics , Pichia/genetics , Amino Acid Sequence , Blotting, Northern , Blotting, Southern , Cloning, Molecular , Gene Deletion , Gene Expression Regulation, Fungal , Genomic Library , Kinetics , Molecular Sequence Data , Restriction Mapping , Saccharomyces cerevisiae/genetics , Sequence Homology, Amino Acid , beta-Galactosidase/analysisABSTRACT
The SSY1 gene of Saccharomyces cerevisiae encodes a member of a large family of amino acid permeases. Compared to the 17 other proteins of this family, however, Ssy1p displays unusual structural features reminiscent of those distinguishing the Snf3p and Rgt2p glucose sensors from the other proteins of the sugar transporter family. We show here that SSY1 is required for transcriptional induction, in response to multiple amino acids, of the AGP1 gene encoding a low-affinity, broad-specificity amino acid permease. Total noninduction of the AGP1 gene in the ssy1Delta mutant is not due to impaired incorporation of inducing amino acids. Conversely, AGP1 is strongly induced by tryptophan in a mutant strain largely deficient in tryptophan uptake, but it remains unexpressed in a mutant that accumulates high levels of tryptophan endogenously. Induction of AGP1 requires Uga35p(Dal81p/DurLp), a transcription factor of the Cys6-Zn2 family previously shown to participate in several nitrogen induction pathways. Induction of AGP1 by amino acids also requires Grr1p, the F-box protein of the SCFGrr1 ubiquitin-protein ligase complex also required for transduction of the glucose signal generated by the Snf3p and Rgt2p glucose sensors. Systematic analysis of amino acid permease genes showed that Ssy1p is involved in transcriptional induction of at least five genes in addition to AGP1. Our results show that the amino acid permease homologue Ssy1p is a sensor of external amino acids, coupling availability of amino acids to transcriptional events. The essential role of Grr1p in this amino acid signaling pathway lends further support to the hypothesis that this protein participates in integrating nutrient availability with the cell cycle.
Subject(s)
Amino Acids/metabolism , Carrier Proteins , Fungal Proteins/metabolism , Genes, Fungal , Membrane Transport Proteins/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Ubiquitin-Protein Ligases , Amino Acid Sequence , Amino Acid Transport Systems , Amino Acids/pharmacology , Base Sequence , Cell Cycle , DNA Primers/genetics , F-Box Proteins , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Fungal/drug effects , Molecular Sequence Data , Mutation , Saccharomyces cerevisiae/drug effects , Sequence Homology, Amino Acid , Signal Transduction , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
The hexose transporter family of Saccharomyces cerevisiae comprises 18 proteins (Hxt1-17, Gal2). Here, we demonstrate that all these proteins, except Hxt12, and additionally three members of the maltose transporter family (Agt1, Ydl247, Yjr160) are able to transport hexoses. In a yeast strain deleted for HXT1-17, GAL2, AGT1, YDL247w and YJR160c, glucose consumption and transport activity were completely abolished. However, as additional deletion of the glucose sensor gene SNF3 partially restored growth on hexoses, our data indicate the existence of even more proteins able to transport hexoses in yeast.
Subject(s)
Gene Deletion , Hexoses/metabolism , Monosaccharide Transport Proteins/genetics , Saccharomyces cerevisiae/metabolism , Base Sequence , Cloning, Molecular , DNA Primers , Monosaccharide Transport Proteins/metabolism , Saccharomyces cerevisiae/geneticsABSTRACT
Pyruvate, a precursor for several amino acids, can be synthesized from phosphoenolpyruvate by pyruvate kinase. Nevertheless, pyk1 pyk2 mutants of Saccharomyces cerevisiae devoid of pyruvate kinase activity grew normally on ethanol in defined media, indicating the presence of an alternative route for pyruvate synthesis. A candidate for this role is malic enzyme, which catalyzes the oxidative decarboxylation of malate to pyruvate. Disruption of open reading frame YKL029c, which is homologous to malic enzyme genes from other organisms, abolished malic enzyme activity in extracts of glucose-grown cells. Conversely, overexpression of YKL029c/MAE1 from the MET25 promoter resulted in an up to 33-fold increase of malic enzyme activity. Growth studies with mutants demonstrated that presence of either Pyk1p or Mae1p is required for growth on ethanol. Mutants lacking both enzymes could be rescued by addition of alanine or pyruvate to ethanol cultures. Disruption of MAE1 alone did not result in a clear phenotype. Regulation of MAE1 was studied by determining enzyme activities and MAE1 mRNA levels in wild-type cultures and by measuring beta-galactosidase activities in a strain carrying a MAE1::lacZ fusion. Both in shake flask cultures and in carbon-limited chemostat cultures, MAE1 was constitutively expressed. A three- to fourfold induction was observed during anaerobic growth on glucose. Subcellular fractionation experiments indicated that malic enzyme in S. cerevisiae is a mitochondrial enzyme. Its regulation and localization suggest a role in the provision of intramitochondrial NADPH or pyruvate under anaerobic growth conditions. However, since null mutants could still grow anaerobically, this function is apparently not essential.
Subject(s)
Genes, Fungal/genetics , Malate Dehydrogenase/genetics , Mitochondria/enzymology , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Ethanol/metabolism , Gene Expression Regulation, Fungal , Malate Dehydrogenase/metabolism , Mutation , Pyruvate Kinase/genetics , RNA, Fungal/analysis , RNA, Messenger/analysis , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/growth & development , Sequence Homology, Amino AcidABSTRACT
After addition of high concentrations of glucose, rates of high-affinity glucose uptake in Saccharomyces cerevisiae decrease rapidly. We found that the high-affinity hexose transporters Hxt6 and Hxt7 are subject to glucose-induced proteolytic degradation (catabolite inactivation). Degradation occurs in the vacuole, as Hxt6/7 were stabilized in proteinase A-deficient mutant cells. Degradation was independent of the proteasome. The half-life of Hxt6 and Hxt7 strongly increased in end4, ren1 and act1 mutant strains, indicating that the proteins are delivered to the vacuole by endocytosis. Moreover, both proteins were also stabilized in mutants defective in ubiquitination. However, the initial signal that triggers catabolite inactivation is not relayed via the glucose sensors Snf3 and Rgt2.
Subject(s)
Endocytosis , Fungal Proteins/antagonists & inhibitors , Monosaccharide Transport Proteins/antagonists & inhibitors , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Vacuoles/metabolism , Base Sequence , DNA Primers , Glucose/metabolism , Hydrolysis , Membrane Proteins/metabolism , Monosaccharide Transport Proteins/metabolismABSTRACT
Transport across the plasma membrane is the first, obligatory step of hexose utilization. In yeast cells the uptake of hexoses is mediated by a large family of related transporter proteins. In baker's yeast Saccharomyces cerevisiae the genes of 20 different hexose transporter-related proteins have been identified. Six of these transmembrane proteins mediate the metabolically relevant uptake of glucose, fructose and mannose for growth, two others catalyze the transport of only small amounts of these sugars, one protein is a galactose transporter but also able to transport glucose, two transporters act as glucose sensors, two others are involved in the pleiotropic drug resistance process, and the functions of the remaining hexose transporter-related proteins are not yet known. The catabolic hexose transporters exhibit different affinities for their substrates, and expression of their corresponding genes is controlled by the glucose sensors according to the availability of carbon sources. In contrast, milk yeast Kluyveromyces lactis contains only a few different hexose transporters. Genes of other monosaccharide transporter-related proteins have been found in fission yeast Schizosaccharomyces pombe and in the xylose-fermenting yeast Pichia stipitis. However, the molecular genetics of hexose transport in many other yeasts remains to be established. The further characterization of this multigene family of hexose transporters should help to elucidate the role of transport in yeast sugar metabolism.
Subject(s)
Calcium-Binding Proteins , Hexoses/metabolism , Monosaccharide Transport Proteins/physiology , Periplasmic Binding Proteins , Yeasts/metabolism , Biological Transport , Monosaccharide Transport Proteins/genetics , Saccharomyces cerevisiae/metabolismABSTRACT
We have characterized the gene YOR347c of Saccharomyces cerevisiae and shown that it encodes a second functional pyruvate kinase isoenzyme, Pyk2p. Overexpression of the YOR347c/PYK2 gene on a multicopy vector restored growth on glucose of a yeast pyruvate kinase 1 (pyk1) mutant strain and could completely substitute for the PYK1-encoded enzymatic activity. PYK2 gene expression is subject to glucose repression. A pyk2 deletion mutant had no obvious growth phenotypes under various conditions, but the growth defects of a pyk1 pyk2 double-deletion strain were even more pronounced than those of a pyk1 single-mutation strain. Pyk2p is active without fructose-1,6-bisphosphate. However, overexpression of PYK2 during growth on ethanol did not cause any of the deleterious effects expected from a futile cycling between pyruvate and phosphoenolpyruvate. The results indicate that the PYK2-encoded pyruvate kinase may be used under conditions of very low glycolytic flux.
Subject(s)
Fructosediphosphates/metabolism , Pyruvate Kinase/genetics , Pyruvate Kinase/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Allosteric Regulation , Amino Acid Sequence , Animals , Base Sequence , Ethanol/metabolism , Gene Deletion , Genes, Fungal , Genotype , Glucose/metabolism , Glycolysis , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Kidney/enzymology , Kinetics , Liver/enzymology , Molecular Sequence Data , Muscle, Skeletal/enzymology , Oligodeoxyribonucleotides , Pyruvate Kinase/chemistry , Rats , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/physiology , Sequence Homology, Amino Acid , Substrate Specificity , beta-Galactosidase/metabolismABSTRACT
In Saccharomyces cerevisiae, there are a large number of genes (HXT1-HXT17/SNF3/RGT2) encoding putative hexose transporters which, together with a galactose permease gene (GAL2), belong to a superfamily of monosaccharide facilitator genes. We have performed a systematic analysis of the HXT1-7 and GAL2 genes and their function in hexose transport. Glucose uptake was below the detection level in the hxt1-7 null strain growing on maltose. Determination of the kinetic parameters of individual hexose transporter-related proteins (Hxtp) expressed in the hxt null background revealed Hxt1p and Hxt3p as low-affinity transporters (Km(glucose) = 50-100 mM), Hxt2p and Hxt4p as moderately low in affinity (Km(glucose) about 10 mM), and Hxt6p, Hxt7p as well as Gal2p as high-affinity transporters (Km(glucosse) = 1-2 mM). However, Hxt2p kinetics in cells grown on low glucose concentrations showed a high-affinity (Km = 1.5 mM) and a low-affinity component (Km = 60 mM). Furthermore, we investigated the involvement of glucose transport in glucose signalling. Glucose repression of MAL2, SUC2 and GAL1 was not dependent on a specific transporter but, instead, the strength of the repression signal was dependent on the level of expression, the properties of the individual transporters and the kind of sugar transported. The strength of the glucose repression signal correlated with the glucose consumption rates in the different strains, indicating that glucose transport limits the provision of a triggering signal rather then being directly involved in the triggering mechanism.
Subject(s)
Fungal Proteins/genetics , Glucose/physiology , Monosaccharide Transport Proteins/genetics , Saccharomyces cerevisiae/physiology , Enzyme Induction , Fungal Proteins/metabolism , Galactokinase/biosynthesis , Galactokinase/genetics , Gene Expression Regulation, Fungal , Glycoside Hydrolases/metabolism , Kinetics , Monosaccharide Transport Proteins/metabolism , Saccharomyces cerevisiae/genetics , Signal Transduction , Transcription, Genetic , alpha-Glucosidases/metabolism , beta-FructofuranosidaseABSTRACT
In this work we used in vitro mutagenesis to modify the allosteric properties of the heterooctameric yeast phosphofructokinase. Specifically, we identified two amino acids involved in the binding of the most potent allosteric activator fructose 2,6-bisphosphate. Thus, Ser724 was replaced by an aspartate and His859 by a serine in each of the enzyme subunits. Whereas the substitutions had no drastic effects when introduced only in one of the two types of subunits, kinetic parameters were modified when both subunits carried the mutation. Thus, the enzyme with His859 --> Ser showed an increase in Ka for binding of the activator, whereas the one with Ser724 --> Asp failed to react to the addition of fructose 2, 6-bisphosphate, at all. The enzymes still responded to other allosteric activators, such as AMP. Stabilities of the mutant subunits were not significantly altered in vivo, as judged from Western blot analysis. Phenotypically, strains expressing the mutant PFK genes showed a pronounced effect on the level of intermediary metabolites after growth on glucose. Mutants not responding to the activator at all (Ser724 --> Asp) also displayed higher generation times on glucose medium. This could be suppressed by increasing the gene dosage of the mutant alleles. These results indicate that fructose 2,6-bisphosphate through its activation of phosphofructokinase plays an important role in regulation of the glycolytic flux.
Subject(s)
Fructosediphosphates/pharmacology , Phosphofructokinase-1/metabolism , Saccharomyces cerevisiae/enzymology , Allosteric Regulation , Allosteric Site , Amino Acid Sequence , Aspartic Acid , Base Sequence , DNA Primers , Enzyme Activation , Glycolysis , Histidine , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligodeoxyribonucleotides , Phosphofructokinase-1/biosynthesis , Phosphofructokinase-1/chemistry , Point Mutation , Polymerase Chain Reaction , Promoter Regions, Genetic , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , SerineABSTRACT
The complete nucleotide sequence of Saccharomyces cerevisiae chromosome X (745 442 bp) reveals a total of 379 open reading frames (ORFs), the coding region covering approximately 75% of the entire sequence. One hundred and eighteen ORFs (31%) correspond to genes previously identified in S. cerevisiae. All other ORFs represent novel putative yeast genes, whose function will have to be determined experimentally. However, 57 of the latter subset (another 15% of the total) encode proteins that show significant analogy to proteins of known function from yeast or other organisms. The remaining ORFs, exhibiting no significant similarity to any known sequence, amount to 54% of the total. General features of chromosome X are also reported, with emphasis on the nucleotide frequency distribution in the environment of the ATG and stop codons, the possible coding capacity of at least some of the small ORFs (<100 codons) and the significance of 46 non-canonical or unpaired nucleotides in the stems of some of the 24 tRNA genes recognized on this chromosome.