Nitrogen catabolite repression in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the expression of all known nitrogen catabolite pathways are regulated by four regulators known as Gln3, Gat1, Dal80, and Deh1. This is known as nitrogen catabolite repression (NCR). They bind to motifs in the promoter region to the consensus sequence 5'GATAA 3'. Gln3 and Gat1 act positively on gene expression whereas Dal80 and Deh1 act negatively. Expression of nitrogen catabolite pathway genes known to be regulated by these four regulators are glutamine, glutamate, proline, urea, arginine. GABA, and allantonie. In addition, the expression of the genes encoding the general amino acid permease and the ammonium permease are also regulated by these four regulatory proteins. Another group of genes whose expression is also regulated by Gln3, Gat1, Dal80, and Deh1 are some proteases, CPS1, PRB1, LAP1, and PEP4, responsible for the degradation of proteins into amino acids thereby providing a nitrogen source to the cell. In this review, all known promoter sequences related to expression of nitrogen catabolite pathways are discussed as well as other regulatory proteins. Overview of metabolic pathways and promotors are presented.     

Nitrogen catabolite repression in Saccharomyces cerevisiae

In Saccharomyces cerevisiae the expression of all known nitrogen catabolite pathways are regulated by four regulators known as Gln3, Gat1, Dal80, and Deh1. This is known as nitrogen catabolite repression (NCR). They bind to motifs in the promoter region to the consensus sequence 5′ GATAA 3′. Gln3 and Gat1 act positively on gene expression whereas Dal80 and Deh1 act negatively. Expression of nitrogen catabolite pathway genes known to be regulated by these four regulators are glutamine, glutamate, proline, urea, arginine, GABA, and allantoine. In addition, the expression of the genes encoding the general amino acid permease and the ammonium permease are also regulated by these four regulatory proteins. Another group of genes whose expression is also regulated by Gln3, Gat1, Dal80, and Deh1 are some protease, CPS1, PRB1, LAP1, and PEP4, responsible for the degradation of proteins into amino acids thereby providing a nitrogen source to the cell. In this review, all known promoter sequences related to expression of nitrogen catabolite pathways are discussed as well as other regulatory proteins. Overview of metabolic pathways and promotors are presented.     

Cloning of hexokinase structural genes from Saccharomyces cerevisiae mutants with regulatory mutations responsible for glucose repression.

The regulatory hexokinase PII mutants isolated previously (K.-D. Entian and K.-U. Fröhlich, J. Bacteriol. 158:29-35, 1984) were characterized further. These mutants were defective in glucose repression. The mutation was thought to be in the hexokinase PII structural gene, but it did not affect the catalytic activity of the enzyme. Hence, a regulatory domain for glucose repression was postulated. For further understanding of this regulatory system, the mutationally altered hexokinase PII proteins were isolated from five mutants obtained independently and characterized by their catalytic constants and bisubstrate kinetics. None of these characteristics differed from those of the wild type, so the catalytic center of the mutant enzymes remained unchanged. The only noticeable difference observed was that the in vivo modified form of hexokinase PII, PIIM, which has been described recently (K.-D. Entian and E. Kopetzki, Eur. J. Biochem. 146:657-662, 1985), was absent from one of these mutants. It is possible that the PIIM modification is directly connected with the triggering of glucose repression. To establish with certainty that the mutation is located in the hexokinase PII structural gene, the genes of these mutants were isolated after transforming a hexokinaseless mutant strain and selecting for concomitant complementation of the nuclear function. Unlike hexokinase PII wild-type transformants, glucose repression was not restored in the hexokinase PII mutant transformants. In addition mating experiments with these transformants followed by tetrad analysis of sporulated diploids gave clear evidence of allelism to the hexokinase PII structural gene.     

New genes involved in carbon catabolite repression and derepression in the yeast Saccharomyces cerevisiae.

A mutation causing resistance to carbon catabolite repression in gene HEX2, mutant allele hex2-3, causes an extreme sensitivity to maltose when in combination with the genes necessary for maltose metabolism. This provided a convenient system for the selective isolation of mutations in genes specifically required for maltose metabolism and other genes involved in general carbon catabolite repression. In addition to reversion of the hex2-3 allele, mutations in three other genes were detected. These genes were called CAT1, CAT3, and MUR1 and in a mutated form abolished maltose inhibition caused by mutant allele hex2-3. Mutant alleles cat1 and cat3 also restored normal repression in the presence of the hex2-3 allele. Segregants having only mutant alleles cat1 or cat3 were obtained by tetrad analysis. These segregants could not grow on nonfermentable carbon sources. Mutant alleles of gene CAT1 were allelic to a mutant allele cat1-1 previously isolated (Zimmermann et al., Mol. Gen. Genet. 151:95-103). Such mutants prevented derepression not only of the maltose catabolizing system, the selected property, but also of glyoxylate shunt and gluconeogenic enzymes. However, respiratory activities and invertase formation were not affected under derepressing conditions. cat3 mutants had the same phenotypic properties as cat1 mutants. This showed that carbon metabolism in yeast cells is under a very complex and ramified control of repressing and derepressing genes, which are interdependent.     

Mutants of Saccharomyces cerevisiae resistant to carbon catabolite repression.

Summary Mutants with defective carbon catabolite repression have been isolated in the yeast Saccharomyces cerevisiae using a selective procedure. This was based on the fact that invertase is a glucose repressible cell wall enzyme which slowly hydrolyses raffinose to yield fructose and that the inhibitory effects of 2-deoxyglucose can be counteracted by fructose. Repressed cells were plated on a raffinose-2-deoxyglucose medium and the resistant cells growing up into colonies were tested for glucose non-repressible invertase and maltase. The yield of regulatory mutants was very high. All were equally derepressed for invertase and maltase, no mutants were obtained with only non-repressible invertase synthesis which was the selected function. A total of 61 mutants isolated in different strains were allele tested and could be attributed to three genes. They were all recessive. Mutants in one gene had reduced hexokinase activities, the other class, located in a centromere linked gene, had elevated hexokinase levels and was inhibited by maltose. Mutants in a third gene were isolated on a 2-deoxyglucose galactose medium and had normal hexokinase levels. A partial derepression was observed for malate dehydrogenase in all mutants. Isocitrate lyase, however, was still fully repressible.     

Recessive mutations conferring resistance to carbon catabolite repression of galactokinase synthesis in Saccharomyces cerevisiae

A total of 37 recessive mutations showing enhanced resistance to the glucose repression of galactokinase synthesis have been isolated by a selection procedure with a GAL81 gal7 double mutant. These mutations were grouped into three different complementation classes. One class, reg1, contains mutants arising from mutations at a site close to, but complementing, the gal3 locus. The reg1 mutant also showed resistance to the glucose repression of invertase synthesis but not to that of alpha-D-glucosidase. The two other classes were identified as arising from recessive mutations at the GAL82 locus and the GAL83 locus, respectively, at which various dominant mutations were isolated previously. When in a constitutive background due to the GAL81 or gal80 mutation, the GAL82 and GAL83 mutations did not show a mutually additive effect on the resistance to glucose repression of galactokinase synthesis, while the reg1 and GAL82 (or GAL83) mutations did. Based upon the specific behavior of cells with various genotypes for the above genes in response to the concentration of galactose and glucose in the medium, we propose a model involving three independent circuits for glucose signals in the regulation of the structural genes for the galactose pathway enzymes.     

Nitrogen catabolite repression of asparaginase II in Saccharomyces cerevisiae

A new procedure was devised for selecting, from lac+ galE strains of Escherichia coli, mutants resistant to galactoside-induced lysis. When applied to trp-lac fusions, our method yields down mutations in the trp promoter.     

Glucose uptake in Saccharomyces cerevisiae grown under anaerobic conditions: effect of null mutations in the hexokinase and glucokinase structural genes.

Glucose uptake was investigated in a set of isogenic strains carrying a single glucose kinase structural gene, the other two kinase genes having been rendered nonfunctional through the construction of null mutations. Any one of the three kinases was sufficient for growth and glucose utilization aerobically or anaerobically. Under anaerobic conditions, substrate inhibition and regulation of carrier activity varied and depended upon the particular kinase present in the cell.     

Effects of reactant heterogeneity and mixing on catabolite repression in cultures of Saccharomyces cerevisiae

An experimental study was conducted into the effect of reactant heterogeneity on glucose-fed continuous cultures of S. cerevisiae, The heterogeneity was altered by varying mixing intensity in the nutrient entry region within a static mixing device. Experimental results confirm simulation predictions based upon a simple growth model, showing that mixing in the entry region can govern macroscopic culture behavior. Specifically, at high dilution rates, the biomass concentration was reduced by mixing patterns that increased the size of regions where glucose exceeded the threshold for catabolite repression. Because the size of such repressive regions is not uniquely determined by reactant segregation, the authors argue that in biological systems (and others involving a threshold response) an alternative measure of mixing quality should be used. Conclusions are drawn concerning the simulation of biological reactors for design purposes, and the importance of nutrient delivery systems to reactor performance.     

Regulation of catalase synthesis in Saccharomyces cerevisiae by carbon catabolite repression.

Summary A number of strains of Saccharomyces cerevisiae, wild type or respiratory deficient, were grown on glucose, galactose or raffinose. Specific activities of catalase T were about tenfold higher in late stationary wild type cells grown on glucose than in wild type cells harvested when glucose had just disappeared completely from the medium, or in respiratory deficient strains (rho^–, mit^–, pet) grown to stationary phase.Catalase A activity is completely absent in wild type cells grown to zero percent glucose or in respiratory deficient cells grown on glucose to stationary phase. High catalase A activity was detected in derepressed wild type cells and in a strain carrying the op 1 (pet 9) mutation, although this strain is unable to grow on nonfermentable carbon sources. All respiratory deficient strains tested have low, but significant catalase A activities after growth on galactose or raffinose.Wild type cells harvested during growth on glucose and rho^–-cells grown on low glucose to stationary phase contain enzymatically inactive catalase A protein. The apoprotein of the enzyme is apparently accumulated in rho^–-cells whereas glucose-repressed wild type cells seem to contain a mixture of apoprotein and heme-containing catalase A monomer.These results show that a source of chemical energy, probably ATP, is required for derepression of yeast catalase from catabolite repression. At least in the case of catalase A, energy produced by respiration is necessary if catabolite repression is caused by glucose. If less repressing sugars are utilized, ATP derived from fermentation appears sufficient for partial derepression. Formation of the active enzyme can apparently be influenced by carbon catabolite repression at different points: (1) at the level of protein synthesis, (2) at the stage of heme incorporation, (3) at the level of formation of the enzymatically active tetramer.     

Nitrogen catabolite repression in a glutamate auxotroph of Saccharomyces cerevisiae

The biosynthesis of asparaginase II in Saccharomyces cerevisiae is subject to nitrogen catabolite repression. In the present study we examined the physiological effects of glutamate auxotrophy on cellular metabolism and on the nitrogen catabolite repression of asparaginase II. Glutamate auxotrophic cells, incubated without a glutamate supplement, had a diminished internal pool of alpha-ketoglutarate and a concomitant inability to equilibrate ammonium ion with alpha-amino nitrogen. In the glutamate auxotroph, asparaginase II biosynthesis exhibited a decreased sensitivity to nitrogen catabolite repression by ammonium ion but normal sensitivity to nitrogen catabolite repression by all amino acids tested.     

Requirement of upstream activation sequences for nitrogen catabolite repression of the allantoin system genes in Saccharomyces cerevisiae.

Synthesis of the transport systems and enzymes mediating uptake and catabolism of nitrogenous compounds is sensitive to nitrogen catabolite repression. In spite of the widespread occurrence of the control process, little is known about its mechanism. We have previously demonstrated that growth of cells on repressive nitrogen sources results in a dramatic decrease in the steady-state levels of mRNA encoded by the allantoin and arginine catabolic pathway genes and of the transport systems associated with allantoin metabolism. The present study identified the upstream activation sequences in the 5'-flanking regions of the allantoin system genes as the cis-acting sites through which nitrogen catabolite repression is exerted.     

Isolation and characterization of dominant mutations resistant to carbon catabolite repression of galactokinase synthesis in Saccharomyces cerevisiae.

Seven dominant mutations showing greatly enhanced resistance to the glucose repression of galactokinase synthesis have been isolated from GAL81 mutants, which have the constitutive phenotype but are still strongly repressible by glucose for the synthesis of the Leloir enzymes. These glucose-resistant mutants were due to semidominant mutations at either of two loci, GAL82 and GAL83. Both loci are unlinked to the GAL81- gal4, gal80, or gal7 X gal10 X gal1 locus or to each other. The GAL83 locus was mapped on chromosome V at a site between arg9 and cho1. The GAL82 and GAL83 mutations produced partial resistance of galactokinase to glucose repression only when one or both of these mutations were combined with a GAL81 or a gal80 mutation. The GAL82 and GAL83 mutations are probably specific for expression of the Leloir pathway and related enzymes, because they do not affect the synthesis of alpha-D-glucosidase, invertase, or isocitrate lyase.