A carbon catabolite repression mutant of Saccharomyces cerevisiae with elevated hexokinase activity: Evidence for regulatory control of hexokinase PII synthesis

Summary Mutants were investigated that had elevated hexokinase activity and had been isolated previously as resistant to carbon catabolite repression (Zimmermann and Scheel 1977). They were allele tested with mutant strains of Lobo and Maitra (1977), which had defects in one or more of the genes coding for glucokinase and unspecific hexokinases. It was shown, that the mutation abolishing carbon catabolite repression had occured in a gene that was not allelic to any of the structural genes coding for hexokinases. This indicated that a regulatory defect was responsible for elevated hexokinase activity. This agreed with observations that hexokinase activities were like wild-type during growth on non-fermentable carbon sources in hex2 mutants. Recombination between the mutant allele hex2 and mutant alleles hxk1 and hxk2, coding for hexokinase PI and PII respectively, clearly demonstrated that only hexokinase PII was elevated in hex2 mutants. When hex2 mutant cells grown on YEP ethanol were shifted to YEP glucose media, hexokinase activity increased after 30min. This increase depended on de novo protein synthesis. hex2 mutants provide evidence, that carbon catabolite repression and synthesis of hexokinase PII are under common regulatory control.     

Glucose repression in Saccharomyces cerevisiae is directly associated with hexose phosphorylation by hexokinases PI and PII

Genetic and biochemical analyses showed that hexokinase PII is mainly responsible for glucose repression in Saccharomyces cerevisiae, indicating a regulatory domain mediating glucose repression. Hexokinase PI/PII hybrids were constructed to identify the supposed regulatory domain and the repression behavior was observed in the respective transformants. The hybrid constructs allowed the identification of a domain (amino acid residues 102-246) associated with the fructose/glucose phosphorylation ratio. This ratio is characteristic of each isoenzyme, therefore this domain probably corresponds to the catalytic domain of hexokinases PI and PII. Glucose repression was associated with the C-terminal part of hexokinase PII, but only these constructs had high catalytic activity whereas opposite constructs were less active. Reduction of hexokinase PII activity by promoter deletion was inversely followed by a decrease in the glucose repression of invertase and maltase. These results did not support the hypothesis that a specific regulatory domain of hexokinase PII exists which is independent of the hexokinase PII catalytic domain. Gene disruptions of hexokinases further decreased repression when hexokinase PI was removed in addition to hexokinase PII. This proved that hexokinase PI also has some function in glucose repression. Stable hexokinase PI overproducers were nearly as effective for glucose repression as hexokinase PII. This showed that hexokinase PI is also capable of mediating glucose repression. All these results demonstrated that catalytically active hexokinases are indispensable for glucose repression. To rule out any further glycolytic reactions necessary for glucose repression, phosphoglucoisomerase activity was gradually reduced. Cells with residual phosphoglucoisomerase activities of less than 10% showed reduced growth on glucose. Even 1% residual activity was sufficient for normal glucose repression, which proved that additional glycolytic reactions are not necessary for glucose repression. To verify the role of hexokinases in glucose repression, the third glucose-phosphorylating enzyme, glucokinase, was stably overexpressed in a hexokinase PI/PII double-null mutant. No strong effect on glucose repression was observed, even in strains with 2.6 U/mg glucose-phosphorylating activity, which is threefold increased compared to wild-type cells. This result indicated that glucose repression is only associated with the activity of hexokinases PI and PII and not with that of glucokinase.     

Saccharomyces cerevisiae mutants provide evidence of hexokinase PII as a bifunctional enzyme with catalytic and regulatory domains for triggering carbon catabolite repression

A selection system has been devised for isolating hexokinase PII structural gene mutants that cause defects in carbon catabolite repression, but retain normal catalytic activity. We used diploid parental strains with homozygotic defects in the hexokinase PI structural gene and with only one functional hexokinase PII allele. Of 3,000 colonies tested, 35 mutants (hex1r) did not repress the synthesis of invertase, maltase, malate dehydrogenase, and respiratory enzymes. These mutants had additional hexokinase PII activity. In contrast to hex1 mutants (Entian et al., Mol. Gen. Genet. 156:99-105, 1977; F.K. Zimmermann and I. Scheel, Mol. Gen. Genet. 154:75-82, 1977), which were allelic to structural gene mutants of hexokinase PII and had no catalytic activity (K.-D. Entian, Mol. Gen. Gent. 178:633-637, 1980), the hex1r mutants sporulated hardly at all or formed aberrant cells. Those ascospores obtained were mostly inviable. As the few viable hex1r segregants were sterile, triploid cells were constructed to demonstrate allelism between hex1r mutants and hexokinase PII structural gene mutants. Metabolite concentrations, growth rate, and ethanol production were the same in hex1r mutants and their corresponding wild-type strains. Recombination of hexokinase and glucokinase alleles gave strains with different specific activities. The defect in carbon catabolite repression was strongly associated with the defect in hexokinase PII and was independent of the glucose phosphorylating capacity. Hence, a secondary effect caused by reduced hexose phosphorylation was not responsible for the repression defect in hex1 mutants. These results, and those with the hex1r mutants isolated, strongly supported our earlier hypothesis that hexokinase PII is a bifunctional enzyme with (i) catalytic activity and (ii) a regulatory component triggering carbon catabolite repression (Entian, Mol. Gen. Genet. 178:633-637, 1980; K.-D. Entian and D. Mecke, J. Biol. Chem. 257:870-874, 1982).     

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.     

Effects of null mutations in the hexokinase genes of Saccharomyces cerevisiae on catabolite repression.

Saccharomyces cerevisiae has two homologous hexokinases, I and II; they are 78% identical at the amino acid level. Either enzyme allows yeast cells to ferment fructose. Mutant strains without any hexokinase can still grow on glucose by using a third enzyme, glucokinase. Hexokinase II has been implicated in the control of catabolite repression in yeasts. We constructed null mutations in both hexokinase genes, HXK1 and HXK2, and studied their effect on the fermentation of fructose and on catabolite repression of three different genes in yeasts: SUC2, CYC1, and GAL10. The results indicate that hxk1 or hxk2 single null mutants can ferment fructose but that hxk1 hxk2 double mutants cannot. The hxk2 single mutant, as well as the double mutant, failed to show catabolite repression in all three systems, while the hxk1 null mutation had little or no effect on catabolite repression.     

Aluminum detoxification mechanism in Pseudomonas fluorescens is dependent on iron

Abstract Hexose phosphorylation was studied in Aspergillus nidulans wild-type and in a fructose non-utilising mutant ( frA ). The data indicate the presence of at least one hexokinase and one glucokinase in wild-type A. nidulans , while the fr A1 mutant lacks hexokinase activity. The A. nidulans gene encoding hexokinase was isolated by complementation of the fr A1 mutation. The absence of hexokinase activity in the fr A1 mutant did not interfere with glucose repression of the enzymes involved in alcohol and l-arabinose catabolism. This suggests that, unlike the situation in yeast where mutation of hexokinase PII abolishes glucose repression, the A. nidulans hexokinase might not be involved in glucose repression.     

The hexokinase isoenzyme PII of Saccharomyces cerevisiae ia a protein kinase

The HXK2 gene product has an important role in controlling carbon catabolite repression in Saccharomyces cerevisiae. We have raised specific antibodies against the hexokinase PII protein and have demonstrated that it is a 58 kDa phosphoprotein with protein kinase activity. The predicted amino acid sequence of the HXK2 gene product has significant homology to the conserved catalytic domain of mammalian and yeast protein kinases. Protein kinase activity was located in a different domain of the protein from the hexose-phosphorylating activity. The hexokinase PII protein level remained unchanged in P2T22D mutant cells (hxk1 HXK2 glk1) growing in a complex medium with glucose. The protein kinase activity of hexokinase PII is regulated by the glucose concentration of the culture medium. Exit from the carbon catabolite repression phase and entry into derepression phase may be controlled, in part, by modulation of the 58 kDa protein kinase activity by changes in cyclic AMP concentration.     

Saccharomyces Cerevisiae Null Mutants in Glucose Phosphorylation: Metabolism and Invertase Expression

A congenic series of Saccharomyces cerevisiae strains has been constructed which carry, in all combinations, null mutations in the three genes for glucose phosphorylation: HXK1, HXK2 and GLK1, coding hexokinase 1 (also called PI or A), hexokinase 2 (PII or B), and glucokinase, respectively: i.e., eight strains, all of which grow on glucose except for the triple mutant. All or several of the strains were characterized in their steady state batch growth with 0.2% or 2% glucose, in aerobic as well as respiration-inhibited conditions, with respect to growth rate, yield, and ethanol formation. Glucose flux values were generally similar for different strains and conditions, provided they contained either hexokinase 1 or hexokinase 2. And their aerobic growth, as known for wild type, was largely fermentative with ca. 1.5 mol ethanol made per mol glucose used. The strain lacking both hexokinases and containing glucokinase was an exception in having reduced flux, a result fitting with its maximal rate of glucose phosphorylation in vitro. Aerobic growth of even the latter strain was largely fermentative (ca. 1 mol ethanol per mol glucose). Invertase expression was determined for a variety of media. All strains with HXK2 showed repression in growth on glucose and the others did not. Derepression in the wild-type strain occurred at ca. 1 mM glucose. The metabolic data do not support- or disprove-a model with HXK2 having only a secondary role in catabolite repression related to more rapid metabolism.     

Cloning and restriction analysis of the hexokinase PII gene of the yeast Saccharomyces cerevisiae

Summary Carbon catabolite repression in yeast depends on catalytic active hexokinase isoenzyme PII (Entian 1980a). A yeast strain lacking hexokinase isoenzymes PI and PII was transformed, using a recombinant pool with inserts of yeast nuclear DNA up to 10 kbp in length. One hundred transformants for hexokinase were obtained. All selected plasmids coded for hexokinase isoenzyme PII, none for hexokinase isoenzyme PI, and carbon catabolite repression was restored in the transformants. Thirty-five independently isolated stable plasmids were investigated further. Analysis with the restriction enzyme EcoRI showed that these plasmids fell into two classes with different restriction behaviour. One representative of each class was amplified in Escherichia coli and transferred back into the yeast hexokinase-deficient strain with concomitant complementation of the nuclear mutation. The two types of insert were analysed in detail with 16 restriction enzymes, having 0–3 cleavage sites on transformant vector YRp7. The plasmids differed from each other by the orientation of the yeast insert in the vector. After yeast transformation with fragments of one plasmid the hexokinase PII gene was localised within a region of 1.65 kbp.     

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.     

Mechanism of inactivation of hexokinase PII of Saccharomyces cerevisiae by D-xylose

The mechanism of inactivation of hexokinase PII of Saccharomyces cerevisiae by D-xylose was characterized. Inactivation was dependent on the presence of MgATP and was irreversible. Inactivation involved phosphorylation of the protein. Observation of the carbon catabolite repression of selected enzymes showed that invertase and maltase synthesis were not repressed when hexokinase PII was phosphorylated.     

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.     

Xylose induced decrease in hexokinase PII activity confers resistance to carbon catabolite repression of invertase synthesis in Saccharomyces carlsbergensis

The relationship between the xylose induced decrease in hexokinase PII activity and the derepression of invertase synthesis in yeast is described. When xylose was added to cells growing in a chemostat under nitrogen limitation, the catabolic repression was supressed as shown by the large increase on invertase levels even if glucose remained high. The glucose phosphorylating-enzymes were separated by hydroxylapatite chromatography and it is shown that the treatment with xylose is accompanied by a loss of 98% hexokinase PII and a 50% of the PI isoenzyme, whereas the levels of glucokinase as well as those of glucose-6-phosphate, fructose-6-phosphate, pyruvate and ATP remained unaffected.The analysis of the enzymes present in cells grown in ethanol, limiting glucose and high glucose, shows that hexokinase PII predominates in cells under catabolic repression, the opposite is true for glucokinase, whereas hexokinase PI remains unaffected.     

The Aspergillus nidulans xprF gene encodes a hexokinase-like protein involved in the regulation of extracellular proteases

The extracellular proteases of Aspergillus nidulans are produced in response to limitation of carbon, nitrogen, or sulfur, even in the absence of exogenous protein. Mutations in the A. nidulans xprF and xprG genes have been shown to result in elevated levels of extracellular protease in response to carbon limitation. The xprF gene was isolated and sequence analysis indicates that it encodes a 615-amino-acid protein, which represents a new type of fungal hexokinase or hexokinase-like protein. In addition to their catalytic role, hexokinases are thought to be involved in triggering carbon catabolite repression. Sequence analysis of the xprF1 and xprF2 alleles showed that both alleles contain nonsense mutations. No loss of glucose or fructose phosphorylating activity was detected in xprF1 or xprF2 mutants. There are two possible explanations for this observation: (1) the xprF gene may encode a minor hexokinase or (2) the xprF gene may encode a protein with no hexose phosphorylating activity. Genetic evidence suggests that the xprF and xprG genes are involved in the same regulatory pathway. Support for this hypothesis was provided by the identification of a new class of xprG(-) mutation that suppresses the xprF1 mutation and results in a protease-deficient phenotype.     

Complete amino acid sequence of the type III isozyme of rat hexokinase, deduced from the cloned cDNA

Clones containing cDNA coding for the Type III isozyme of rat hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) were isolated from a library prepared in lambda gt10 with rat liver mRNA. Three clones were characterized. Their composite sequence includes the entire coding region for Type III hexokinase, 3' untranslated sequence extending into the polyadenylated region, and 80 bp of 5' untranslated sequence. Extensive similarity in sequence of N- and C-terminal halves of the enzyme, previously seen with the Type I isozyme, is consistent with the view that these 100-kDa mammalian hexokinases are the evolutionary result of duplication and fusion of a gene coding for an ancestral hexokinase having a molecular weight of approximately 50 kDa. Extensive similarities are seen between sequences of the Type I and III isozymes, and those reported for mammalian glucokinase (also called Type IV hexokinase) and for the hexokinase and glucokinase of yeast. Residues thought to be involved in catalytic function are highly conserved in all of these enzymes. Based on a quantitative comparison of sequence similarities, it is concluded that the 50-kDa mammalian glucokinase is more closely related to the 100-kDa mammalian enzymes than it is to the 50-kDa enzymes from yeast. One interpretation of this might be that the mammalian glucokinase arose by resplitting of the gene coding for the 100-kDa mammalian hexokinases.     

Proteolysis of hexokinase PII is not the triggering signal of carbon catabolite derepression in Saccharomyces cerevisiae

The role of hexokinase PII in mediating carbon catabolite derepression in yeast has been examined. Hexokinase isoenzyme PII (EC 2.7.1.1) was partially degraded when protease inhibitors were omitted from the buffer used for preparation of cell-free extracts. The hexokinase PII inactivation induced by D-xylose was correlated with derepression of maltase (EC 3.2.1.20) in the wild-type strain Saccharomyces cerevisiae G-517 and in D.308.3, a strain that contains the cloned hexokinase PII gene on a multicopy plasmid. This inactivation was not correlated with the loss of hexokinase PII protein as assayed by immunoblotting. We conclude that during the derepression process there is no release of proteolytic peptides from hexokinase PII.     

Cloning of hexokinase isoenzyme PI from Saccharomyces cerevisiae: PI transformants confirm the unique role of hexokinase isoenzyme PII for glucose repression in yeasts

Summary Hexokinase isoenzyme PI was cloned using a gene pool obtained from a yeast strain having only one functional hexokinase, isoenzyme PI. The gene was characterized using 20 restriction enzymes and located within a region of 2.0 kbp. The PI plasmid strongly hybridized with the PII plasmids isolated previously (Fröhlich et al. 1984). Hence there was a close relationship between the two genes, one of which must have been derived from the other by gene duplication. In conrrast, glucose repression was restored only in hexokinase PII transformants; PI transformants remained non-repressible. This observation provided additional evidence for the hypothesis of Entian (1980) that only hexokinase PII is necessary for glucose repression. Furthermore, glucose phosphorylating activity in PI transformants exceeded that of wild-type cells, giving clear evidence that the phosphorylating capacity is not important for glucose repression.     

Identification,expression and bioactivity of hexokinase in amphioxus: Insights into evolution of vertebrate hexokinase genes

Hexokinase family includes hexokinases I, II, III and IV, that catalyze the phosphorylation of glucose to produce glucose 6-phosphate. Hexokinase IV, also known as glucokinase, is only half size of the other types of hexokinases that contain two hexokinase domains. Despite the enormous progress in the study of hexokinases, the evolutionary relationship between glucokinase and other hexokinases is still uncertain, and the molecular processes leading to the emergence of hexokinases in vertebrates remain controversial. Here we clearly demonstrated the presence of a single hexokinase-like gene in the amphioxus Branchiostoma japonicum, Bjhk, which shows a tissue-specific expression pattern, with the most abundant expression in the hepatic caecum, testis and ovary. The phylogenetic and synteny analyses both reveal that BjHK is the archetype of vertebrate hexokinases IV, i.e. glucokinases. We also found for the first time that recombinant BjHK showed functional enzyme activity resembling vertebrate hexokinases I, II, III and IV. In addition, a native glucokinase activity was detected in the hepatic caecum. Finally, glucokinase activity in the hepatic caecum was markedly reduced by fasting, whereas it was considerably increased by feeding. Altogether, these suggest that Bjhk represents the archetype of glucokinases, from which vertebrate hexokinase gene family was evolved by gene duplication, and that the hepatic caecum plays a role in the control of glucose homeostasis in amphioxus, in favor of the notion that the hepatic caecum is a tissue homologous to liver.     

Lack of carbon catabolite inactivation in a mutant of Saccharomyces cerevisiae with reduced hexokinase activity

Summary A mutant of Saccharomyces cerevisiae with reduced hexokinase activity and deficient in carbon catabolite inactivation is described. The reason for this lack of inactivation is not a lowered concentration of glycolysis metabolites or other low molecular effectors such as glucose, and ATP. The results point to the hexose phosphorylation step as initiator for carbon catabolite inactivation. It appears that one of the hexokinase isoenzymes, altered in the mutant, initiates the inactivation by conformational change. Repression of enzymes that are subject to carbon catabolite inactivation, is normal in the mutant. This indicates that inactivation and repression of those enzymes proceed in different ways, even though they may share common intermediate reactions.