Genetic and biochemical evidence for hexokinase PII as a key enzyme involved in carbon catabolite repression in yeast

Summary Mutants with reduced hexokinase activity previously isolated as resistant to carbon catabolite repression of invertase and maltase (Zimmermann and Scheel, 1977) were allele tested with mutant strains of Lobo and Maitra (1977) which had defects in one or several of the genes coding for glucokinase and the two unspecific hexokinases. It could be demonstrated, that the mutation abolishing carbon catabolite repression had occurred in a gene allelic to the structural gene of hexokinase PII. Moreover, the defective mutant allele for hexokinase PII isolated by Lobo and Maitra (1977) was also defective in carbon catabolite repression. Neither glucokinase nor hexokinase PI showed any effect on this regulatory system. Biochemical analysis in crude extracts also showed altered kinetic properties of hexokinases in the hex1 mutants. The results directly support the hypothesis previously put forward, that one of the hexokinases is not only active as a catalytic, but also as a regulatory protein.     

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).     

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.     

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.     

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.     

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.     

A defect in carbon catabolite repression associated with uncontrollable and excessive maltose uptake

Summary The previously isolated recessive mutant allele hex2-3 of Saccharomyces cerevisiae caused a defect in carbon catabolite repression of maltase, invertase, malate dehydrogenase, and respiration but at the same time led to an extreme sensitivity to maltose (Zimmermann and Scheel, 1977; Entian and Zimmermann, 1980). Addition of maltose to a growing culture of a hex2-3 mutant resulted within 60 to 90 min in an inhibition of growth, glycolysis, and de novo protein synthesis. This was not accompanied by any abnormal levels of glycolysis metabolites or glycolytic enzyme activities. However, inhibitory effects coincided with a dramatic increase in intracellular glucose up to 150 mM relative to cell water as opposed to 2.5 mM in wild-type cells. This abnormal behavior is interpreted as a result of an uncontrolled maltose uptake in hex2 mutants, which in combination with increasing maltase activity results in an accumulation of intracellular glucose. Obviously the amount of available glucose surpassed glycolytic capacity in hex2 mutants.Properties of mutant alleles hex2 and hex1 (see Entian and Zimmermann, 1980) clearly show, that specific gene functions are involved in adapting the rate of sugar uptake into the cell to the actual glycolytic capacity.     

Extragenic suppressors of yeast glucose derepression mutants leading to constitutive synthesis of several glucose-repressible enzymes

Saccharomyces cerevisiae regulatory genes CAT1 and CAT3 constitute a positive control circuit necessary for derepression of gluconeogenic and disaccharide-utilizing enzymes. Mutations within these genes are epistatic to hxk2 and hex2, which cause defects in glucose repression. cat1 and cat3 mutants are unable to grow in the presence of nonfermentable carbon sources or maltose. Stable gene disruptions were constructed inside these genes, and the resulting growth deficiencies were used for selecting epistatic mutations. The revertants obtained were tested for glucose repression, and those showing altered regulatory properties were further investigated. Most revertants belonged to a single complementation group called cat4. This recessive mutation caused a defect in glucose repression of invertase, maltase, and iso-1-cytochrome c. Additionally, hexokinase activity was increased. Gluconeogenic enzymes are still normally repressible in cat4 mutants. The occurrence of recombination of cat1::HIS3 and cat3::LEU2 with some cat4 alleles allowed significant growth in the presence of ethanol, which could be attributed to a partial derepression of gluconeogenic enzymes. The cat4 complementation group was tested for allelism with hxk2, hex2, cat80, cid1, cyc8, and tup1 mutations, which were previously described as affecting glucose repression. Allelism tests and tetrad analysis clearly proved that the cat4 complementation group is a new class of mutant alleles affecting carbon source-dependent gene expression.     

Physiological properties of Saccharomyces cerevisiae from which hexokinase II has been deleted

Hexokinase II is an enzyme central to glucose metabolism and glucose repression in the yeast Saccharomyces cerevisiae. Deletion of HXK2, the gene which encodes hexokinase II, dramatically changed the physiology of S. cerevisiae. The hxk2-null mutant strain displayed fully oxidative growth at high glucose concentrations in early exponential batch cultures, resulting in an initial absence of fermentative products such as ethanol, a postponed and shortened diauxic shift, and higher biomass yields. Several intracellular changes were associated with the deletion of hexokinase II. The hxk2 mutant had a higher mitochondrial H(+)-ATPase activity and a lower pyruvate decarboxylase activity, which coincided with an intracellular accumulation of pyruvate in the hxk2 mutant. The concentrations of adenine nucleotides, glucose-6-phosphate, and fructose-6-phosphate are comparable in the wild type and the hxk2 mutant. In contrast, the concentration of fructose-1,6-bisphosphate, an allosteric activator of pyruvate kinase, is clearly lower in the hxk2 mutant than in the wild type. The results suggest a redirection of carbon flux in the hxk2 mutant to the production of biomass as a consequence of reduced glucose repression.     

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.     

Construction of industrial baker's yeast strains able to assimilate maltose under catabolite repression conditions

Spore progeny from an industrial baker's yeast strain were mutagenized with UV and mutants resistant to 2-deoxyglucose isolated. One of these mutants (10a12–13) showed high levels of maltase (-glucosidase) and external invertase, and assimilated maltose when growing under catabolite repression conditions. This mutant was not allelic to any of the catabolite repression mutants tested cat4, cat80, cid1, cyc8, hex2, hxk2 and tup1. Mutant 10a12–13 was crossed with appropriate strains to construct hybrids that were also able to assimilate maltose in the presence of glucose. These hybrids may be useful in fermentation processes where both glucose and maltose are present.     

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.     

The residual enzymatic phosphorylation activity of hexokinase II mutants is correlated with glucose repression in Saccharomyces cerevisiae.

Saccharomyces cerevisiae mutants containing different point mutations in the HXK2 gene were used to study the relationship between phosphorylation by hexokinase II and glucose repression in yeast cells. Mutants showing different levels of hexokinase activity were examined for the degree of glucose repression as indicated by the levels of invertase activity. The levels of hexokinase activity and invertase activity showed a strong inverse correlation, with a few exceptions attributable to very unstable hexokinase II proteins. The in vivo hexokinase II activity was determined by measuring growth rates, using fructose as a carbon source. This in vivo hexokinase II activity was similarly inversely correlated with invertase activity. Several hxk2 alleles were transferred to multicopy plasmids to study the effects of increasing the amounts of mutant proteins. The cells that contained the multicopy plasmids exhibited less invertase and more hexokinase activity, further strengthening the correlation. These results strongly support the hypothesis that the phosphorylation activity of hexokinase II is correlated with glucose repression.     

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

Summary 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.     

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.     

Genetics of yeast hexokinase

Two independent isolates of Saccharomyces cerevisiae lacking hexokinase activity (EC 2.7.1.1) are described. Both mutant strains grow on glucose but are unable to grow on fructose, and contain two mutant genes hxk1 and hxk2 each. The mutations are recessive and noncomplementing. Genetic analysis suggests that these two unlinked genes hxk1 and hxk2 determine, independently of each other, the synthesis of hexokinase isozymes P1 and P2, respectively. hxk1 is located on chromosome VIR distal to met10, and hxk2 is on chromosome IIIR distal to MAL2. Of four hexokinase-positive spontaneous reversions, one is very tightly linked to hxk1 and the other three to the hxk2 locus. The reverted enzymes are considerably more thermolabile than the respective wild-type enzymes, and in one case show altered immunological properties. Data are presented which suggest that the hxk1 and hxk2 mutations are missense mutations in the structural genes of hexokinase P1 and hexokinase P2, respectively. These are presumably the only enzymes that allow S. cerevisiae to grow on fructose.     

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.     

The regulation of hexokinase and phosphoglucomutase activity in Aspergillus nidulans.

Summary The levels of glucose-6-phosphate and 6-phosphogluconate dehydrogenase in wildtype cells of Aspergillus nidulans varied with the carbon and nitrogen source. In general, hexokinase activity did not vary with carbon or nitrogen source. The ammonium derepressed mutant amrA1 had only 50% of the wildtype level of hexokinase. Phosphoglucomutase activity was low in wildtype cells grown with nitrate, but high in cells grown with ammonium when glucose was the carbon source. A non-inducible mutant, nirA ^-1, in the regulatory gene for nitrate reductase, had high phosphoglucomutase activity when grown with nitrate or ammonium. A constitutive mutant nirA ^c1, in the regulatory gene for nitrate reductase had low phosphoglucomutase activity when grown with nitrate or ammonium. The mutants nir ^-1 and nirA ^c1 are recessive and semi-dominant respectively for abnormal phosphoglucomutase activity.     

Physiological Properties of Saccharomyces cerevisiae from Which Hexokinase II Has Been Deleted

Hexokinase II is an enzyme central to glucose metabolism and glucose repression in the yeast Saccharomyces cerevisiae. Deletion of HXK2, the gene which encodes hexokinase II, dramatically changed the physiology of S. cerevisiae. The hxk2-null mutant strain displayed fully oxidative growth at high glucose concentrations in early exponential batch cultures, resulting in an initial absence of fermentative products such as ethanol, a postponed and shortened diauxic shift, and higher biomass yields. Several intracellular changes were associated with the deletion of hexokinase II. The hxk2 mutant had a higher mitochondrial H⁺-ATPase activity and a lower pyruvate decarboxylase activity, which coincided with an intracellular accumulation of pyruvate in the hxk2 mutant. The concentrations of adenine nucleotides, glucose-6-phosphate, and fructose-6-phosphate are comparable in the wild type and the hxk2 mutant. In contrast, the concentration of fructose-1,6-bisphosphate, an allosteric activator of pyruvate kinase, is clearly lower in the hxk2 mutant than in the wild type. The results suggest a redirection of carbon flux in the hxk2 mutant to the production of biomass as a consequence of reduced glucose repression.     

Cloning of genes that complement yeast hexokinase and glucokinase mutants.

Genes complementing the glucose-negative fructose-negative Saccharomyces cerevisiae triple mutant strain (hxkl hxk2 glk1), which lacks hexokinase PI, hexokinase PII, and glucokinase, were obtained from a pool of yeast DNA in the multicopy plasmid YEp13.