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.     

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.     

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.     

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.     

Characteristics of Saccharomyces cerevisiae gal1 Delta and gal1 Delta hxk2 Delta mutants expressing recombinant proteins from the GAL promoter

Galactose can be used not only as an inducer of the GAL promoters, but also as a carbon source by Saccharomyces cerevisiae, which makes recombinant fermentation processes that use GAL promoters complicated and expensive. To overcome this problem during the cultivation of the recombinant strain expressing human serum albumin (HSA) from the GAL10 promoter, a gal1 Delta mutant strain was constructed and its induction kinetics investigated. As expected, the gal1 Delta strain did not use galactose, and showed high levels of HSA expression, even at extremely low galactose concentrations (0.05-0.1 g/L). However, the gal1 Delta strain produced much more ethanol, in a complex medium containing glucose, than the GAL1 strain. To improve the physiological properties of the gal1 Delta mutant strain as a host for heterologous protein production, a null mutation of either MIG1 or HXK2 was introduced into the gal1 Delta mutant strain, generating gal1 Delta mig1 Delta and gal1 Delta hxk2 Delta double strains. The gal1 Delta hxk2 Delta strain showed a decreased rate of ethanol synthesis, with an accelerated rate of ethanol consumption, compared to the gal1 Delta strain, whereas the gal1 Delta mig1 Delta strain showed similar patterns to the gal1 Delta strain. Furthermore, the gal1 Delta hxk2 Delta strain secreted much more recombinant proteins (HSA and HSA fusion proteins) than the other strains. The results suggest that the gal1 Delta hxk2 Delta strain would be useful for the large-scale production of heterologous proteins from the GAL10 promoter in S. cerevisiae.     

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.     

N-acetylglucosamine kinase, HXK1 contributes to white–opaque morphological transition in Candida albicans

Morphological transition (yeast-hyphal and white–opaque) is an important biological process in the life cycle of pathogenic yeast, Candida albicans and is a major determinant of virulence. Earlier reports show that the amino sugar, N-acetylglucosamine (GlcNAc) induces white to opaque switching in this pathogen. We report here a new contributor to this switching phenomenon, namely N-acetylglucosamine kinase or HXK1, the first enzyme of the GlcNAc catabolic cascade. Microarray profile analysis of wild type vs. hxk1 mutant cells grown under switching inducing condition showed upregulation of opaque specific and cell wall specific genes along genes involved in the oxidative metabolism. Further, our qRT-PCR and immunoblot analysis revealed that the expression levels of Wor1, a master regulator of the white–opaque switching phenomenon remained unaltered during this HXK1 mediated transition. Thus the derepression of opaque specific gene expression observed in hxk1 mutant could be uncoupled to the expression of WOR1. Moreover, this regulation via HXK1 is independent of Ras1, a major regulator of morphogenetic transition and probably independent of MTL locus too. These results extend our understanding of multifarious roles of metabolic enzymes like Hxk1 and suggest an adaptive mechanism during host–pathogen interactions.     

Genetics of Yeast Glucokinase

Mutants of Saccharomyces cerevisiae lacking glucokinase (EC 2.7.1.2) have no discernible phenotypic difference from the wild-type strain; in a hexokinaseless background, however, they are unable to grow on any sugar except galactose. Reversion studies with glucokinase mutants indicate that the yeast S. cerevisiae has no other enzyme for phosphorylating glucose except the two hexokinases, P1 and P2, and glucokinase. Spontaneous revertants of hxk1 hxk2 glk1 strains collected on glucose regain any one of these three enzymes. The majority of glucokinase revertants synthesize species of enzyme activity that are kinetically or otherwise indistinguishable from the wild-type enzyme. In a few cases the reverted enzyme is very perceptibly altered in properties with a Km for glucose two orders of magnitude higher than that of the enzyme from the wild-type parent. These recessive, noncomplementing mutants, thus, define a single structural gene GLK1 of glucokinase. Yeast diploids lacking all of the three enzymes for glucose phosphorylation fail to sporulate. Heterozygosity of either of the hexokinase genes HXK1 or HXK2, but not GLK1, restores sporulation. The location of GLK1 on chromosome III was indicated by loss of this chromosome when hexokinaseless diploids heterozygous for glk1 were selected for resistance to 2-deoxyglucose; the homologue of chromosome III carrying GLK1, the mating-type allele and other nutritional markers on this chromosome was lost. Meiotic mapping of glucokinase executed with heterozygosity of one of the hexokinases indicated that the gene GLK1 defining the structure of glucokinase protein is located on the left arm of chromosome III 24 cM to the left of his4 in the order: leu2--his4--glk1. --Only two of 206 independent glucokinase mutants are nonsense ochre, both of which map at one end of the gene. In hxk1 only one of 130 isolates is a nonsense mutation, whereas in hxk2 none has been found among 220 independent mutants. These results raise the possibility that the protein products of these genes have some other essential function. --An earlier mapping result for hxk2 has been corrected. The new location is on the left arm of chromosome VII, 17 cM distal to ade5 in the order: lys5--ade5--hxk2.     

Isolation and characterization of mutations in the HXK2 gene of Saccharomyces cerevisiae.

Several hundred new mutations in the gene (HXK2) encoding hexokinase II of Saccharomyces cerevisiae were isolated, and a subset of them was mapped, resulting in a fine-structure genetic map. Among the mutations that were sequenced, 35 were independent missense mutations. The mutations were obtained by mutagenesis of cloned HXK2 DNA carried on a low-copy-number plasmid vector and screened for a number of different phenotypes in yeast strains bearing chromosomal hxk1 and hxk2 null mutations. Some of these mutants were characterized both in vivo and in vitro; they displayed a wide spectrum of residual hexokinase activities, as indicated by three assays: in vitro enzyme activity, ability to grow on glucose and fructose, and ability to repress invertase production when growing on glucose. Of those that failed to support growth on fructose, only a small minority made normal-size, stable, and inactive protein. Analysis of the amino acid changes in these mutants in light of the crystallographically determined three-dimensional structure of hexokinase II suggests important roles in structure or catalysis for six amino acid residues, only two of which are near the active site.     

The role of HEXOKINASE1 in Arabidopsis leaf growth

Plant Molecular Biology - Here, we used a hxk1 mutant in the Col-0 background. We demonstrated that HXK1 regulates cell proliferation and expansion early during leaf development, and that HXK1 is...     

Procyclic Trypanosoma brucei do not use Krebs cycle activity for energy generation

The importance of a functional Krebs cycle for energy generation in the procyclic stage of Trypanosoma brucei was investigated under physiological conditions during logarithmic phase growth of a pleomorphic parasite strain. Wild type procyclic cells and mutants with targeted deletion of the gene coding for aconitase were derived by synchronous in vitro differentiation from wild type and mutant (Delta aco::NEO/Delta aco::HYG) bloodstream stage parasites, respectively, where aconitase is not expressed and is dispensable. No differences in intracellular levels of glycolytic and Krebs cycle intermediates were found in procyclic wild type and mutant cells, except for citrate that accumulated up to 90-fold in the mutants, confirming the absence of aconitase activity. Surprisingly, deletion of aconitase did not change differentiation nor the growth rate or the intracellular ATP/ADP ratio in those cells. Metabolic studies using radioactively labeled substrates and NMR analysis demonstrated that glucose and proline were not degraded via the Krebs cycle to CO(2). Instead, glucose was degraded to acetate, succinate, and alanine, whereas proline was degraded to succinate. Importantly, there was absolutely no difference in the metabolic products released by wild type and aconitase knockout parasites, and both were for survival strictly dependent on respiration via the mitochondrial electron transport chain. Hence, although the Krebs cycle enzymes are present, procyclic T. brucei do not use Krebs cycle activity for energy generation, but the mitochondrial respiratory chain is essential for survival and growth. We therefore propose a revised model of the energy metabolism of procyclic T. brucei.     

Distinct modulations of the hexokinase1-mediated glucose response and hexokinase1-independent processes by HYS1/CPR5 in Arabidopsis

The Arabidopsis mutant hypersenescence 1 (hys1), that is allelic to constitutive expresser of pathogenesis-related genes 5 (cpr5), displays phenotypes related to glucose signalling and defence responses. In the present study, it is shown that the hys1 mutation boosts the inhibitory effects of glucose upon the greening of seedlings and reduces the antagonistic activities of ethylene and cytokinin toward this inhibition. Neither the glucose content nor the sensitivities to ethylene, cytokinin, and abscisic acid were found to differ between wild-type and hys1 seedlings. However, disruption of the gene encoding hexokinase1 (HXK1), which acts as a glucose sensor, partially suppressed the glucose hypersensitive phenotype of the hys1 mutant. These results thus suggest that the hys1 mutation promotes a process associated with the HXK1-mediated glucose response during greening. By contrast, additional hys1 phenotypes, including an increase in salicylic acid (SA), production of abnormal trichomes, and early senescence, were not suppressed by the loss of HXK1. Surprisingly, the hxk1 and hys1 mutations acted synergistically towards an increased SA accumulation. Hence, HYS1/CPR5 appears to be a versatile protein that modulates both the HXK1-mediated glucose response and various HXK1-indepndent processes that are involved in growth control. A possible role for HYS1/CPR5 as a component of the networks that regulate growth control is discussed.     

Role of rice cytosolic hexokinase OsHXK7 in sugar signaling and metabolism

We characterized the function of the rice cytosolic hexokinase Os HXK7(Oryza sativa Hexokinase7),which is highly upregulated when seeds germinate under O_2-deficient conditions. According to transient expression assays that used the promoter:luciferase fusion construct,Os HXK7 enhanced the glucose(Glc)-dependent repression of a rice a-amylase gene(RAmy3D) in the mesophyll protoplasts of maize,but its catalytically inactive mutant alleles did not. Consistently,the expression of Os HXK7,but not its catalytically inactive alleles,complemented the Arabidopsis glucose insensitive2-1(gin2-1) mutant,thereby resulting in the wild type characteristics of Glc-dependent repression,seedling development,and plant growth. Interestingly,Os HXK7-mediated Glc-dependent repression was abolished in the O_2-deficient mesophyll protoplasts of maize. This result provides compelling evidence that Os HXK7 functions in sugar signaling via a glycolysis-dependent manner under normal conditions,but its signaling role is suppressed when O_2 is deficient. The germination of two null Os HXK7 mutants,oshxk7-1 and oshxk7-2,was affected by O_2 deficiency,but overexpression enhanced germination in rice. This result suggests the distinct role that OsH XK7 plays in sugar metabolism and efficient germination by enforcing glycolysis-mediated fermentation in O_2-deficient rice.     

Glucose Signaling in Yeast Is Partially Mimicked by Galactose and Does Not Require the Tps1 Protein

Glucose produces multiple effects inSaccharomyces cerevisiae,as it controls the expression of many genes and the activity of various enzymes. However, the elements involved in glucose signaling are not well characterized. In this work the capacity of galactose to bring about the same effects than glucose has been assessed. Galactose mimics glucose only partially; it is suggested that it does not interact with a “sensor” in the plasma membrane and that it produces a weaker intracellular signal than glucose. To examine whether trehalose-6P synthase (Tps1) is required to transduce the glucose signal, we have constructed atps1 hxk2/tps1 HXK2strain which, at difference of atps1strain, grows on glucose, and, at difference of atps1 hxk2strain, still possess the Hxk2 protein, possibly involved in glucose repression. From the response of this strain to glucose, we conclude that Tps1 does not play a prominent role in glucose signaling.