Physiological role of glucose-phosphorylating enzymes in Saccharomyces cerevisiae

Lobo, Zita; Maitra, P. K.

doi:10.1016/0003-9861(77)90544-6

Cited by 80 publications

(49 citation statements)

References 8 publications

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“…2) Glk1. Each of these enzymes can support growth on glucose, while either of the two hexokinases is required for growth on fructose (Lobo & Maitra, 1977;Gancedo et al, 1977). Hxk2, in addition, is mainly required for catabolite repression, which prevents the expression of genes involved in catabolism of less preferred carbon sources in the presence of a more favourable nutrient such as glucose (Entian, 1980).…”

Section: Introductionmentioning

confidence: 99%

The Botrytis cinerea hexokinase, Hxk1, but not the glucokinase, Glk1, is required for normal growth and sugar metabolism, and for pathogenicity on fruits

Rui

Hahn

2007

Microbiology

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Hexose kinases play a central role in the initiation of sugar metabolism of living organisms and have also been implicated in carbon catabolite repression in yeasts and plants. In this study, the genes encoding glucokinase (Glk1) and hexokinase (Hxk1) from the plant-pathogenic ascomycete Botrytis cinerea were isolated and functionally characterized. Glk1-deficient mutants were indistinguishable from the wild-type in all growth parameters tested. In contrast, Dhxk1 mutants lacking Hxk1 showed a pleiotropic growth defect. On artificial media, vegetative growth was retarded, and conidia formation strongly reduced. No or only marginal growth of Dhxk1 mutants was observed when fructose, galactose, sucrose or sorbitol were used as carbon sources, and fructose inhibited growth of the mutant in the presence of other carbon sources. B. cinerea mutants containing hxk1 alleles with point mutations leading to enzymically inactive enzymes showed phenotypes similar to the Dhxk1 disruption mutant, indicating that loss of hexose phosphorylation activity of Hxk1 is solely responsible for the pleiotropic growth defect. Virulence of the Dhxk1 mutants was dependent on the plant tissue: on leaves, lesion formation was only slightly retarded compared to the wild-type, whereas only small lesions were formed on apples, strawberries and tomatoes. The low virulence of Dhxk1 mutants on fruits was correlated with their high contents of sugars, in particular fructose. Heterologous expression of Hxk1 and Glk1 in yeast allowed their enzymic characterization, revealing kinetic properties similar to other fungal hexokinases and glucokinases. Both Dglk1 and Dhxk1 mutants showed normal glucose repression of secreted lipase 1 activity, indicating that, in contrast to yeast, B. cinerea hexose kinases are not involved in carbon catabolite repression. INTRODUCTIONHexose kinases are crucial enzymes for most living cells, by catalysing the intracellular trapping and feeding into metabolism of the hexoses glucose and fructose after their uptake. In addition, hexokinases have been shown in yeast, plants and mammals to participate in glucose signalling (De Winde et al., 1996;Moore et al., 2003;Efrat et al., 1994). Saccharomyces cerevisiae has three enzymes catalysing hexose phosphorylation, namely hexokinases (EC 2 . 7 . 1 . 1) Hxk1 and Hxk2, and glucokinase (EC 2 . 7 . 1 . 2) Glk1. Each of these enzymes can support growth on glucose, while either of the two hexokinases is required for growth on fructose (Lobo & Maitra, 1977;Gancedo et al., 1977). Hxk2, in addition, is mainly required for catabolite repression, which prevents the expression of genes involved in catabolism of less preferred carbon sources in the presence of a more favourable nutrient such as glucose (Entian, 1980). In the presence of glucose, Hxk2 interacts with the carbon catabolite repressor Mig1 and thereby is translocated into the nucleus. In the nucleus, the Hxk2-Mig1 complex seems to form a repressor complex that binds to the promoters of carbon-catabolite-repressible genes (Ahuatzi ...

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Section: Introductionmentioning

confidence: 99%

The Botrytis cinerea hexokinase, Hxk1, but not the glucokinase, Glk1, is required for normal growth and sugar metabolism, and for pathogenicity on fruits

Rui

Hahn

2007

Microbiology

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“…However, the HXK2 gene product appears to play the main role during glucose phosphorylation in i o, because it is the isoenzyme that predominates in growth on glucose [1,2]. The two other isoenzymes are also singly adequate for growth on glucose, but their normal expression is highest on other carbon sources [2,3]. Furthermore, various genetic and biochemical evidence has shown that the hexokinase 2 protein (Hxk2p) is also important in regulating glucose repression [4,5] ; in particular, a HXK2 mutant gene (HXK2∆K(M"'), with a 30 bp deletion between nucleotides j19 and j48, has been described in which glucose repression is absent but Hxk2 catalytic activity is intact [6].…”

Section: Introductionmentioning

confidence: 99%

The hexokinase 2 protein regulates the expression of the GLK1, HXK1 and HXK2 genes of Saccharomyces cerevisiae

Rodríguez

Herrero

et al. 2001

Biochemical Journal

188

145

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The key glycolytic HXK2 gene, coding for the enzyme hexokinase 2 (Hxk2p), is expressed when cells of the yeast Saccharomyces cere isiae are grown on a fermentable medium using glucose, fructose or mannose as a carbon source. After shifting the cells to a non-fermentable carbon source, the HXK2 gene is repressed and the HXK1 and GLK1 genes are rapidly de-repressed, producing the enzymes hexokinase 1 (Hxk1p) and glucokinase (Glk1p) respectively. Because the in i o functions of the Hxk1p and Glk1p enzymes have remained a mystery so far, we have investigated this glucose-induced regulatory process. Here we demonstrate the involvement of Hxk2p in the glucose-induced repression of the HXK1 and GLK1 genes and the glucoseinduced expression of the HXK2 gene. We have also demonstrated the involvement of Hxk1p as a negative factor in the expression of the GLK1 and HXK2 genes. Further experimental evidence, using mutant cells expressing a truncated version of

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“…It functions in vivo both in glycolysis (16,17) and in catabolite repression (6,20). Among the mutations that cause failures in glucose repression (25), some have reduced levels of hexokinase activity; these mutations were determined to be alleles of the structural gene of hexokinase II, HXK2 (6).…”

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confidence: 99%

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

Ma¹,

Bloom²,

Walsh³

et al. 1989

Mol. Cell. Biol.

100

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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 H is correlated with glucose repression.Hexokinase II is one of the two hexokinase isoenzymes of the budding yeast Saccharomyces cerevisiae. It functions in vivo both in glycolysis (16,17) and in catabolite repression (6,20). Among the mutations that cause failures in glucose repression (25), some have reduced levels of hexokinase activity; these mutations were determined to be alleles of the structural gene of hexokinase II, HXK2 (6). Biochemical analysis of one of the mutants (10) indicated that hexokinase II was indeed defective. The synthesis of invertase from the SUC3 gene (reviewed in references 3 and 4) was derepressed in these mutants even when cells were grown in medium with a high glucose concentration. These results suggest that hexokinase II mediates glucose repression in yeast cells. Mutations in another gene, HEX2 (which may be the same as REG] [22]), also cause failures in glucose repression, but hex2 strains have levels of hexokinase II activity higher than the wild-type level (7).It is difficult to interpret these results because mutations that either reduce (hxk2) or increase (hex2) hexokinase II catalytic activity can cause failures of glucose repression. To determine whether hexokinase II protein is required for glucose repression or derepression, we constructed null mutations in the HXKI (encoding hexokinase I) and HXK2 genes and studied their effects on glucose repression (20). The result that hxk2 null mutants do not show glucose repression indicates that hexokinase II is indeed required for glucose repression. Although hexokinase I is normally not required, it can partially suppress the hxk2 defect in glucose repression if overproduced. Entian and Frohlich (8) isolated mutations in the HXK2gene that cause defects in glucose repression yet still retain hexose phosphorylation activity. On the basis of these * Corresponding author. results and the conformational change observed in yeast hexokinases upon binding ...

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Physiological role of glucose-phosphorylating enzymes in Saccharomyces cerevisiae

Cited by 80 publications

References 8 publications

The Botrytis cinerea hexokinase, Hxk1, but not the glucokinase, Glk1, is required for normal growth and sugar metabolism, and for pathogenicity on fruits

The Botrytis cinerea hexokinase, Hxk1, but not the glucokinase, Glk1, is required for normal growth and sugar metabolism, and for pathogenicity on fruits

The hexokinase 2 protein regulates the expression of the GLK1, HXK1 and HXK2 genes of Saccharomyces cerevisiae

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

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