Constitutive glucose-induced activation of the Ras-cAMP pathway and aberrant stationary-phase entry on a glucose-containing medium in the Saccharomyces cerevisiae glucose-repression mutant hex2 (original) (raw)
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European Journal of Biochemistry, 1984
Glucose-induced inactivation of the gluconeogenetic enzymes fi-uctose-I ,6-bisphosphatase, cytoplasmic mala te dehydrogenase and phosphoenolpyruvate carboxykinase was tested in yeast mutants defective in adenylate cyclase (cj,rl mutation) and in the CAMP-binding subunit of CAMP-dependent protein kinase (hi.j.1 mutation). In the mutant AM7-I 1 D ( q r 1 mutation), glucose-induced CAMP overshoot was absent, and no significant inactivation of the gluconeogenetic enzymes was detected, thus supporting the role ofeAMP in the process. Moreover. in the mutant AM9-8B ( h q l mutation), no CAMP-dependent protein kinase activity was evidenced, and. in addition. a normal inactivation pattern was observed, thus indicating that other mechanisms evoked by glucose inight be required in the process. In the double mutant AM7-11DR-4 ( q r l /w;i~I mutations), no inactivating effect was triggered by the sugar: this suggests that CAMP exerts some additional effect o n the process, besides the activation of CAMP-dependent protein kinase. Furthermore, in AM7-11 D, extracellular CAMP triggered about 50% of inactivation of fructose-I ,6-bisphosphatase; this effect was largely reversed in acetate medium plus cqcloheximide even after 150 min of incubation. However, an extensive and essentially irreversible inactivation was evidenced i n the presence of glucose plus CAMP, whereas glucose alone was only slightly effective. Thereforc, the reversiblc effect of CAMP, which probably corresponds to enzyme phosphorylation, seeins to be required for the irrcvcrsible, probably proteolytic, glucose-stimulated inactivation of this enzyme. Cytoplasmic inalatc dehydrogenase and phosphoeriolpyruvate carboxykinase in AM7-11 D were also inactivated by CAMP, and much morc by glucose plus CAMP, whereas glucose was practically ineffective. However, reversibility of the effect was not detected. and, in addition, no phosphorylation of phosphoenolpyruvate carboxykinase could be evidenced. Thereforc, the sugar quite probably stimulates proteolysis of these enzymes, but the mechanism ofcAMP in their degt-adation has still to be defined.
FEMS Yeast Research, 2001
Glucose-induced cAMP signalling in Saccharomyces cerevisiae requires extracellular glucose detection via the Gpr1-Gpa2 G-protein coupled receptor system and intracellular glucose-sensing that depends on glucose uptake and phosphorylation. The glucose uptake requirement can be fulfilled by any glucose carrier including the Gal2 permease or by intracellular hydrolysis of maltose. Hence, the glucose carriers do not seem to play a regulatory role in cAMP signalling. Also the glucose carrier homologues, Snf3 and Rgt2, are not required for glucose-induced cAMP synthesis. Although no further metabolism beyond glucose phosphorylation is required, neither Glu6P nor ATP appears to act as metabolic trigger for cAMP signalling. This indicates that a regulatory function may be associated with the hexose kinases. Consistently, intracellular acidification, another known trigger of cAMP synthesis, can bypass the glucose uptake requirement but not the absence of a functional hexose kinase. This may indicate that intracellular acidification can boost a downstream effect that amplifies the residual signal transmitted via the hexose kinases when glucose uptake is too low. ß
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1992
Addition of glucose and other sugars to derepressed cells of the fungus Fusarium oxysporum var. Zini triggered activation of the plasma membrane H+-ATPase within 5 min. Glucose was the best activator while galactose and lactose had a lesser effect. The activation was not prevented by previous addition of cycloheximide and it was fully reversible when the glucose was removed. The activation process in uiuo also caused changes in the kinetic properties of the enzyme. The non-activated enzyme had an apparent K, of about 3.2 m M for ATP whereas the activated enzyme showed an apparent K,,, of 0.26 mM. In addition, the pH optimum of the H+-ATPase changed from 6.0 to 7.5 upon activation. The activated enzyme was more sensitive to inhibition by vanadate. When F. oxysporum was cultivated in media containing glucose as the major carbon source, enhanced M+-ATPase activity was largely confined to the period corresponding to the lag phase, i.e. just before the start of acidification of the medium. This suggests that the activation process might play a role in the onset of extracellular acidification. Addition of glucose to F. oxysporum var. Zini cells also caused an increase in the cAMP level. No reliable increase could be demonstrated for the other sugars. Addition of proton ionophores such as DNP and CCCP at pH 5-0 caused both a large increase in the intracellular level of cAMP and in the activity of the plasma membrane H+-ATPase. Inhibition of the DNP-induced increase in the cAMP level by acridine orange also resulted in inhibition of the activation of plasma membrane H+-ATPase. These results suggest a possible causal relationship between the activity of F. oxysporum var. Zini plasma membrane H+-ATPase and the intracellular level of CAMP.
European journal of biochemistry / FEBS, 1987
Changes in the concentration of several metabolites and enzymes related to carbohydrate metabolism were measured during the growth of Saccharomyces cerevisiae on a mineral medium containing glucose as the limiting nutrient. When about 50% of the original glucose was used the exponential phase ended and the culture entered a 'transition' phase before the complete exhaustion of glucose. In this transition phase several metabolic changes occurred. cAMP, that decreased along growth, reached a constant value of about 0.7 nmol/g dry weight. A pronounced drop in fructose-6-phosphate-2-kinase activity and in the concentration of fructose 2,6-bisphosphate and fructose 1,6-bisphosphate was observed accompanied by a less marked decrease in hexose monophosphates. Trehalase activity also dropped and reached a minimal value at the onset of the stationary phase when synthesis of trehalose began. Glycogen concentration and glycogen synthase activity increased sharply during the transition p...
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2002
The aim of this study was to investigate the effects of an overactivation of the cAMP/protein kinase A signaling pathway on the energetic metabolism of growing yeast. By using a cAMP-permeant mutant strain, we show that the rise in intracellular cAMP activates both anabolic and catabolic pathways. Indeed, different physiological patterns were observed with respect to the growth condition: (i) When cells were grown with a limiting amount of lactate, cAMP addition markedly increased the growth rate, whereas it only slightly increased the mitochondrial and cellular protein content. In parallel, the respiratory rate increased and the growth yield, as assessed by direct microcalorimetry, was not significantly modified by cAMP. (ii) Under conditions where the growth rate was already optimal (high lactate concentration), exogenous cAMP led to a proliferation of well-coupled mitochondria within cells and to an accumulation of cellular and mitochondrial proteins. This phenomenon was associated with a rise in the respiratory activity, thus leading to a drop in the growth yield. (iii) Under conditions of catabolic repression (high glucose concentration), cAMP addition markedly increased the fermentation rate and decreased the growth yield. It is concluded that overactivation of the cAMP/PKA pathway leads to uncoupling between biomass synthesis and catabolism, under conditions where an optimal growth rate is sustained by either a fermentative or a respiratory metabolism. D : S 0 0 0 5 -2 7 2 8 ( 0 2 ) 0 0 2 4 0 -2
Proceedings of The National Academy of Sciences, 1993
The systems which control the levels of the gluconeogenic enzymes in Saccharomyces cerevisiae have been bypassed to ascertain their physiological significance. The coding regions of the genes FBP1 and PCK1, which encode fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, have been put under the control of the promoter of ADC1 (alcohol dehydrogenase I), a gene not repressed by glucose, and introduced into yeast in multicopy plasmids. The transformed yeast cells show high levels of the gluconeogenic enzymes during growth on glucose. Generation time and growth yield of yeast expressing either fructose-1,6-bisphosphatase or phosphoenolpyruvate carboxykinase are not significantly different from those of the wild-type strain. For a strain expressing both enzymes the increase in generation time is about 20% and the decrease in growth yield around 30%. The concentration of ATP is about 1.5 mM in the growing cells of the different strains. The extent of in vivo cycling was measured by 13 C NMR in cell-free extracts from yeast growing on [6-13 C]glucose. Cycling between fructose-6-phosphate and fructose-1,6-bisphosphate is <2%, most likely due to the very strong inhibition of fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate. Cycling between phosphoenolpyruvate and pyruvate is low, but a precise figure could not be obtained due to poor equilibration of label between carbons 2 and 3 of oxaloacetate.
Microbiology, 1991
Summary Addition of glucose or related fermentable sugars to derepressed cells of the yeast Saccharomyces cerevisiae triggers a RAS-protein-mediated cAMP signal, which induces a protein phosphorylation cascade. Yeast strains without a functional CDC25 gene were deficient in basal cAMP synthesis and in the glucose-induced cAMP signal. Addition of dinitrophenol, which in wild-type strains strongly stimulates in vivo cAMP synthesis by lowering intracellular pH, did not enhance the cAMP level. cdc25 disruption mutants, in which the basal cAMP level was restored by the RAS2 va119 oncogene or by disruption of the gene (PDE2) coding for the high-affinity phosphodiesterase, were still deficient in the glucose- and acidification-induced cAMP responses. These results indicate that the CDC25 gene product is required not only for basal cAMP synthesis in yeast but also for specific activation of cAMP synthesis by the signal transmission pathway leading from glucose to adenyl cyclase. They also s...
Molecular Microbiology, 1999
In the yeast Saccharomyces cerevisiae, the addition of glucose to derepressed cells and intracellular acid-i®cation trigger a rapid increase in the cAMP level within 1 min. We have identi®ed a mutation in the genetic background of several related`wild-type' laboratory yeast strains (e.g. ENY.cat80-7A, CEN.PK2-1C) that largely prevents both cAMP responses, and we have called it lcr1 (for lack of cAMP responses). Subsequent analysis showed that lcr1 was allelic to CYR1/CDC35, encoding adenylate cyclase, and that it contained an A to T substitution at position 5627. This corresponds to a K1876M substitution near the end of the catalytic domain in adenylate cyclase. Introduction of the A5627T mutation into the CYR1 gene of a W303-1A wild-type strain largely eliminated glucose-and acidi®cation-induced cAMP signalling and also the transient cAMP increase that occurs in the lag phase of growth. Hence, lysine 1876 of adenylate cyclase is essential for cAMP responses in vivo. Lysine 1876 is conserved in Schizosaccharomyces pombe adenylate cyclase. Mn 2 -dependent adenylate cyclase activity in isolated plasma membranes of the cyr1 met1876 (lcr1) strain was similar to that in the isogenic wild-type strain, but GTP/Mg 2 -dependent activity was strongly reduced, consistent with the absence of signalling through adenylate cyclase in vivo. Glucose-induced activation of trehalase was reduced and mobilization of trehalose and glycogen and loss of stress resistance were delayed in the cyr1 met1876 (lcr1) mutant. During exponential growth on glucose, there was little effect on these protein kinase A (PKA) targets, indicating that the importance of glucose-induced cAMP signalling is restricted to the transition from gluconeogenic/respiratory to fermentative growth. Inhibition of growth by weak acids was reduced, consistent with prevention of the intracellular acidi®cation effect on cAMP by the cyr1 met1876 (lcr1) mutation. The mutation partially suppressed the effect of RAS2 val19 and GPA2 val132 on several PKA targets. These results demonstrate the usefulness of the cyr1 met1876 (lcr1) mutation for epistasis studies on the signalling function of the cAMP pathway.