Studies on the mechanism of the glucose‐induced cAMP signal in glycolysis and glucose repression mutants of the yeast Saccharomyces cerevisiae

Abstract

When glucose is added to cells of the yeast Succharomyces cerevisiae grown on non-fermentable carbon sources, a cAMP signal is induced which triggers a protein phosphorylation cascade. Addition of glucose or fructose to cells of a phosphoglucose isomerase mutant also induced the cAMP signal indicating that metabolization of the sugar beyond the sugar phosphate step is not necessary. Glucose 6-phosphate might stimulate the triggering reaction since induction with fructose shows a significant delay. Experiments with double and triple mutants in hexokinase 1, hexokinase 2 or glucokinase indicated that the presence of one of the three kinases was both necessary and enough for induction of the cAMP signal by glucose and the presence of one of the two hexokinases necessary and enough for induction by fructose. The product of the kinase reaction itself however does not appear to be the trigger of the reaction: when the increase in the level of glucose 6-phosphate and fructose 6-phosphate was measured as a function of time after addition of different glucose concentrations, no correlation was observed with the increase in the cAMP level. From the dependence of the cAMP increase on the external concentration of glucose, a rough estimate was obtained of the K , of the triggering reaction: about 25 mM. This value clearly fits with the K , of the low-affinity glucose carrier (about 20 mM) and differs by at least an order of magnitude from the K , values of the high-affinity glucose carrier and the three kinases. The present results situate the primary triggering reaction at the level of transport-associated phosphorylation. The main (= low-affinity) glucose carrier appears to be the receptor while association of the corresponding kinase is needed for induction of the signal. Since it is known that the presence of the kinases influences the characteristics of sugar transport, no definite conclusion can be given on whether the necessity of the kinases reflects the need for a certain type of transport or the need for phosphorylation of the sugar. The increase in the level of fructose 1,6-bisphosphate, on the other hand, correlated very well with the cAMP increase. However, it clearly lagged behind the cAMP increase, confirming the previously suggested importance of the cAMP signal for the stimulation of glycolytic flux at the level of phosphofructokinase 1. The importance of the cAMP signal for the stimulation of phosphofructokinase 1 also provides an explanation for the transient overshoot in the levels of glucose 6-phosphate and fructose 6-phosphate which are observed after addition of glucose to derepressed yeast cells. Addition of glucose to glucoserepressed wild-type cells triggers no or just a very weak cAMP signal, indicating that one of the intermediates in the induction sequence must be glucose repressible. This conclusion was confirmed by experiments with wild-type cells grown on either galactose or maltose (which have less glucose repression) and with cells of the hxk2 mutant which is deficient in glucose repression...

The Ca2+ Homeostasis Defects in a pgm2Δ Strain of Saccharomyces cerevisiae Are Caused by Excessive Vacuolar Ca2+ Uptake Mediated by the Ca2+-ATPase Pmc1p

“…Ca 2ϩ Uptake Assays in Isolated Membranes-To assay for Ca 2ϩ transport activity in sucrose gradient fractions, 0.7 ml of O-Buffer (10 mM HEPES-NaOH, pH 6.7, 150 mM KCl, 5 mM MgCl 2 , 0.5 mM ATP (pre-buffered to pH 6.7), 5 mM NaN 3 , 0.5 Ci/ml 45 CaCl 2 (9.6 mCi/mg)) was added to 0.3 ml of gradient fraction and incubated for 12 min at 25°C (36). The entire 1-ml sample was then collected by filtration through a pre-washed nitrocellulose filter (Millipore, HAWP02500) and washed twice with 5 ml of ice-cold wash buffer (10 mM HEPES-NaOH, pH 7.5, 150 mM KCl) (36).…”

“…The entire 1-ml sample was then collected by filtration through a pre-washed nitrocellulose filter (Millipore, HAWP02500) and washed twice with 5 ml of ice-cold wash buffer (10 mM HEPES-NaOH, pH 7.5, 150 mM KCl) (36). Membrane-sequestered 45 Ca 2ϩ was then determined by liquid scintillation counting. Where indicated, Ca 2ϩ transport by Vcx1p was inhibited by the addition of 25 M carbonyl cyanide m-chlorophenylhydrazone (Sigma).…”

“…The medium was then supplemented with 45 Ca 2ϩ , and growth was continued to a cell density of 0.5-1 A 600 /ml. The cells were then harvested by centrifugation at 4000 ϫ g for 5 min, washed, and resuspended in fresh YPGal supplemented with 50 mM CaCl 2 .…”

“…YPD and YPGal media were routinely buffered to pH 5.5 with 40 mM MES-Tris. To prepare membranes for in vitro 45 Ca 2ϩ uptake assays, cells were grown in Yeast Mito medium containing 6.7 g of yeast nitrogen base (Difco) and 0.3% yeast extract (Difco)/liter in the presence of 2% glucose or 2% galactose (YMMG) (33). Liquid cultures were grown for a minimum of five generations to Յ1.0 A 600 units/ml before harvesting.…”

Signalling pathways leading to transcriptional regulation of genes involved in the activation of glycolysis in yeast

“…Evidence has been found for the involvement of multiple signalling pathways in sensing glucose (Beullens et al, 1988;Ö zcan and Johnston, 1995;Griffioen et al, 1996;Pernambuco et al, 1996;Yin et al, 1996). Among these, the main glucose repression pathway and the Ras-adenylate cyclase pathway have been characterized in some detail (Thevelein, 1994, and references therein).…”