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...

Recently a new family of membrane proteins comprising the bovine lens fibre major intrinsic protein, soybean nodulin‐26 protein and the Escherichia coli glycerol facilitator has been described [M.E. Baker and M.H. Saier, Jr (1990) Cell, 60, 185–186]. These proteins have six putative membrane spanning domains and one (probably intracellular) intermembrane fragment is particularly well conserved. We have identified a new member of this family in the yeast Saccharomyces cerevisiae. It also possesses the six transmembrane domains and the highly conserved intermembrane sequence. In contrast to the other three proteins which are all approximately 280 amino acids long, the yeast protein has an N‐terminal extension of approximately 250 amino acids, which contains a string of 17 asparagine residues and a C‐terminal extension of approximately 150 amino acids. The gene, which we called FPS1 (for fdp1 suppressor), suppresses in single copy the growth defect on fermentable sugars of the yeast fdp1 mutant but it is not allelic to FDP1. The deficiency of the fdp1 mutant in glucose‐induced RAS‐mediated cAMP signalling and in rapid glucose‐induced changes in the activity of certain enzymes was not restored. Deletion of FPS1 does not cause any of the phenotypic deficiencies of the fdp1 mutant.

Addition of glucose or related fermentable sugars to derepressed cells of the yeast Saccharomyces cerevisiae triggers a RAS-mediated cyclic AMP (cAMP) signal that induces a protein phosphorylation cascade. In yeast mutants (tpklwl, tpk2", and tpk3w') containing reduced activity of cAMP-dependent protein kinase, fermentable sugars, as opposed to nonfermentable carbon sources, induced a permanent hyperaccumulation of cAMP. This finding confirms previous conclusions that fermentable sugars are specific stimulators of cAMP synthesis in yeast cells. Despite the huge cAMP levels present in these mutants, deletion of the gene (BCYI) coding for the regulatory subunit of cAMP-dependent protein kinase severely reduced hyperaccumulation of cAMP. Glucose-induced hyperaccumulation of cAMP was also observed in exponential-phase glucose-grown cells of the tpklwl and tpk2w' strains but not the tpk3w' strain even though addition of glucose to glucose-repressed wild-type cells did not induce a cAMP signal. Investigation of mitochondrial respiration by in vivo 31P nuclear magnetic resonance spectroscopy showed the tpklwl and tpk2wl strains, but not the tpk3Wl strain, to be defective in glucose repression. These results are consistent with the idea that the signal transmission pathway from glucose to adenyl cyclase contains a glucose-repressible protein. They also show that a certain level of cAMP-dependent protein phosphorylation is required for glucose repression. Investigation of the glucose-induced cAMP signal and glucose-induced activation of trehalase in derepressed cells of strains containing only one of the wild-type TPK genes indicates that the transient nature of the cAMP signal is due to feedback inhibition by cAMP-dependent protein kinase.Cells of the yeast Saccharomyces cerevisiae contain two RAS genes, RAS] and RAS2, which are homologs of the mammalian ras genes H-ras, K-ras, and N-ras (21). The RAS proteins are plasma membrane-bound G proteins that are generally considered to be signal transmitters. The yeast RAS proteins were shown to act as controlling elements of adenyl cyclase (21). The product of the yeast gene CDC25 is required for RAS activity and hence for cyclic AMP (cAMP) synthesis in vivo (2). Addition of glucose or related fermentable sugars to derepressed yeast cells triggers a transient increase in the cAMP level which is mediated by the CDC25 and RAS proteins (12, 13). The hormonelike effect of glucose on adenyl cyclase, which resembles the well-known hormone effects on adenyl cyclase in mammalian cells, induces a protein phosphorylation cascade that mobilizes the storage sugar trehalose and stimulates the switch of metabolism from gluconeogenic to fermentative (16).In yeast cells, three genes, TPKJ, TPK2, and TPK3, code for the catalytic subunit of cAMP-dependent protein kinase (19). The presence of one of the three is enough for viability. Recently, yeast mutants tpkl"', tpk2"', and tpk3"' that show greatly reduced activity of cAMP-dependent protein kinase were isolated (14). In these strains, two ...

Addition of plasma membrane depolarizing agents, such as dinitrophenol (DNP) and azide, to cells of Saccharomyces cerevisiae under aerobic conditions, is known to cause an increase in the cAMP level within 15 s. We found that both compounds lowered the intracellular pH (measured by in vivo 32P-NMR) drastically within the same time period. Plasma membrane depolarization, however, was much slower: DNP and azide had no effect on the membrane potential during, respectively, the first 2 min and the first 10 min after addition. Apparently, the intracellular pH of yeast is much more sensitive to perturbation than the membrane potential. The effect of both compounds on the cAMP level was highly dependent on the extracellular pH: when the latter was raised, the effect disappeared completely between pH 6 and 7. A similar dependence on the extracellular pH was observed for the lowering of intracellular pH. Addition of organic acids, such as acetate and butyrate, at low pH and under aerobic conditions, also caused an immediate increase in the cAMP level and an immediate drop in the intracellular pH. These results suggest that agents such as DNP and azide do not raise the cAMP level in yeast cells because of their membrane depolarizing properties but because they lower the intracellular pH. Under anaerobic conditions, DNP, azide and organic acids were much less effective in increasing the cAMP level. Addition of a small amount of glucose, however, restored their capacity to enhance the cAMP level. This suggests that under anaerobic conditions and in the absence of glucose the ATP level is a limiting factor for cAMP synthesis.

Addition of glucose to derepressed cells of the yeast Saccharomyces cerevisiae is known to cause a rapid, transient increase in the cAMP level, which lasts for 1-2min and induces a CAMPdependent protein phosphorylation cascade. The glucose-induced cAMP signal cannot be explained solely on the basis of an increased ATP level. Transient membrane depolarization and transient intracellular acidification have been suggested as possible triggers for the cAMP peak. Addition of glucose to cells in which the plasma membrane had been depolarized still produced the increase in the cAMP level excluding membrane depolarization as the possible trigger. Using in vivo P NMR-spectroscopy we followed phosphate metabolism and the time course of the drop in the intracellular pH after addition of glucose with a time resolution of 15 s. Under aerobic conditions the initial pH and ATP level were high. On addition of glucose, they both showed a rapid, transient drop, which lasted for about 30 s. Under anaerobic conditions, the initial pH and ATP level were low and on addition of glucose they both increased relatively slowly compared to aerobic conditions. Several conditions were found in which the pH drop which occurs under aerobic conditions could be blocked completely without effect on the cAMP signal or without completely preventing it: addition of NH4Cl together with glucose at high extracellular pH and addition of a low concentration of glucose before a high concentration. Also, when glucose was added twice to the same cells no consistent relationship was observed between the pH drop and the cAMP peak. These results appear to exclude transient intracellular acidification as the trigger for the cAMP signal. Hence, we conclude that the effect of glucose cannot be explained on the basis of effects known to be caused by the membrane depolarizing compounds which cause increases in the cAMP level. A new, more specific kind of interaction appears to be involved.