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...

Addition of glucose to Saccharomyces cerevisiae cells grown on a nonfermentable carbon source triggers a cyclic AMP (cAMP) signal, which induces a protein phosphorylation cascade. In a yeast strain lacking functional RAS] and RAS2 genes and containing a bcy mutation to suppress the lethality of RAS deficiency, the cAMP signal was absent. Addition of dinitrophenol, which stimulates in vivo cAMP synthesis by lowering intracellular pH, also did not enhance the cAMP level. A bcy control strain, with functional RAS genes present, showed cAMP responses similar to those of a wild-type strain. In disruption mutants containing either a functional RAS] gene or a functional RAS2 gene, the cAMP signal was not significantly different from the one in wild-type cells, indicating that RAS function cannot be a limiting factor for cAMP synthesis during induction of the signal. Compared with wild-type cells, the cAMP signal decreased in intensity with increasing temperature in a ras2 disruption mutant. When the mutant RAS2V'-l9, which carries the equivalent of the human H-rasVa~2 oncogene, was grown under conditions in which RAS] expression is repressed, the cAMP signal was absent. The oncogene product is known to be deficient in GTPase activity. However, the amino acid change at position 19 (or 12 in the corresponding human oncogene product) might also have other effects, such as abolishing receptor interaction. Such an additional effect probably provides a better explanation for the lack of signal transmission than the impaired GTPase activity. When the RAS2val-19 mutant was grown under conditions in which RAS] is expressed, the cAMP signal was present but significantly delayed compared with the signal in wild-type cells. This indicates that oncogenic RAS proteins inhibit normal functioning of wild-type RAS proteins in vivo and also that in spite of the presence of the RAS2va'-9 oncogene, adenyl cyclase is not maximally stimulated in vivo. Expression of only the RAS2Va'-l9 gene product also prevented most of the stimulation of cAMP synthesis by dinitrophenol, indicating that lowered intracellular pH does not act directly on adenyl cyclase but on a step earlier in the activation pathway of the enzyme. The results obtained with the control bcy strain, the RAS2va'-I9 strain under conditions in which RAS] is expressed, and with dinitrophenol show that the inability of the oncogene product to mediate the cAMP signal is not due to feedback inhibition by the high protein kinase activity in strains containing the RAS2Va-l9 oncogene. Hence, the present results show that the RAS proteins in S. cerevisiae are involved in the transmission of the glucose-induced cAMP signal and that the oncogenic RAS protein is unable to act as a signal transducer. The RAS proteins in S. cerevisiae apparently act similarly to the G. proteins of mammalian adenyl cyclase, but instead of being involved in hormone signal transmission, they function in a nutrient-induced signal transmission pathway. (23,31) and transient intracellular acidification (10,36,46) we...

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 glucose to derepressed cells of the yeast Saccharomyces cerevisiae induces a transient, specific cAMP signal. Intracellular acidification in these cells, as caused by addition of protonophores like 2,4-dinitrophenol (DNP) causes a large, lasting increase in the cAMP level. The effect of glucose and DNP was investigated in glucose-repressed wild type cells and in cells of two mutants which are deficient in derepression of glucose-repressible proteins, cat1 and cat3. Addition of glucose to cells of the cat3 mutant caused a transient increase in the cAMP level whereas cells of the cat1 mutant and in most cases also repressed wild type cells did not respond to glucose addition with a cAMP increase. The glucose-induced cAMP increase in cat3 cells and the cAMP increase occasionally present in repressed wild type cells however could be prevented completely by addition of a very low level of glucose in advance. In derepressed wild type cells this does not prevent the specific glucose-induced cAMP signal at all. These results indicate that repressed cells do not show a true glucose-induced cAMP signal. When DNP was added to glucose-repressed wild type cells or to cells of the cat1 and cat3 mutants no cAMP increase was observed. Addition of a very low level of glucose before the DNP restored the cAMP increase which points to lack of ATP as the cause for the absence of the DNP effect. These data show that intracellular acidification is able to enhance the cAMP level in repressed cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Saccharomyces cerevisiae bypl-3 mutants displayed a long lag phase when shifted from a nonfermentable carbon source to a medium containing glucose. The bypl-3 mutation also caused several defects in regulatory phenomena which occur during the transition from the derepressed state to the repressed state. As opposed to wild-type cells, the addition of glucose to cells of the bypl-3 mutant grown on nonfermentable carbon sources did not induce a cyclic AMP signal. Fructose-2,6-bisphosphate formation and inactivation of fructose-1,6-bisphosphatase were severely delayed, but trehalase activation was not affected. In addition, the induction of pyruvate decarboxylase both at the level of activity and that of transcription was very slow compared with that in wild-type cells. These pleiotropic defects in glucose-induced regulatory phenomena might be responsible for the very long lag phase of bypl-3 cells and the inability of ascospores to initiate growth after germination on glucose media. Screening of a yeast gene library for clones complementing the bypl-3 phenotype resulted in the isolation of a truncated form of the previously described zinc finger transcription repressor MIGi. The entire MIG) gene and the truncated form suppressed even on a single-copy vector the growth initiation defect but not the regulatory abnormalities of the bypl-3 mutant. MIG) is not allelic to bypl-3.The addition of glucose to derepressed cells of the yeast Saccharomyces cerevisiae causes a series of rapid changes in enzyme activity due to posttranslational modification (12, 20) followed by slower changes which are due to repression (6) or induction (e.g., reference 17) at the transcriptional level. Identification of the glucose-induced signaling pathways triggering these events has attracted a lot of attention. Evidence has been obtained that cyclic AMP (cAMP)-dependent protein phosphorylation is involved in rapid inactivation of fructose-1,6-bisphosphatase, isocitrate lyase, and the galactose and high-affinity glucose carrier and in rapid activation of trehalase and phosphofructokinase 2 (22). The addition of glucose to derepressed yeast cells causes a rapid signal-like spike in the cAMP level (21). Incubation of temperature sensitive mutants in cAMP synthesis at the restrictive temperature or cAMP-requiring mutants in the absence of cAMP abolishes the glucose-induced cAMP signal and also the inactivation of fructose-1,6-bisphosphatase (27) and the activation of trehalase and phosphofructokinase 2 (7). This has been taken as evidence that the glucose-induced cAMP signal triggers phosphorylation of the enzymes by causing activation of cAMP-dependent protein kinase. Under these conditions, however, not only is the cAMP signal abolished, but the basal cAMP level in the cells is also much lower than that in wild-type cells. It has never been investigated whether the absence of the cAMP * Corresponding author. monophosphates and sedoheptulose-7-phosphate, a metabolite of the pentosephosphate pathway, accumulate at unusually high levels (4). Howe...