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J M Gancedo

Publications and source records attributed to J M Gancedo.

At least 37 records · Page 2Linked to original sources
PubMed1992

Regulatory regions in the yeast FBP1 and PCK1 genes.

By deletion analysis of the fusion genes FBP1-lacZ and PCK1-lacZ we have identified a number of strong regulatory regions in the genes FBP1 and PCK1 which encode fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase. Lack of expression of beta-galactosidase in fusions lacking sequences from the coding regions suggests the existence of downstream activating elements. Both promoters have several UAS and URS regions as well as sites implicated in catabolite repression. We have found in both genes consensus sequences for the binding of the same regulatory proteins, such as yAP1, MIG1 or the complex HAP2/HAP3/HAP4. Neither deletion nor overexpression of the MIG1 gene affected the regulated expression of the FBP1 or PCK1 genes.

Base Sequence↗
PubMed1991

Regions in the promoter of the yeast FBP1 gene implicated in catabolite repression may bind the product of the regulatory gene MIG1.

We have identified in the promoter of the yeast FBP1 gene two sites able to bind nuclear proteins. These sites have a nucleotide sequence strongly similar to that of sites which bind the regulatory protein MIG1 in the promoters of GAL4 and SUC2. Deletions performed in the FBP1 promoter showed that one of the sites contributes to catabolite repression of this gene. In this same promoter, another region was identified with a strong effect on the catabolite repression of FBP1. In this region a sequence similar to the consensus for the binding site of the MIG1 protein was also present.

Base Sequence↗
PubMed1988

Regulation of yeast fructose-1,6-bisphosphatase in strains containing multicopy plasmids coding for this enzyme.

The structural gene for yeast fructose-1,6-bisphosphatase (FbPase) has been cloned by complementation of a strain carrying the fbp1-1 mutation and lacking FbPase activity. By Northern analysis it could be shown that the fbp1-1 mutation does not interfere with the transcription of the gene although no normal active enzyme is produced. Strains transformed with the cloned gene on a multicopy plasmid overproduce FbPase but are still subject to strong catabolite repression. Catabolite inactivation, however, is slowed down in such strains.

Blotting, Northern↗
PubMed1987

Internal acidification and cAMP increase are not correlated in Saccharomyces cerevisiae.

Addition of glucose to a yeast suspension can produce both an increase in the level of cAMP and a decrease in the intracellular pH. This observation led to the idea that internal acidification triggers the cAMP increase. We have tested this hypothesis using different approaches. To study the effect of sugar metabolism on internal pH we added to the yeast either glucose or a sugar, like xylose, that cannot be phosphorylated. We also utilized yeast strains lacking hexose kinases or phosphoglucose isomerase. We found that phosphorylation of the sugar added is a requisite for internal acidification but not for the cAMP increase. Internal acidification is due to an imbalance between the rate of the metabolic reactions that generate protons and the rate at which protons can be pumped out of the cell. We have manipulated the excretion of protons by using yeast harvested at different phases of growth and resuspended in a medium with or without added K+. Addition of glucose produced a marked drop in internal pH only when the yeast was harvested in the stationary phase of growth and transferred to a medium without added K+. In contrast an increase in cAMP was observed in all situations. We conclude that in yeast there is no correlation between internal acidification and cAMP increase.

Carbohydrate Metabolism↗
PubMed1985

Phosphorylation of 3-O-methyl-D-glucose and catabolite repression in yeast.

The glucose analog, 3-O-methyl-D-glucose, inhibited growth of yeast on non-fermentable carbon sources. The sugar was phosphorylated by the yeast and also in vitro by a commercial preparation of yeast hexokinase. The chromatographic behaviour of the phosphorylated product was identical in both cases. This suggests that 3-O-methyl-D-glucose is phosphorylated to form 3-O-methyl-D-glucose 6-phosphate. The inhibition of the growth appears to be due to interference with the derepression of several enzymes necessary to grow on non-fermentable carbon sources. Spontaneous mutants whose growth was unaffected by 3-O-methyl-D-glucose were isolated. In these mutants there was no significant accumulation of the phosphorylated ester and the derepression of the enzymes tested was not affected by the glucose analog.

3-O-Methylglucose↗
PubMed1984

Catabolite repression in yeasts is not associated with low levels of cAMP.

relationship between levels of cAMP and catabolite repression in yeasts has been investigated. Strains of Saccharomyces cerevisiae, Schizosaccharomyces pombe and Kluyveromyces fragilis were used. The yeasts were grown on different carbon sources to attain various degrees of repression. Galactose repressed as much as glucose, while maltose was less effective. Full derepression was achieved with ethanol. The enzymes tested were fructose-bisphosphatase, malate dehydrogenase, glutamate dehydrogenase (NAD dependent), cytochrome oxidase and isocitrate lyase (this last enzyme was found to be absent in Schizosaccharomyces). The levels of cAMP were 2-3 times higher in the repressed conditions than in the derepressed ones. It is therefore concluded that in yeasts catabolite repression is not mediated by a lowering of the intracellular concentration of cAMP.

Cyclic AMP↗
PubMed1984

Metabolic imbalance in a Saccharomyces cerevisiae mutant unable to grow on fermentable hexoses.

A mutant of Saccharomyces cerevisiae unable to grow on fermentable hexoses has been studied. The mutant grew normally on galactose or maltose. It was also able to grow on a medium containing glucose or fructose with a 25-fold excess of D-xylose. Assay of the glycolytic enzymes in vitro did not show differences between the parental and the mutant strains. Upon addition of fructose, metabolites up to triose phosphates accumulated and the ATP dropped to low levels. It is proposed that an imbalance between the initial and final segments of glycolysis that depletes the cell of ATP produces the observed phenotype.

Adenosine Triphosphate↗
PubMed1983

Fructose 2,6-bisphosphate activates the cAMP-dependent phosphorylation of yeast fructose-1,6-bisphosphatase in vitro.

Fructose-1,6-bisphosphatase purified from Saccharomyces cerevisiae is phosphorylated in vitro by a cAMP-dependent protein kinase. The phosphorylation reaction incorporates 1 mol of phosphate/mol of enzyme and is greatly stimulated by fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate acts upon fructose-1,6-bisphosphatase, not on the protein kinase. The phosphorylation of fructose 1,6-bisphosphatase lowers its activity by about 50%. The characteristics of the phosphorylation reaction in vitro show that this modification is responsible for the inactivation of fructose-1,6-bisphosphatase observed in vivo.

Cyclic AMP↗
PubMed1982

Calculation of half-lives of proteins in vivo. Heterogeneity in the rate of degradation of yeast proteins.

A method is given for the calculation of half-lives of proteins in vivo from the measurement of the decrease of radioactivity in pulse-labelled proteins with time. This method could be particularly useful for the study of the degradation of proteins in cells that have a low growth rate. The method applied to growing yeast indicates that there are two major classes of protein. The class with low turnover constitutes the bulk of yeast protein and has a half-life of 160 h in a medium with glucose or galactose and of 50 h in a medium with ethanol. The class of proteins with high turnover (half-life between 0.8 and 2.4 hours) represents from 1% of total protein in yeast growing on glucose to 7% in yeast growing on ethanol. It is shown that some proteins which are depressed during growth on ethanol or induced during growth on galactose are particularly susceptible to degradation in a medium which contains glucose. It is proposed that protein degradation is regulated by a coarse control at the level of protease activity and a fine control on the susceptibility of individual proteins to proteases.

Ethanol↗
PubMed1982

Inactivation of yeast fructose-1,6-bisphosphatase. In vivo phosphorylation of the enzyme.

Incorporation of 32P into yeast fructose-1,6-bisphosphatase (EC 3.1.3.11) was observed after addition of glucose to a cell suspension incubated with (32P)orthophosphoric acid. The 32P counts were coincident with the enzyme band when immunoprecipitates were subjected to sodium dodecyl sulfate disc gel electrophoresis. The incorporation of phosphate was associated with a decrease in enzyme activity. Approximately 1 mol of phosphate was incorporated/mol of enzyme. The phosphate is bound to the enzyme in a phosphoester linkage with a serine residue. Release of 32P accompanying enzyme reactivation was observed both in vivo and in cell-free extracts.

Fructose-Bisphosphatase↗
PubMed1982

Phosphorylation and inactivation of yeast fructose-bisphosphatase in vivo by glucose and by proton ionophores. A possible role for cAMP.

Addition of glucose to yeast cells causes a phosphorylation and an inactivation of the gluconeogenic enzyme fructose-bisphosphatase [Mazón, M.J., Gancedo, J.M., and Gancedo, C. (1982) J. Biol. Chem. 257, 1128-1130]. We report here that the addition of the proton ionophores 2,4-dinitrophenol and carbonylcyanide m-chlorophenylhydrazone to yeast cells produces the same effect as that of glucose. Both glucose and ionophores produced: (a) phosphorylation and inactivation of fructose-bisphosphatase, (b) an immediate rise in the intracellular concentration of cAMP, (c) an instant inhibition of the transport of amino acids driven by the membrane potential. It is proposed that the effect of glucose on fructose-bisphosphatase involves as a first step the depolarization of the plasma membrane resulting in an increase of the intracellular concentration of cAMP. This in turn would stimulate phosphorylation of fructose-bisphosphatase.

Biological Transport, Active↗
PubMed1980

Turnover of yeast fructose-bisphosphatase in different metabolic conditions.

Earlier work demonstrated that addition of glucose to yeast growing on noncarbohydrate carbon sources sharply reduces the levels of fructose bisphosphatase. This report indicates that the decrease in the levels of fructose bisphosphatase is accompanied by a parallel decrease of cross-reacting material to specific antibody to fructose bisphosphatase. Use of the specific antibody shows that the loss of activity is irreversible and that its reapperance requires synthesis of protein de novo. The protein is highly stable during growth in ethanol (half life about 90 h). Addition of glucose increases the rate of degradation abut 200-fold. It is shown that the values of the rates of synthesis and degradation of fructose bisphosphatase vary with the metabolic situation of the yeast.

Drug Stability↗
PubMed1979

Effect of metabolic conditions on protein turnover in yeast.

1. In yeast growing on ethanol a turnover rate of up to 2%/h was measured. As much as 80% of the protein was subject to turnover, and no marked heterogeneity in the rate of degradation of protein was observed. When the yeast grew on glucose, the protein was degraded at a lower rate (0.5-1%/h). 2. Starvation for a nitrogen source increased the rate of protein degradation severalfold, whereas deprivation of phosphate had only a marginal effect (30% increase). Removal of glucose from a medium containing 50mM-phosphate did not cause marked changes in the rate of protein degradation. In contrast, when the media were low in phosphate (0.1 mM) removal of glucose increased the rate of turnover 2-4-fold. 3. Protein degradation proceeded unimpaired when the intracellular concentration of ATP decreased from 4 to 1 mM, but stopped completely when it decreased below 0.3 mM.

Adenosine Triphosphate↗
PubMed1979

Inactivation of gluconeogenic enzymes in glycolytic mutants of Saccharomyces cerevisiae.

Yeast mutants blocked at different steps of the glycolytic pathways have been used to study the inactivation of several gluconeogenic enzymes upon addition of sugars. While phosphorylation of the sugars appears a requisite for the inactivation of fructose 1,6-bisphosphatase and phosphoenol-pyruvate carboxykinase, malate dehydrogenase is inactivated by fructose in mutants lacking hexokinase. The normal inactivation elicited by glucose in a mutant lacking phosphofructokinase indicates that the process does not require metabolism of the sugar beyond hexose monophosphates. A possible role for ATP in the inactivation process is suggested.

Fructose-Bisphosphatase↗