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J M Gancedo

Publications and source records attributed to J M Gancedo.

At least 19 recordsLinked to original sources
PubMed2000

Mth1 receives the signal given by the glucose sensors Snf3 and Rgt2 in Saccharomyces cerevisiae.

We have determined that the mutant genes DGT1-1 and BPC1-1, which impair glucose transport and catabolite repression in Saccharomyces cerevisiae, are allelic forms of MTH1. Deletion of MTH1 had only slight effects on the expression of HXT1 or SNF3, but increased expression of HXT2 in the absence of glucose. A two-hybrid screen revealed that the Mth1 protein interacts with the cytoplasmic tails of the glucose sensors Snf3 and Rgt2. This interaction was affected by mutations in Mth1 and by the concentration of glucose in the medium. A double mutant, snf3 rgt2, recovered sensitivity to glucose when MTH1 was deleted, thus showing that glucose signalling may occur independently of Snf3 and Rgt2. A model for the possible mode of action of Snf3 and Rgt2 is presented.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Glucose signaling in yeast is partially mimicked by galactose and does not require the Tps1 protein.

Glucose produces multiple effects in Saccharomyces cerevisiae, as it controls the expression of many genes and the activity of various enzymes. However, the elements involved in glucose signaling are not well characterized. In this work the capacity of galactose to bring about the same effects than glucose has been assessed. Galactose mimics glucose only partially; it is suggested that it does not interact with a "sensor" in the plasma membrane and that it produces a weaker intracellular signal than glucose. To examine whether trehalose-6P synthase (Tps1) is required to transduce the glucose signal, we have constructed a tps1 hxk2/tps1 HXK2 strain which, at difference of a tps1 strain, grows on glucose, and, at difference of a tps1 hxk2 strain, still possess the Hxk2 protein, possibly involved in glucose repression. From the response of this strain to glucose, we conclude that Tps1 does not play a prominent role in glucose signaling.

Cyclic AMP↗
PubMed1999

Cyclic AMP can decrease expression of genes subject to catabolite repression in Saccharomyces cerevisiae.

External cyclic AMP (cAMP) hindered the derepression of gluconeogenic enzymes in a pde2 mutant of Saccharomyces cerevisiae, but it did not prevent invertase derepression. cAMP reduced nearly 20-fold the transcription driven by upstream activation sequence (UAS1FBP1) from FBP1, encoding fructose-1,6-bisphosphatase; it decreased 2-fold the activation of transcription by UAS2FBP1. Nuclear extracts from cells derepressed in the presence of cAMP were impaired in the formation of specific UASFBP1-protein complexes in band shift experiments. cAMP does not appear to act through the repressing protein Mig1. Control of FBP1 transcription through cAMP is redundant with other regulatory mechanisms.

Carboxy-Lyases↗
PubMed1998

A 27 kDa protein binds to a positive and a negative regulatory sequence in the promoter of the ICL1 gene from Saccharomyces cerevisiae.

IsocitrateICL1, is one of the key enzymes of the glyoxylate pathway, which operates as an anaplerotic route for replenishing the tricarboxylic acid cycle; it is required for growth of Saccharomyces cerevisiae on carbon sources such as ethanol, but is dispensable when fermentable carbon sources are available. The positive regulation of the ICL1 gene by an upstream activating sequence (UAS) element located between -397 and -388 has been previously reported. In this paper we show that the ICL1 promoter sequence 5'-AGTCCGGACTAGCATCCCAG-3' located between -261 and -242 contains an upstream repressing sequence (URS) element. We have identified and partially purified a 27 kDa protein that binds specifically to both the UAS and URS sequences of the ICL1 promoter. For both UAS and URS, binding requires the protein Snf1 (Cat1), a protein kinase essential for the derepression of genes repressed by glucose. Binding does not take place with extracts from glucose-grown strains, unless they lack Mig1, a negative regulatory protein involved in glucose repression.

Chromatography, Affinity↗
PubMed1998

Functional analysis of upstream activating elements in the promoter of the FBP1 gene from Saccharomyces cerevisiae.

We have investigated the effect of different carbon sources and of different mutations on the capacity of two elements, UAS1 and UAS2, from the promoter of the FBP1 gene to form specific DNA-protein complexes and to activate expression of a reporter gene. The complexes are observed with nuclear extracts from yeast derepressed on glycerol or ethanol. When hxk2 mutants are grown on glucose the nuclear extracts are able to complex UAS1 but not UAS2, while for wild-type cells grown on galactose only the complex with UAS2 is formed. In contrast, in vivo the operation of both UASs is high in ethanol, moderate to low in glycerol, and negligible in galactose; no expression is observed in glucose even in a hxk2 background. There is no effect of a MIG1 deletion, either in the formation of DNA-protein complexes or on the expression of reporter genes.

Base Sequence↗
PubMed1998

Yeast carbon catabolite repression.

Glucose and related sugars repress the transcription of genes encoding enzymes required for the utilization of alternative carbon sources; some of these genes are also repressed by other sugars such as galactose, and the process is known as catabolite repression. The different sugars produce signals which modify the conformation of certain proteins that, in turn, directly or through a regulatory cascade affect the expression of the genes subject to catabolite repression. These genes are not all controlled by a single set of regulatory proteins, but there are different circuits of repression for different groups of genes. However, the protein kinase Snf1/Cat1 is shared by the various circuits and is therefore a central element in the regulatory process. Snf1 is not operative in the presence of glucose, and preliminary evidence suggests that Snf1 is in a dephosphorylated state under these conditions. However, the enzymes that phosphorylate and dephosphorylate Snf1 have not been identified, and it is not known how the presence of glucose may affect their activity. What has been established is that Snf1 remains active in mutants lacking either the proteins Grr1/Cat80 or Hxk2 or the Glc7 complex, which functions as a protein phosphatase. One of the main roles of Snf1 is to relieve repression by the Mig1 complex, but it is also required for the operation of transcription factors such as Adr1 and possibly other factors that are still unidentified. Although our knowledge of catabolite repression is still very incomplete, it is possible in certain cases to propose a partial model of the way in which the different elements involved in catabolite repression may be integrated.

Amino Acid Sequence↗
PubMed1996

The regulatory characteristics of yeast fructose-1,6-bisphosphatase confer only a small selective advantage.

The question of how the loss of regulatory mechanisms for a metabolic enzyme would affect the fitness of the corresponding organism has been addressed. For this, the fructose-1,6-bisphosphatase (FbPase) from Saccharomyces cerevisiae has been taken as a model. Yeast strains in which different controls on FbPase (catabolite repression and inactivation; inhibition by fructose-2,6-bisphosphate and AMP) have been removed have been constructed. These strains express during growth on glucose either the native yeast FbPase, the Escherichia coli FbPase which is insensitive to inhibition by fructose-2,6-bisphosphate, or a mutated E. coli FbPase with low sensitivity to AMP. Expression of the heterologous FbPases increases the fermentation rate of the yeast and its generation time, while it decreases its growth yield. In the strain containing high levels of an unregulated bacterial FbPase, cycling between fructose-6-phosphate and fructose-1,6-bisphosphate reaches 14%. It is shown that the regulatory mechanisms of FbPase provide a slight but definite competitive advantage during growth in mixed cultures.

Adenosine Monophosphate↗
PubMed1995

Analysis of positive elements sensitive to glucose in the promoter of the FBP1 gene from yeast.

We have identified in the promoter of the FBP1 gene from Saccharomyces cerevisiae, which codes for fructose-1,6-bisphosphatase, two elements which can form specific DNA.protein complexes and which confer glucose-repressed expression to an heterologous reporter gene. Complex formation and activation of transcription by either element require a functional CAT1 gene and are not blocked by a hap2-1 mutation, although this mutation interferes with maximal expression of the FBP1 gene. A sequence from one of the elements acts as a weak upstream activating sequence, but its activity can be stimulated up to 10-fold by neighboring sequences. A further element of the promoter has been characterized, which forms a specific DNA.protein complex only when a nuclear extract from derepressed cells is used. This element does not activate transcription in a heterologous promoter. The DNA sequences of the three elements involved in protein binding, defined by DNase I footprinting, have no homology with consensus sequences for known activating factors.

Base Sequence↗
PubMed1995

Expression of a yeast gene can be blocked by insertion of short yeast DNA fragments between a UAS and the TATA box.

We have constructed a plasmid, pOV10, which facilitates the introduction of putative upstream activating sequences (UAS) or upstream repressing sequences (URS) from yeast genes into plasmids containing CYCl-lacZ fusions. We have observed that the insertion of yeast sequences from 155 to 195 bp between the UAS and the TATA box of a CYCl-lacZ fusion gene can block beta-galactosidase expression. It is suggested that this block is related to the formation of nucleosomes on the DNA.

Cloning, Molecular↗
PubMed1994

The levels of yeast gluconeogenic mRNAs respond to environmental factors.

The FBP1 and PCK1 genes encode the gluconeogenic enzymes fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, respectively. In the yeast, Saccharomyces cerevisiae, the corresponding mRNAs are present at low levels during growth on glucose, but are present at elevated levels during growth on gluconeogenic carbon sources. We demonstrate that the levels of the FBP1 and PCK1 mRNAs are acutely sensitive to the addition of glucose to the medium and that the levels of these mRNAs decrease rapidly when glucose is added to the medium at a concentration of only 0.005%. At this concentration, glucose blocks FBP1 and PCK1 transcription, but has no effect on iso-1 cytochrome c (CYC1) mRNA levels. Glucose also increases the rate of degradation of the PCK1 mRNA approximately twofold, but only has a slight effect upon FBP1 mRNA turnover. We show that the levels of the FBP1 and PCK1 mRNAs are also sensitive to other environmental factors. The levels of these mRNAs decrease transiently in response to a decrease of the pH from pH 7.5 to pH 6.5 in the medium, or to a mild temperature shock (from 24 degrees C to 36 degrees C). The latter response appears to be mediated by accelerated mRNA decay.

Base Sequence↗
PubMed1994

Use of Yarrowia lipolytica hexokinase for the quantitative determination of trehalose 6-phosphate.

This paper describes a procedure for the quantitative determination of trehalose 6-phosphate (T6P) based on its ability to inhibit hexokinase from Yarrowia lipolytica. The assay is linear between 1 nmol and at least 8 nmol. The concentration of T6P in wild-type Saccharomyces cerevisiae (0.15 mM) and in ras2 mutants (0.25 mM) remained unchanged in the exponential or stationary phase of growth or after heat shock. A tps1 mutant affected in T6P synthase did not show detectable T6P. Heat shock increased the concentration of T6P in Schizosaccharomyces pombe from 0.43 to 0.75 mM.

Hexokinase↗
PubMed1994

Catabolite inactivation of heterologous fructose-1,6-bisphosphatases and fructose-1,6-bisphosphatase-beta-galactosidase fusion proteins in Saccharomyces cerevisiae.

Fructose-1,6-bisphosphatase (FruP2ase) from Saccharomyces cerevisiae is rapidly inactivated upon addition of glucose to a culture growing on non-sugar carbon sources. Under the same conditions the FruP2ases from Schizosaccharomyces pombe or Escherichia coli expressed in S. cerevisiae were not affected. A chimaeric protein containing the first 178 amino acids from the N-terminal half of S. cerevisiae FruP2ase fused to E. coli beta-galactosidase was susceptible to catabolite inactivation. Elimination of a putative destruction box, RAELVNLVG ... KK .... K., beginning at amino acid 60 did not prevent catabolite inactivation. Similarly a change of the vacuole-targeting sequence QKKLD, amino acids 80-84, to QKNSD did not affect significantly the course of inactivation of beta-galactosidase. A fusion protein carrying only the first 138 amino acids from FruP2ase was inactivated at a higher rate than the one carrying the first 178, suggesting the existence of a protective region between amino acids 138 and 178. A fusion protein carrying the first 81 amino acids from FruP2ase was inactivated by glucose at a similar rate to the one carrying the 178 amino acids, but one with only the first 18 amino acids was resistant to catabolite inactivation. Inactivation of FruP2ase in mutants ubr1 that lack a protein required for ubiquitin-dependent proteolysis, or pra1 that lack vacuolar protease A, proceeded as in a wild type. Our results suggest that at least two domains of FruP2ase may mark beta-galactosidase for catabolite inactivation and that FruP2ase can be inactivated by a mechanism independent of transfer to the vacuole.

Amino Acid Sequence↗
PubMed1993

Trehalose-6-phosphate, a new regulator of yeast glycolysis that inhibits hexokinases.

Trehalose-6-phosphate (P) competitively inhibited the hexokinases from Saccharomyces cerevisiae. The strongest inhibition was observed upon hexokinase II, with a Ki of 40 microM, while in the case of hexokinase I the Ki was 200 microM. Glucokinase was not inhibited by trehalose-6-P up to 5 mM. This inhibition appears to have physiological significance, since the intracellular levels of trehalose-6-P were about 0.2 mM. Hexokinases from other organisms were also inhibited, while glucokinases were unaffected. The hexokinase from the yeast, Yarrowia lipolytica, was particularly sensitive to the inhibition by trehalose-6-P: when assayed with 2 mM fructose an apparent Ki of 5 microM was calculated. Two S. cerevisiae mutants with abnormal levels of trehalose-6-P exhibited defects in glucose metabolism. It is concluded that trehalose-6-P plays an important role in the regulation of the first steps of yeast glycolysis, mainly through the inhibition of hexokinase II.

Binding, Competitive↗
PubMed1993

Futile cycles in Saccharomyces cerevisiae strains expressing the gluconeogenic enzymes during growth on glucose.

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 13C NMR in cell-free extracts from yeast growing on [6-13C]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.

Adenine Nucleotides↗
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↗