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

6 pages, 6 figures, 1 table.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.This work was supported by grants PB94-0091-C02-01 and PB94-0091-C02-02 from the DGICYT.Peer reviewe

Transcriptional responses to glucose in Saccharomyces cerevisiae strains lacking a functional protein kinase A

Background The pattern of gene transcripts in the yeast Saccharomyces cerevisiae is strongly affected by the presence of glucose. An increased activity of protein kinase A (PKA), triggered by a rise in the intracellular concentration of cAMP, can account for many of the effects of glucose on transcription. In S. cerevisiae three genes, TPK1, TPK2, and TPK3, encode catalytic subunits of PKA. The lack of viability of tpk1 tpk2 tpk3 triple mutants may be suppressed by mutations such as yak1 or msn2/msn4. To investigate the requirement for PKA in glucose control of gene expression, we have compared the effects of glucose on global transcription in a wild-type strain and in two strains devoid of PKA activity, tpk1 tpk2 tpk3 yak1 and tpk1 tpk2 tpk3 msn2 msn4. Results We have identified different classes of genes that can be induced -or repressed- by glucose in the absence of PKA. Representative examples are genes required for glucose utilization and genes involved in the metabolism of other carbon sources, respectively. Among the genes responding to glucose in strains devoid of PKA some are also controlled by a redundant signalling pathway involving PKA activation, while others are not affected when PKA is activated through an increase in cAMP concentration. On the other hand, among genes that do not respond to glucose in the absence of PKA, some give a full response to increased cAMP levels, even in the absence of glucose, while others appear to require the cooperation of different signalling pathways. We show also that, for a number of genes controlled by glucose through a PKA-dependent pathway, the changes in mRNA levels are transient. We found that, in cells grown in gluconeogenic conditions, expression of a small number of genes, mainly connected with the response to stress, is reduced in the strains lacking PKA. Conclusions In S. cerevisiae, the transcriptional responses to glucose are triggered by a variety of pathways, alone or in combination, in which PKA is often involved. Redundant signalling pathways confer a greater robustness to the response to glucose, while cooperative pathways provide a greater flexibility.BT/BiotechnologyApplied Science

Carbon catabolite repression in yeast

Now known as FEBS Journal: Open access backfiles of content older than 1 year.Control of gene expression is a basic regulatory mechanism of living organisms. In microorganisms, glucose or other rapidly metabolizable carbon sources repress the expression of genes that code for enzymes related to the metabolism of other carbon sources. This phenomenon, known as catabolite repression, allows microorganisms to cope effectively with changes in the carbon sources present in their environment. In the case of Escherichia coli, a model to explain at the molecular level the mechanism of catabolite repression has been worked out (Ullmann, 1985; Saier, 1989), although some of its elements remain unidentified.Work in the author’s laboratory has been supported by grants from the EEC [BAP 0389. E JR] and the Spanish Dirección General de Investigación Científica y Técnica (PB87-0294).Peer Reviewe

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.This work has benefited from a grant of the Spanish Comisibn Asesora de Investigación Científica y Técnica

Inactivation of Gluconegenic Enzymes in Glycolytic Mutants of Saccharomyces cervisiae

Yeast mutants blocked at different steps of the glycolytic pathway 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 phosphoeenol-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.This work was partially sup- ported by the Comisión Asesora de Investigación Científica y Técnica.Peer reviewe

Fructose-1,6-diphosphatase, phosphofructokinase and glucose-6-phosphate dehydrogenase from fermenting and non fermenting yeasts

1. Levels of phosphofructokinase, glucose-6-phosphate dehydrogenase and fructose-1,6-diphosphatase activities have been compared in different yeasts belonging to glucose fermenting and non-fermenting groups grown in different conditions. 2. Phosphofructokinase was present in all the fermentative species tested. On the contrary its level was not measurable in any of the aerobic yeasts tested with the exception of Pichia species. 3. No significant variations were observed in the values of glucose-6-phosphate dehydrogenase from the two groups of yeasts. 4. The synthesis of fructose-1,6-diphosphatase was repressed in both groups, by growth in sugar carbon sources. However, a remarkable difference in the sensitivity of the fructose-1,6-diphosphatase from both groups towards inhibition by AMP was observed. The enzyme from all fermentative yeasts tested showed a strong inhibition by AMP (1 mM producing about 80% inhibition) while the enzyme from aerobic yeasts showed different responses, inhibition ranging from 10% in Rhodotorula and Sporobolomyces, to 90% in Pichia

Concentrations of intermediary metabolites in yeast

[EN]: The concentrations of some intermediary metabolites in yeast in different metabolic situations have been tabulated from data available in the literature. A critical examination of the extraction procedures and their influence on the values obtained is performed.[FR]: Les concentrations d'un grand nombre de métabolites de la levure soumise à de différentes conditions ont été rassemblées à partir des valeurs publiées dans la littérature. Un examen critique des procédés d'extraction et de leur influence sur les valeurs obtenues a été également effectué

55 years together—our life with yeasts

The authors look back at their life together in yeast research, the influences that shaped it, certain challenges and changes in laboratory and funding policies.Peer reviewe

Catabolite repression mutants of yeast

The mechanism of catabolite repression in yeast is not well understood, although it has been established that cAMP does not play a role similar to that found in Escherichia coli. To identify the elements implicated in catabolite repression in yeast, a variety of mutants affected in this process have been isolated by different research groups. A systematic review of the results reported in the literature is presented. The conclusion that can be drawn is that the mechanism of catabolite repression is a complex one, with no single gene controlling all the genes subject to repression. The expression of a given gene or set of genes is controlled by several regulatory genes, but it is not yet known whether these genes act cooperatively or sequentially.Work in the laboratory of the authors has been supported by grants of the Comisi6n Asesora de lnvestigación Científica y Técnica