RESUMO
Aging is characterized by a gradual decline in physiological integrity, which impairs functionality and increases susceptibility to mortality. Dietary restriction, mimicking nutrient scarcity without causing malnutrition, is an intervention known to decelerate the aging process. While various hypotheses have been proposed to elucidate how dietary restriction influences aging, the underlying mechanisms remain incompletely understood. This project aimed to investigate the role of the primary regulator of the general amino acid control (GAAC) pathway, the transcription factor Gcn4, in the aging process of S. cerevisiae cells. Under conditions of amino acid deprivation, which activate Gcn4, the deletion of GCN4 led to a diverse array of physiological changes in the cells. Notably, the absence of Gcn4 resulted in heightened mitochondrial activity, likely contributing to the observed increase in reactive oxygen species (ROS) accumulation. Furthermore, these mutant gcn4Δ cells exhibited reduced ethanol production despite maintaining similar glucose consumption rates, suggesting a pivotal role for Gcn4 in regulating the Crabtree effect. Additionally, there was a marked reduction in trehalose, the storage carbohydrate, within the mutant cells compared to the wild-type strain. The intracellular content of free amino acids also exhibited disparities between the wild-type and GCN4-deficient strains. Taken together, our findings indicate that the absence of GCN4 disrupts cellular homeostasis, triggering significant alterations in interconnected intracellular metabolic pathways. These disruptions have far-reaching metabolic consequences that ultimately culminate in a shortened lifespan.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Longevidade , Biossíntese de Proteínas , Aminoácidos/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas Fúngicas/genéticaRESUMO
Gene regulation in yeast occurs at the transcription level, i.e. the basal level of expression is very low and increased transcription requires gene-specific transcription factors allowing the recruitment of basal transcriptional machinery. Saccharomyces cerevisiae BAP2 gene encodes the permease responsible for most uptake of leucine, valine and isoleucine, amino acids that this yeast can use as nitrogen sources. Moreover, BAP2 expression is known to be induced by the presence of amino acids such as leucine. In this context, the results presented in this paper show that BAP2 is an inducible gene in the presence of nitrogen-non-preferred source proline but exhibits high constitutive non-inducible expression in nitrogen-preferred source ammonium. BAP2 expression is regulated by the SPS sensor system and transcription factors Leu3, Gcn4 and Dal81. This can be achieved or not through a direct binding to the promoter depending on the quality of the nitrogen source. We further demonstrate here that an interaction occurs in vivo between Uga3 â the transcriptional activator responsible for γ-aminobutyric acid (GABA)-dependent induction of the GABA genes â and the regulatory region of the BAP2 gene, which leads to an increase in BAP2 transcription.
Assuntos
Sistemas de Transporte de Aminoácidos/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Nitrogênio/metabolismo , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genéticaRESUMO
Yeast cells can use γ-aminobutyric acid (GABA), a non-protein amino acid, as a nitrogen source that is mainly imported by the permease Uga4 and catabolized by the enzymes GABA transaminase and succinate-semialdehyde dehydrogenase, encoded by the UGA1 and UGA2 genes, respectively. The three UGA genes are inducible by GABA and subject to nitrogen catabolite repression. Hence, their regulation occurs through two mechanisms, one dependent on the inducer and the other on nitrogen source quality. The aim of this work was to better understand the molecular mechanisms of transcription factors acting on different regulatory elements present in UGA promoters, such as Uga3, Dal81, Leu3 and the GATA factors, and to establish the mechanism of the concerted action between them. We found that Gat1 plays an important role in the induction of UGA4 transcription by GABA and that Gzf3 has an effect in cells grown in a poor nitrogen source such as proline and that this effect is positive on UGA4 expression. We also found that Gln3 and Dal80 affect the interaction of Uga3 and Dal81 on UGA promoters. Moreover, our results indicated that the repressing activity of Leu3 on UGA4 and UGA1 occurs through Dal80 since we demonstrated that Leu3 facilitates Dal80 interaction with DNA. However, when the expression of GATA factors is null or negligible, Leu3 functions as an activator.
Assuntos
Proteínas da Membrana Plasmática de Transporte de GABA/genética , Fatores de Transcrição GATA/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transativadores/metabolismo , Ácido gama-Aminobutírico/metabolismo , Fatores de Transcrição GATA/genética , Redes Reguladoras de Genes , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Transativadores/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Yeast can use a wide variety of nitrogen compounds. However, the ability to synthesize enzymes and permeases for catabolism of poor nitrogen sources is limited in the presence of a rich one. This general mechanism of transcriptional control is called nitrogen catabolite repression. Poor nitrogen sources, such as leucine, γ-aminobutyric acid (GABA), and allantoin, enable growth after the synthesis of pathway-specific catabolic enzymes and permeases. This synthesis occurs only under conditions of nitrogen limitation and in the presence of a pathway-specific signal. In this work we studied the temporal order in the induction of AGP1, BAP2, UGA4, and DAL7, genes that are involved in the catabolism and use of leucine, GABA, and allantoin, three poor nitrogen sources. We found that when these amino acids are available, cells will express AGP1 and BAP2 in the first place, then DAL7, and at last UGA4. Dal81, a general positive regulator of genes involved in nitrogen utilization related to the metabolisms of GABA, leucine, and allantoin, plays a central role in this coordinated regulation.
RESUMO
Yeast cells are able to adapt their metabolism according to the quality of both carbon and nitrogen sources available in the environment. Saccharomyces cerevisiae UGA4 gene encodes a permease capable of transporting γ-aminobutyric acid (GABA) into the cells. Yeast uses this amino acid as a nitrogen source or as a carbon skeleton that enters the tricarboxylic acid cycle. The quality of the carbon source modulates UGA4 expression through two parallel pathways, each one acting on different regulatory elements, the UAS(GATA) and the UAS(GABA). In the presence of a fermentable carbon source, UGA4 expression is induced by GABA while in the presence of a non-fermentable carbon source this expression is GABA-independent. The aim of this work was to study the mechanisms responsible for the differences in the profiles of UGA4 expression in both growth conditions. We found that although the subcellular localization of Gln3 depends on the carbon source and UGA4 expression depends on Tor1 and Snf1, Gln3 localization does not depend on these kinases. We also found that the phosphorylation of Gln3 is mediated by two systems activated by a non-fermentable carbon source, involving the Snf1 kinase and an unidentified TORC1-regulated kinase. We also found that the activity of the main transcription factors responsible for UGA4 induction by GABA varies depending on the quality of the carbon source. In a fermentable carbon source such as glucose, the negative GATA factor Dal80 binds to UGA4 promoter; only after the addition of the inducer, the positive factors Uga3, Dal81 and Gln3 interact with the promoter removing Dal80 and leading to gene induction. In contrast, in the non-fermentable carbon source acetate the negative GATA factor remains bound to UGA4 promoter in the presence or absence of GABA, the positive factors are not detected bound in any of these conditions and in consequence, UGA4 is not induced.
Assuntos
Carbono/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Ácido gama-Aminobutírico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fermentação , Glucose/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/farmacologia , Ácido gama-Aminobutírico/genética , Ácido gama-Aminobutírico/farmacologiaRESUMO
γ-Aminobutyric acid (GABA) transport and catabolism in Saccharomyces cerevisiae are subject to a complex transcriptional control that depends on the nutritional status of the cells. The expression of the genes that form the UGA regulon is inducible by GABA and sensitive to nitrogen catabolite repression (NCR). GABA induction of these genes is mediated by Uga3 and Dal81 transcription factors, whereas GATA factors are responsible for NCR. Here, we show how members of the UGA regulon share the activation mechanism. Our results show that both Uga3 and Dal81 interact with UGA genes in a GABA-dependent manner, and that they depend on each other for the interaction with their target promoters and the transcriptional activation. The typical DNA-binding domain Zn(II)(2)-Cys(6) of Dal81 is unnecessary for its activity and Uga3 acts as a bridge between Dal81 and DNA. Both the trans-activation activity of the GATA factor Gln3 and the repressive activity of the GATA factor Dal80 are exerted by their interaction with UGA promoters in response to GABA, indicating that Uga3, Dal81, Gln3 and Dal80 all act in concert to induce the expression of UGA genes. So, an interplay between the factors responsible for GABA induction and those responsible for NCR in the regulation of the UGA genes is proposed here.
Assuntos
Fatores de Transcrição GATA/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Ácido gama-Aminobutírico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição GATA/genética , Regulação Fúngica da Expressão Gênica , Regulon , Proteínas Repressoras/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The three genes that form the UGA regulon in Saccharomyces cerevisiae are responsible for the transport and degradation of γ-aminobutyric acid (GABA) in this organism. Despite the differences in the sequence of their promoters, these genes similarly respond to GABA stimuli. The expression of UGA1, UGA2 and UGA4 depends on GABA induction and nitrogen catabolite repression (NCR). The induction of these genes requires the action of at least two positive proteins, the specific Uga3 and the pleiotropic Uga35/Dal81 transcription factors. Here we show that all the members of the UGA regulon, as was already demonstrated for UGA4, are negatively regulated by extracellular amino acids through the SPS amino acid sensor. We also show that this negative effect is caused by a low availability of Uga35/Dal81 transcription factor and that Leu3 transcription factor negatively regulates UGA4 and UGA1 expression but it does not affect UGA2 expression.
Assuntos
4-Aminobutirato Transaminase/genética , Regulação Fúngica da Expressão Gênica , Leucina/metabolismo , Regulon , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Succinato-Semialdeído Desidrogenase (NADP+)/genética , Regulação para Baixo , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The Asr gene family (named after abscisic acid, stress and ripening), currently classified as a novel group of the LEA superfamily, is exclusively present in the genomes of seed plants, except for the Brassicaceae family. It is associated with water-deficit stress and is involved in adaptation to dry climates. Motivated by separate reports depicting ASR proteins as either transcription factors or chaperones, we decided to determine the intracellular localization of ASR proteins. For that purpose, we employed an in vivo eukaryotic expression system, the heterologous model Saccharomyces cerevisiae, including wild type strains as well as mutants in which the variant ASR1 previously proved to be functionally protective against osmotic stress. Our methodology involved immunofluorescence-based confocal microscopy, without artificially altering the native structure of the protein under study. Results show that, in both normal and osmotic stress conditions, recombinant ASR1 turned out to localize mainly to the cytoplasm, irrespective of the genotype used, revealing a scattered distribution in the form of dots or granules. The results are discussed in terms of a plausible dual (cytoplasmic and nuclear) role of ASR proteins.
RESUMO
The Saccharomyces cerevisiae UGA4 gene encodes a permease capable of importing gamma-aminobutyric acid (GABA) and delta-aminolevulinic acid (ALA) into the cell. GABA-dependent induction of this permease requires at least two positive-acting proteins, the specific factor Uga3 and the pleiotropic factor Uga35/Dal81. UGA4 is subjected to a very complex regulation, and its induction is affected by the presence of extracellular amino acids; this effect is mediated by the plasma membrane amino acid sensor SPS. Our results show that leucine affects UGA4 induction and that the SPS sensor and the downstream effectors Stp1 and Stp2 participate in this regulation. Moreover, we found that the Uga3 and Uga35/Dal81 transcription factors bind to the UGA4 promoter in a GABA-dependent manner and that this binding is impaired by the presence of leucine. We also found that the Leu3 transcription factor negatively regulates UGA4 transcription, although this seems to be through an indirect mechanism.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Leucina/farmacologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Ácido gama-Aminobutírico/farmacologia , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Fúngicos/genética , Modelos Genéticos , Mutação/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacosRESUMO
The Saccharomyces cerevisiae UGA4 gene, which encodes the gamma-aminobutyric acid (GABA) and delta-aminolaevulinic acid (ALA) permease, is well known to be regulated by the nitrogen source. Its expression levels are low in the presence of a rich nitrogen source but are higher when a poor nitrogen source is used. In addition, GABA can induce UGA4 expression when cells are grown with proline but not when they are grown with ammonium. Although vast amounts of evidence have been gathered about UGA4 regulation by nitrogen, little is known about its regulation by the carbon source. Using glucose and acetate as rich and poor carbon source respectively, this work aimed to shed light on hitherto unclear aspects of the regulation of this gene. In poor nitrogen conditions, cells grown with acetate were found to have higher UGA4 basal expression levels than those grown with glucose, and did not show UGA4 induction in response to GABA. Analysis of the expression and subcellular localization of the transcription factors that regulate UGA4 as well as partial deletions and site-directed mutations of the UGA4 promoter region suggested that there are two parallel pathways that act in regulating this gene by the carbon source. Furthermore, the results demonstrate the existence of a new factor operating in UGA4 regulation.