RESUMO
Glucose is the preferred nutrient for most living cells and is also a signaling molecule that modulates several cellular processes. Glucose regulates the expression of glucose permease genes in yeasts through signaling pathways dependent on plasma membrane glucose sensors. In the yeast Kluyveromyces lactis, sufficient levels of glucose induction of the low-affinity glucose transporter RAG1 gene also depends on a functional glycolysis, suggesting additional intracellular signaling. We have found that the expression of RAG1 gene is also induced by hypoxia in the presence of glucose, indicating that glucose and oxygen signaling pathways are interconnected. In this study we investigated the molecular mechanisms underlying this crosstalk. By analyzing RAG1 expression in various K. lactis mutants, we found that the bHLH transcriptional activator Sck1 is required for the hypoxic induction of RAG1 gene. The RAG1 promoter region essential for its hypoxic induction was identified by promoter deletion experiments. Taken together, these results show that the RAG1 glucose permease gene is synergistically induced by hypoxia and glucose and highlighted a novel role for the transcriptional activator Sck1 as a key mediator in this mechanism.
Assuntos
Proteínas Fúngicas/genética , Proteínas Facilitadoras de Transporte de Glucose/genética , Glucose/metabolismo , Kluyveromyces/genética , Fatores de Transcrição/genética , Anaerobiose , Regulação Fúngica da Expressão Gênica , Glicólise , Kluyveromyces/metabolismo , Mutação , Transdução de SinaisRESUMO
In the yeast Kluyveromyces lactis, the inactivation of structural or regulatory glycolytic and fermentative genes generates obligate respiratory mutants which can be characterized by sensitivity to the mitochondrial drug antimycin A on glucose medium (Rag(-) phenotype). Rag(-) mutations can occasionally be generated by the inactivation of genes not evidently related to glycolysis or fermentation. One such gene is the hypoxic regulatory gene KlMGA2. In this work, we report a study of the many defects, in addition to the Rag(-) phenotype, generated by KlMGA2 deletion. We analyzed the fermentative and respiratory metabolism, mitochondrial functioning and morphology in the Klmga2Δ strain. We also examined alterations in the regulation of the expression of lipid biosynthetic genes, in particular fatty acids, ergosterol and cardiolipin, under hypoxic and cold stress and the phenotypic suppression by unsaturated fatty acids of the deleted strain. Results indicate that, despite the fact that the deleted mutant strain had a typical glycolytic/fermentative phenotype and KlMGA2 is a hypoxic regulatory gene, the deletion of this gene generated defects linked to mitochondrial functions suggesting new roles of this protein in the general regulation and cellular fitness of K. lactis. Supplementation of unsaturated fatty acids suppressed or modified these defects suggesting that KlMga2 modulates membrane functioning or membrane-associated functions, both cytoplasmic and mitochondrial.
Assuntos
Proteínas de Bactérias/genética , Ácidos Graxos Insaturados/metabolismo , Fermentação/genética , Glucose/metabolismo , Kluyveromyces/metabolismo , Consumo de Oxigênio/genética , Fatores de Transcrição/genética , Antifúngicos/farmacologia , Antimicina A/farmacologia , Cardiolipinas/metabolismo , Hipóxia Celular/fisiologia , Resposta ao Choque Frio/fisiologia , Ergosterol/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Glicólise/genética , Kluyveromyces/efeitos dos fármacos , Kluyveromyces/genética , Proteínas de Membrana/genética , Mitocôndrias/metabolismo , Transcrição Gênica/genéticaRESUMO
In the yeast Kluyveromyces lactis, the pyruvate decarboxylase gene KlPDC1 is strongly regulated at the transcription level by different environmental factors. Sugars and hypoxia act as inducers of transcription, while ethanol acts as a repressor. Their effects are mediated by gene products, some of which have been characterized. KlPDC1 transcription is also strongly repressed by its product--KlPdc1--through a mechanism called autoregulation. We performed a genetic screen that allowed us to select and identify the regulatory gene RAG3 as a major factor in the transcriptional activity of the KlPDC1 promoter in the absence of the KlPdc1 protein, i.e. in the autoregulatory mechanism. We also showed that the two proteins Rag3 and KlPdc1 interact, co-localize in the cell and that KlPdc1 may control Rag3 nuclear localization.
Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Homeostase/genética , Kluyveromyces/enzimologia , Piruvato Descarboxilase/genética , Alelos , Proteínas Fúngicas/metabolismo , Genes Reporter , Genótipo , Kluyveromyces/genética , Kluyveromyces/ultraestrutura , Modelos Biológicos , Regiões Promotoras Genéticas/genética , Mapeamento de Interação de Proteínas , Piruvato Descarboxilase/metabolismo , Deleção de Sequência , Transcrição GênicaRESUMO
In the respiratory yeast Kluyveromyces lactis, little is known about the factors regulating the metabolic response to oxygen shortage. After searching for homologues of characterized Saccharomyces cerevisiae regulators of the hypoxic response, we identified a gene that we named KlMGA2, which is homologous to MGA2. The deletion of KlMGA2 strongly reduced both the fermentative and respiratory growth rate and altered fatty acid composition and the unsaturation index of membranes. The reciprocal heterologous expression of MGA2 and KlMGA2 in the corresponding deletion mutant strains suggested that Mga2 and KlMga2 are functional homologues. KlMGA2 transcription was induced by hypoxia and the glucose sensor Rag4 mediated the hypoxic induction of KlMGA2. Transcription of lipid biosynthetic genes KlOLE1, KlERG1, KlFAS1 and KlATF1 was induced by hypoxia and was dependent on KlMga2, except for KlOLE1. Rag4 was required for hypoxic induction of transcription for both KlMga2-dependent (KlERG1) and KlMga2-independent (KlOLE1) structural genes.