RESUMEN
Studies of replicative and chronological lifespan in Saccharomyces cerevisiae have advanced understanding of longevity in all eukaryotes. Chronological lifespan in this species is defined as the age-dependent viability of nondividing cells. To date this parameter has only been estimated under calorie restriction, mimicked by starvation. Because postmitotic cells in higher eukaryotes often do not starve, we developed a model yeast system to study cells as they age in the absence of calorie restriction. Yeast cells were encapsulated in a matrix consisting of calcium alginate to form â¼3 mm beads that were packed into bioreactors and fed ad libitum. Under these conditions cells ceased to divide, became heat shock and zymolyase resistant, yet retained high fermentative capacity. Over the course of 17 d, immobilized yeast cells maintained >95% viability, whereas the viability of starving, freely suspended (planktonic) cells decreased to <10%. Immobilized cells exhibited a stable pattern of gene expression that differed markedly from growing or starving planktonic cells, highly expressing genes in glycolysis, cell wall remodeling, and stress resistance, but decreasing transcription of genes in the tricarboxylic acid cycle, and genes that regulate the cell cycle, including master cyclins CDC28 and CLN1. Stress resistance transcription factor MSN4 and its upstream effector RIM15 are conspicuously up-regulated in the immobilized state, and an immobilized rim15 knockout strain fails to exhibit the long-lived, growth-arrested phenotype, suggesting that altered regulation of the Rim15-mediated nutrient-sensing pathway plays an important role in extending yeast chronological lifespan under calorie-unrestricted conditions.
Asunto(s)
Regulación Fúngica de la Expresión Génica/fisiología , Longevidad/fisiología , Modelos Biológicos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Alginatos , Puntos de Control del Ciclo Celular/fisiología , Células Inmovilizadas/metabolismo , Células Inmovilizadas/fisiología , Citometría de Flujo , Ácido Glucurónico , Ácidos Hexurónicos , Análisis por Micromatrices , Reproducción/fisiologíaRESUMEN
BACKGROUND: Interspecific hybridization occurs in every eukaryotic kingdom. While hybrid progeny are frequently at a selective disadvantage, in some instances their increased genome size and complexity may result in greater stress resistance than their ancestors, which can be adaptively advantageous at the edges of their ancestors' ranges. While this phenomenon has been repeatedly documented in the field, the response of hybrid populations to long-term selection has not often been explored in the lab. To fill this knowledge gap we crossed the two most distantly related members of the Saccharomyces sensu stricto group, S. cerevisiae and S. uvarum, and established a mixed population of homoploid and aneuploid hybrids to study how different types of selection impact hybrid genome structure. RESULTS: As temperature was raised incrementally from 31°C to 46.5°C over 500 generations of continuous culture, selection favored loss of the S. uvarum genome, although the kinetics of genome loss differed among independent replicates. Temperature-selected isolates exhibited greater inherent and induced thermal tolerance than parental species and founding hybrids, and also exhibited ethanol resistance. In contrast, as exogenous ethanol was increased from 0% to 14% over 500 generations of continuous culture, selection favored euploid S. cerevisiae x S. uvarum hybrids. Ethanol-selected isolates were more ethanol tolerant than S. uvarum and one of the founding hybrids, but did not exhibit resistance to temperature stress. Relative to parental and founding hybrids, temperature-selected strains showed heritable differences in cell wall structure in the forms of increased resistance to zymolyase digestion and Micafungin, which targets cell wall biosynthesis. CONCLUSIONS: This is the first study to show experimentally that the genomic fate of newly-formed interspecific hybrids depends on the type of selection they encounter during the course of evolution, underscoring the importance of the ecological theatre in determining the outcome of the evolutionary play.
Asunto(s)
Adaptación Biológica/genética , Genoma Fúngico/genética , Hibridación Genética/genética , Saccharomyces/genética , Selección Genética/genética , Hibridación Genómica Comparativa , Etanol , Cariotipo , Densidad de Población , Especificidad de la Especie , TemperaturaRESUMEN
We have developed a method for preparing high-quality total RNA from Ca-alginate-encapsulated Saccharomyces cerevisiae that is suitable for microarray analysis. Encapsulated cells were harvested from immobilized cell reactors and flash-frozen in liquid nitrogen. Following low-temperature mechanical disruption, cells were freed from Ca-alginate by reverse ionotropic gelation and purified by centrifugation, and then total RNA was extracted using hot acid phenol. The yield and quality of the RNA were consistently high; the RNA was free of contaminating alginate, and in microarray analysis it performed as well as RNA isolated from planktonic cells.
Asunto(s)
Alginatos/química , Calcio/química , Perfilación de la Expresión Génica , ARN de Hongos/aislamiento & purificación , Saccharomyces cerevisiae/genética , Ácido Glucurónico/química , Ácidos Hexurónicos/químicaRESUMEN
In Saccharomyces cerevisiae the HXK2 gene, which encodes the glycolytic enzyme hexokinase II, is involved in the regulatory mechanism known as 'glucose repression'. Its deletion leads to fully respiratory growth at high glucose concentrations where the wild type ferments profusely. Here we describe that deletion of the HXK2 gene resulted in a 75% reduction in fermentative capacity. Using regulation analysis we found that the fluxes through most glycolytic and fermentative enzymes were regulated cooperatively by changes in their capacities (Vmax) and by changes in the way they interacted with the rest of the metabolism. Glucose transport and phosphofructokinase were regulated purely at the metabolic level. The reduction of fermentative capacity in the mutant was accompanied by a remarkable resilience of the remaining capacity to nutrient starvation. After starvation, the fermentative capacity of the hxk2Delta mutant was similar to that of the wild type. Based on our results and previous reports, we suggest an inverse correlation between glucose repression and the resilience of fermentative capacity towards nutrient starvation. Only a limited number of glycolytic enzyme activities changed upon starvation of the hxk2Delta mutant and we discuss to what extent this could explain the stability of the fermentative capacity.
Asunto(s)
Glucosa/metabolismo , Hexoquinasa/genética , Hexoquinasa/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Fermentación , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Glucólisis , Proteínas de Saccharomyces cerevisiaeRESUMEN
A novel method dissecting the regulation of a cellular function into direct metabolic regulation and hierarchical (e.g., gene-expression) regulation is applied to yeast starved for nitrogen or carbon. Upon nitrogen starvation glucose influx is down-regulated hierarchically. Upon carbon starvation it is down-regulated both metabolically and hierarchically. The method is expounded in terms of its implications for diverse types of regulation. It is also fine-tuned for cases where isoenzymes catalyze the flux through a single metabolic step.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Glucosa/metabolismo , Respuesta al Choque Térmico , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Medios de Cultivo , Genómica , Nitrógeno/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
A widely accepted mechanism for selective degradation of plasma membrane proteins is via ubiquitination and/or phosphorylation events. Such a regulated degradation has previously been suggested to rely on the presence of a specific SINNDAKSS sequence within the protein. Modification of a partly conserved SINNDAKSS-like sequence in the C-terminal tail of the Pho84 phosphate transporter, in combination with C-terminal fusion of green fluorescent protein or a MYC epitope, were used to evaluate the presence of this sequence and its role in the regulated degradation. The functional Pho84 mutants in which this SINNDAKSS-like sequence was altered or truncated were subjected to degradation like that of the wild type, suggesting that degradation of the Pho84 protein is regulated by factors other than properties of this sequence.
Asunto(s)
Simportadores de Protón-Fosfato/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas Fluorescentes Verdes , Cinética , Proteínas Luminiscentes/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Simportadores de Protón-Fosfato/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Green fluorescent protein (GFP) from Aequorea victoria was used as an in vivo reporter protein when fused to the C-terminus of the Jen1 lactate permease of Saccharomyces cerevisiae. The Jen1 protein tagged with GFP is a functional lactate transporter with a cellular abundance of 1670 molecules/cell, and a catalytic-centre activity of 123 s(-1). It is expressed and tagged to the plasma membrane under induction conditions. The factors involved in proper localization and turnover of Jen1p were revealed by expression of the Jen1p-GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways. The chimaeric protein Jen1p-GFP is targeted to the plasma membrane via a Sec6-dependent process; upon treatment with glucose, it is endocytosed via END3 and targeted for degradation in the vacuole. Experiments performed in a Deltadoa4 mutant strain showed that ubiquitination is associated with the turnover of the permease.
Asunto(s)
Proteínas Luminiscentes/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Simportadores/genética , Western Blotting , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Regulación Enzimológica de la Expresión Génica , Glucosa/farmacología , Proteínas Fluorescentes Verdes , Concentración de Iones de Hidrógeno , Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/biosíntesis , Espectrometría de Fluorescencia , Simportadores/biosíntesisRESUMEN
In glucose-limited aerobic chemostat cultures of a wild-type Saccharomyces cerevisiae and a derived hxk2 null strain, metabolic fluxes were identical. However, the concentrations of intracellular metabolites, especially fructose 1,6-bisphosphate, and hexose-phosphorylating activities differed. Interestingly, the hxk2 null strain showed a higher maximal growth rate and higher Crabtree threshold dilution rate, revealing a higher oxidative capacity for this strain. After a pulse of glucose, aerobic glucose-limited cultures of wild-type S. cerevisiae displayed an overshoot in the intracellular concentrations of glucose 6-phosphate, fructose 6-phosphate, and fructose 1,6-bisphosphate before a new steady state was established, in contrast to the hxk2 null strain which reached a new steady state without overshoot of these metabolites. At low dilution rates the overshoot of intracellular metabolites in the wild-type strain coincided with the immediate production of ethanol after the glucose pulse. In contrast, in the hxk2 null strain the production of ethanol started gradually. However, in spite of the initial differences in ethanol production and dynamic behaviour of the intracellular metabolites, the steady-state fluxes after transition from glucose limitation to glucose excess were not significantly different in the wild-type strain and the hxk2 null strain at any dilution rate.