RESUMEN
Optimized nutrient utilization is crucial for the progression of microorganisms in competing communities. Here we investigate how different budding yeast species and ecological isolates have established divergent preferences for two alternative sugar substrates: Glucose, which is fermented preferentially by yeast, and galactose, which is alternatively used upon induction of the relevant GAL metabolic genes. We quantified the dose-dependent induction of the GAL1 gene encoding the central galactokinase enzyme and found that a very large diversification exists between different yeast ecotypes and species. The sensitivity of GAL1 induction correlates with the growth performance of the respective yeasts with the alternative sugar. We further define some of the mechanisms, which have established different glucose/galactose consumption strategies in representative yeast strains by modulating the activity of the Gal3 inducer. (1) Optimal galactose consumers, such as Saccharomyces uvarum, contain a hyperactive GAL3 promoter, sustaining highly sensitive GAL1 expression, which is not further improved upon repetitive galactose encounters. (2) Desensitized galactose consumers, such as S. cerevisiae Y12, contain a less sensitive Gal3 sensor, causing a shift of the galactose response towards higher sugar concentrations even in galactose experienced cells. (3) Galactose insensitive sugar consumers, such as S. cerevisiae DBVPG6044, contain an interrupted GAL3 gene, causing extremely reluctant galactose consumption, which is, however, improved upon repeated galactose availability. In summary, different yeast strains and natural isolates have evolved galactose utilization strategies, which cover the whole range of possible sensitivities by modulating the expression and/or activity of the inducible galactose sensor Gal3.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Azúcares/metabolismo , Galactosa/genética , Galactosa/metabolismo , Genes Fúngicos , Glucosa/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Aging is considered the deterioration of physiological functions along with an increased mortality rate. This scientific review focuses on the central importance of genomic instability during the aging process, encompassing a range of cellular and molecular changes that occur with advancing age. In particular, this revision addresses the genetic and epigenetic alterations that contribute to genomic instability, such as telomere shortening, DNA damage accumulation, and decreased DNA repair capacity. Furthermore, the review explores the epigenetic changes that occur with aging, including modifications to histones, DNA methylation patterns, and the role of non-coding RNAs. Finally, the review discusses the organization of chromatin and its contribution to genomic instability, including heterochromatin loss, chromatin remodeling, and changes in nucleosome and histone abundance. In conclusion, this review highlights the fundamental role that genomic instability plays in the aging process and underscores the need for continued research into these complex biological mechanisms.
Asunto(s)
Cromatina , Inestabilidad Genómica , Humanos , Nucleosomas , Epigénesis Genética , Histonas/genéticaRESUMEN
Pleiotropic drug resistance arises by the enhanced extrusion of bioactive molecules and is present in a wide range of organisms, ranging from fungi to human cells. A key feature of this adaptation is the sensitive detection of intracellular xenobiotics by transcriptional activators, activating expression of multiple drug exporters. Here, we investigated the selectivity and sensitivity of the budding yeast (Saccharomyces cerevisiae) multidrug response to better understand how differential drug recognition leads to specific activation of drug exporter genes and to drug resistance. Applying live-cell luciferase reporters, we demonstrate that the SNQ2, PDR5, PDR15, and YOR1 transporter genes respond to different mycotoxins, menadione, and hydrogen peroxide in a distinguishable manner and with characteristic amplitudes, dynamics, and sensitivities. These responses correlated with differential sensitivities of the respective transporter mutants to the specific xenobiotics. We further establish a binary vector system, enabling quantitative determination of xenobiotic-transcription factor (TF) interactions in real time. Applying this system we found that the TFs Pdr1, Pdr3, Yrr1, Stb5, and Pdr8 have largely different drug recognition patterns. We noted that Pdr1 is the most promiscuous activator, whereas Yrr1 and Stb5 are selective for ochratoxin A and hydrogen peroxide, respectively. We also show that Pdr1 is rapidly degraded after xenobiotic exposure, which leads to a desensitization of the Pdr1-specific response upon repeated activation. The findings of our work indicate that in the yeast multidrug system, several transcriptional activators with distinguishable selectivities trigger differential activation of the transporter genes.
Asunto(s)
Antifúngicos/farmacología , Proteínas de Unión al ADN/antagonistas & inhibidores , Farmacorresistencia Fúngica/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Factores de Transcripción/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Peróxido de Hidrógeno/farmacología , Micotoxinas/farmacología , Ocratoxinas/farmacología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relación Estructura-Actividad , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Vitamina K 3/farmacologíaRESUMEN
The regulation of gene expression is a fundamental process enabling cells to respond to internal and external stimuli or to execute developmental programs. Changes in gene expression are highly dynamic and depend on many intrinsic and extrinsic factors. In this review, we highlight the dynamic nature of transient gene expression changes to better understand cell physiology and development in general. We will start by comparing recent in vivo procedures to capture gene expression in real time. Intrinsic factors modulating gene expression dynamics will then be discussed, focusing on chromatin modifications. Furthermore, we will dissect how cell physiology or age impacts on dynamic gene regulation and especially discuss molecular insights into acquired transcriptional memory. Finally, this review will give an update on the mechanisms of heterogeneous gene expression among genetically identical individual cells. We will mainly focus on state-of-the-art developments in the yeast model but also cover higher eukaryotic systems.
Asunto(s)
Fenómenos Fisiológicos Celulares/genética , Expresión Génica , Heterogeneidad Genética , Biología Molecular , Transcripción Genética/genética , Animales , Regulación del Desarrollo de la Expresión Génica , Humanos , Biología Molecular/métodos , Biología Molecular/tendencias , Imagen Molecular/métodos , Imagen Molecular/tendencias , Análisis de la Célula Individual/métodos , Análisis de la Célula Individual/tendenciasRESUMEN
Peroxisomes are dynamic organelles and the sole location for fatty acid ß-oxidation in yeast cells. Here, we report that peroxisomal function is crucial for the adaptation to salt stress, especially upon sugar limitation. Upon stress, multiple layers of control regulate the activity and the number of peroxisomes. Activated Hog1 MAP kinase triggers the induction of genes encoding enzymes for fatty acid activation, peroxisomal import and ß-oxidation through the Adr1 transcriptional activator, which transiently associates with genes encoding fatty acid metabolic enzymes in a stress- and Hog1-dependent manner. Moreover, Na+ and Li+ stress increases the number of peroxisomes per cell in a Hog1-independent manner, which depends instead of the retrograde pathway and the dynamin related GTPases Dnm1 and Vps1. The strong activation of the Faa1 fatty acyl-CoA synthetase, which specifically localizes to lipid particles and peroxisomes, indicates that adaptation to salt stress requires the enhanced mobilization of fatty acids from internal lipid stores. Furthermore, the activation of mitochondrial respiration during stress depends on peroxisomes, mitochondrial acetyl-carnitine uptake is essential for salt resistance and the number of peroxisomes attached to the mitochondrial network increases during salt adaptation, which altogether indicates that stress-induced peroxisomal ß-oxidation triggers enhanced respiration upon salt shock.
Asunto(s)
Peroxisomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Estrés Fisiológico/fisiología , Acilcoenzima A/metabolismo , Coenzima A Ligasas/metabolismo , Ácidos Grasos/metabolismo , Metabolismo de los Lípidos , Lípidos/biosíntesis , Mitocondrias/metabolismo , Presión Osmótica/fisiología , Oxidación-Reducción , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Cloruro de Sodio/metabolismoRESUMEN
Here, we review and update the recent advances in the metabolic control during the adaptive response of budding yeast to hyperosmotic and salt stress, which is one of the best understood signaling events at the molecular level. This environmental stress can be easily applied and hence has been exploited in the past to generate an impressively detailed and comprehensive model of cellular adaptation. It is clear now that this stress modulates a great number of different physiological functions of the cell, which altogether contribute to cellular survival and adaptation. Primary defense mechanisms are the massive induction of stress tolerance genes in the nucleus, the activation of cation transport at the plasma membrane, or the production and intracellular accumulation of osmolytes. At the same time and in a coordinated manner, the cell shuts down the expression of housekeeping genes, delays the progression of the cell cycle, inhibits genomic replication, and modulates translation efficiency to optimize the response and to avoid cellular damage. To this fascinating interplay of cellular functions directly regulated by the stress, we have to add yet another layer of control, which is physiologically relevant for stress tolerance. Salt stress induces an immediate metabolic readjustment, which includes the up-regulation of peroxisomal biomass and activity in a coordinated manner with the reinforcement of mitochondrial respiratory metabolism. Our recent findings are consistent with a model, where salt stress triggers a metabolic shift from fermentation to respiration fueled by the enhanced peroxisomal oxidation of fatty acids. We discuss here the regulatory details of this stress-induced metabolic shift and its possible roles in the context of the previously known adaptive functions.
Asunto(s)
Adaptación Biológica , Metabolismo de los Lípidos , Sales (Química)/metabolismo , Estrés Fisiológico , Levaduras/fisiología , Transducción de SeñalRESUMEN
Stress triggers complex transcriptional responses, which include both gene activation and repression. We used time-resolved reporter assays in living yeast cells to gain insights into the coordination of positive and negative control of gene expression upon salt stress. We found that the repression of "housekeeping" genes coincides with the transient activation of defense genes and that the timing of this expression pattern depends on the severity of the stress. Moreover, we identified mutants that caused an alteration in the kinetics of this transcriptional control. Loss of function of the vacuolar H(+)-ATPase (vma1) or a defect in the biosynthesis of the osmolyte glycerol (gpd1) caused a prolonged repression of housekeeping genes and a delay in gene activation at inducible loci. Both mutants have a defect in the relocation of RNA polymerase II complexes at stress defense genes. Accordingly salt-activated transcription is delayed and less efficient upon partially respiratory growth conditions in which glycerol production is significantly reduced. Furthermore, the loss of Hog1 MAP kinase function aggravates the loss of RNA polymerase II from housekeeping loci, which apparently do not accumulate at inducible genes. Additionally the Def1 RNA polymerase II degradation factor, but not a high pool of nuclear polymerase II complexes, is needed for efficient stress-induced gene activation. The data presented here indicate that the finely tuned transcriptional control upon salt stress is dependent on physiological functions of the cell, such as the intracellular ion balance, the protective accumulation of osmolyte molecules, and the RNA polymerase II turnover.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Glicerol-3-Fosfato Deshidrogenasa (NAD+)/genética , ATPasas de Translocación de Protón/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Tolerancia a la Sal/genética , Transcripción Genética , Perfilación de la Expresión Génica , Genes Reporteros , Glicerol/metabolismo , Glicerol-3-Fosfato Deshidrogenasa (NAD+)/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Presión Osmótica , ATPasas de Translocación de Protón/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Salinidad , Transducción de Señal , Cloruro de Sodio/farmacología , Estrés FisiológicoRESUMEN
Gene expression regulation by intracellular stimulus-activated protein kinases is essential for cell adaptation to environmental changes. There are three PKA catalytic subunits in Saccharomyces cerevisiae: Tpk1, Tpk2, and Tpk3 and one regulatory subunit: Bcy1. Previously, it has been demonstrated that Tpk1 and Tpk2 are associated with coding regions and promoters of target genes in a carbon source and oxidative stress dependent manner. Here we studied five genes, ALD6, SED1, HSP42, RPS29B, and RPL1B whose expression is regulated by saline stress. We found that PKA catalytic and regulatory subunits are associated with both coding regions and promoters of the analyzed genes in a stress dependent manner. Tpk1 and Tpk2 recruitment was completely abolished in catalytic inactive mutants. BCY1 deletion changed the binding kinetic to chromatin of each Tpk isoform and this strain displayed a deregulated gene expression in response to osmotic stress. In addition, yeast mutants with high PKA activity exhibit sustained association to target genes of chromatin-remodeling complexes such as Snf2-catalytic subunit of the SWI/SNF complex and Arp8-component of INO80 complex, leading to upregulation of gene expression during osmotic stress. Tpk1 accumulation in the nucleus was stimulated upon osmotic stress, while the nuclear localization of Tpk2 and Bcy1 showed no change. We found that each PKA subunit is transported into the nucleus by a different ß-karyopherin pathway. Moreover, ß-karyopherin mutant strains abolished the chromatin association of Tpk1 or Tpk2, suggesting that nuclear localization of PKA catalytic subunits is required for its association to target genes and properly gene expression.
Asunto(s)
Cromatina/enzimología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación Fúngica de la Expresión Génica/fisiología , Proteínas de Saccharomyces cerevisiae/biosíntesis , Saccharomyces cerevisiae/enzimología , Estrés Fisiológico/fisiología , Cromatina/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Mot3 and Rox1 are transcriptional repressors of hypoxic genes. Both factors recently have been found to be involved in the adaptive response to hyperosmotic stress, with an important function in the adjustment of ergosterol biosynthesis. Here, we determine the gene expression profile of a mot3 rox1 double mutant under acute osmostress at the genomic scale in order to identify the target genes affected by both transcription factors upon stress. Unexpectedly, we find a specific subgroup of osmostress-inducible genes to be under positive control of Mot3. These Mot3-activated stress genes also depend on the general stress activators Msn2 and Msn4. We confirm that both Mot3 and Msn4 bind directly to some promoter regions of this gene group. Furthermore, osmostress-induced binding of the Msn2 and Msn4 factors to these target promoters is severely affected by the loss of Mot3 function. The genes repressed by Mot3 and Rox1 preferentially encode proteins of the cell wall and plasma membrane. Cell conjugation was the most significantly enriched biological process which was negatively regulated by both factors and by osmotic stress. The mating response was repressed by salt stress dependent on Mot3 and Rox1 function. Taking our findings together, the Mot3 transcriptional regulator has unanticipated diverse functions in the cellular adjustment to osmotic stress, including transcriptional activation and modulation of mating efficiency.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/fisiología , Proteínas de Unión al ADN/metabolismo , Técnicas de Inactivación de Genes , Genes Fúngicos , Genes del Tipo Sexual de los Hongos , Presión Osmótica , Unión Proteica , Proteínas Represoras/metabolismo , Proteínas Represoras/fisiología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Tolerancia a la Sal , Estrés Fisiológico , Factores de Transcripción/metabolismo , Activación Transcripcional , TranscriptomaRESUMEN
Exposure of yeast to high osmolarity induces a transient activation of the Hog1 stress-activated protein kinase (SAPK), which is required for cell survival under these conditions. However, sustained activation of the SAPK results in a severe growth defect. We found that prolonged SAPK activation leads to cell death, which is not observed in nma111 cells, by causing accumulation of reactive oxygen species (ROS). Mutations of the SCF(CDC4) ubiquitin ligase complex suppress cell death by preventing the degradation of Msn2 and Msn4 transcription factors. Accumulation of Msn2 and Msn4 leads to the induction of PNC1, which is an activator of the Sir2 histone acetylase. Sir2 is involved in protection against Hog1-induced cell death and can suppress Hog1-induced ROS accumulation. Therefore, cell death seems to be dictated by the balance of ROS induced by Hog1 and the protective effect of Sir2.
Asunto(s)
Apoptosis , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sirtuina 2/metabolismo , Estrés Fisiológico , Apoptosis/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Expresión Génica , Regulación Fúngica de la Expresión Génica , Mutación/genética , Nicotinamidasa/genética , Nicotinamidasa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Ligasas SKP Cullina F-box/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Antifungal resistance is a growing concern as it poses a significant threat to public health. Fungal infections are a significant cause of morbidity and mortality, especially in immunocompromised individuals. The limited number of antifungal agents and the emergence of resistance have led to a critical need to understand the mechanisms of antifungal drug resistance. This review provides an overview of the importance of antifungal resistance, the classes of antifungal agents, and their mode of action. It highlights the molecular mechanisms of antifungal drug resistance, including alterations in drug modification, activation, and availability. In addition, the review discusses the response to drugs via the regulation of multidrug efflux systems and antifungal drug-target interactions. We emphasize the importance of understanding the molecular mechanisms of antifungal drug resistance to develop effective strategies to combat the emergence of resistance and highlight the need for continued research to identify new targets for antifungal drug development and explore alternative therapeutic options to overcome resistance. Overall, an understanding of antifungal drug resistance and its mechanisms will be indispensable for the field of antifungal drug development and clinical management of fungal infections.
RESUMEN
Virus infection involves the manipulation of key host cell functions by specialized virulence proteins. The Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) small accessory proteins ORF3a and ORF7a have been implicated in favoring virus replication and spreading by inhibiting the autophagic flux within the host cell. Here, we apply yeast models to gain insights into the physiological functions of both SARS-CoV-2 small open reading frames (ORFs). ORF3a and ORF7a can be stably overexpressed in yeast cells, producing a decrease in cellular fitness. Both proteins show a distinguishable intracellular localization. ORF3a localizes to the vacuolar membrane, whereas ORF7a targets the endoplasmic reticulum. Overexpression of ORF3a and ORF7a leads to the accumulation of Atg8 specific autophagosomes. However, the underlying mechanism is different for each viral protein as assessed by the quantification of the autophagic degradation of Atg8-GFP fusion proteins, which is inhibited by ORF3a and stimulated by ORF7a. Overexpression of both SARS-CoV-2 ORFs decreases cellular fitness upon starvation conditions, where autophagic processes become essential. These data confirm previous findings on SARS-CoV-2 ORF3a and ORF7a manipulating autophagic flux in mammalian cell models and are in agreement with a model where both small ORFs have synergistic functions in stimulating intracellular autophagosome accumulation, ORF3a by inhibiting autophagosome processing at the vacuole and ORF7a by promoting autophagosome formation at the ER. ORF3a has an additional function in Ca2+ homeostasis. The overexpression of ORF3a confers calcineurin-dependent Ca2+ tolerance and activates a Ca2+ sensitive FKS2-luciferase reporter, suggesting a possible ORF3a-mediated Ca2+ efflux from the vacuole. Taken together, we show that viral accessory proteins can be functionally investigated in yeast cells and that SARS-CoV-2 ORF3a and ORF7a proteins interfere with autophagosome formation and processing as well as with Ca2+ homeostasis from distinct cellular targets.
RESUMEN
Hyperosmotic stress triggers a complex adaptive response that is dominantly regulated by the Hog1 MAP kinase in yeast. Here we characterize a novel physiological determinant of osmostress tolerance, which involves the Hog1-dependent transcriptional downregulation of ergosterol biosynthesis genes (ERG). Yeast cells considerably lower their sterol content in response to high osmolarity. The transcriptional repressors Mot3 and Rox1 are essential for this response. Both factors together with Hog1 are required to rapidly and transiently shut down transcription of ERG2 and ERG11 upon osmoshock. Mot3 abundance and its binding to the ERG2 promoter is stimulated by osmostress in a Hog1-dependent manner. As an additional layer of control, the expression of the main transcriptional activator of ERG gene expression, Ecm22, is negatively regulated by Hog1 and Mot3/Rox1 upon salt shock. Oxidative stress also triggers repression of ERG2, 11 transcription and a profound decrease in total sterol levels. However, this response was only partially dependent on Mot3/Rox1 and Hog1. Finally, we show that the upc2-1 mutation confers stress insensitive hyperaccumulation of ergosterol, overexpression of ERG2, 11 and severe sensitivity to salt and oxidative stress. Our results indicate that transcriptional control of ergosterol biosynthesis is an important physiological target of stress signalling.
Asunto(s)
Ergosterol/biosíntesis , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo , Proteínas Represoras/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Cloruro de Sodio/farmacología , Factores de Transcripción/genéticaRESUMEN
A destabilized version of firefly luciferase was used in living yeast cells as a real-time reporter for gene expression. This highly sensitive and non-invasive system can be simultaneously used upon many different experimental conditions in small culture aliquots. This allows the dose-response behaviour of gene expression driven by any yeast promoter to be reported and can be used to quantify important parameters, such as the threshold, sensitivity, response time, maximal activity and synthesis rate for a given stimulus. We applied the luciferase assay to the nutrient-regulated GAL1 promoter and the stress-responsive GRE2 promoter. We find that luciferase expression driven by the GAL1 promoter responds dynamically to growing galactose concentrations, with increasing synthesis rates determined by the light increment in the initial linear phase of activation. In the case of the GRE2 promoter, we demonstrate that the very short-lived version of luciferase used here is an excellent tool to quantitatively describe transient transcriptional activation. The luciferase expression controlled by the GRE2 promoter responds dynamically to a gradual increase of osmotic or oxidative stress stimuli, which is mainly based on the progressive increase of the time the promoter remains active. Finally, we determined the dose-response behaviour of a single transcription factor binding site in a synthetic promoter context, using the stress response element (STRE) as an example. Taken together, the luciferase assay described here is an attractive tool to rapidly and precisely determine and compare kinetic parameters of gene expression.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Expresión Génica , Genes Reporteros , Genética Microbiana/métodos , Luciferasas/metabolismo , Saccharomyces cerevisiae/genética , Fusión Artificial Génica , Galactosa/metabolismo , Luciferasas/genética , Presión Osmótica , Estrés Oxidativo , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/metabolismoRESUMEN
Hyperosmotic stress triggers a great variety of adaptive responses in eukaryotic cells that affect many different physiological functions. Here we investigate the role of the mitochondria during osmostress adaptation in budding yeast. Mitochondrial function is generally required for proper salt and osmotic stress adaptation because mutants with defects in many different mitochondrial components show hypersensitivity to increased NaCl and KCl concentrations. Mitochondrial protein abundance rapidly increases upon osmoshock in a selective manner, because it affects Calvin cycle enzymes (Sdh2 and Cit1) and components of the electron transport chain (Cox6) but not the ATP synthase complex (Atp5). Transcription of the SDH2, CIT1, and COX6 genes is severalfold induced within the first minutes of osmotic shock, dependent to various degree on the Hog1 and Snf1 protein kinases. Mitochondrial succinate dehydrogenase enzyme activity is stimulated upon osmostress in a Snf1-dependent manner. The osmosensitivity of mitochondrial mutants is not caused by impaired stress-activated transcription or by a general depletion of the cellular ATP pool during osmostress. We finally show that the growth defect of mitochondrial mutants in high salt medium can be partially rescued by supplementation of glutathione. Additionally, mitochondrial defects cause the hyperaccumulation of reactive oxygen species during salt stress. Our results indicate that the antioxidant function of the mitochondria might play an important role in adaptation to hyperosmotic stress.
Asunto(s)
Adaptación Fisiológica/genética , Mitocondrias/fisiología , Proteínas Mitocondriales/genética , Presión Osmótica , Saccharomyces cerevisiae/fisiología , Regulación de la Expresión Génica , Proteínas Mutantes/fisiología , Proteínas de Saccharomyces cerevisiae , Estrés FisiológicoRESUMEN
Cells respond to external stimuli with transient gene expression changes in order to adapt to environmental alterations. However, the dose response profile of gene induction upon a given stress depends on many intrinsic and extrinsic factors. Here we show that the accurate quantification of dose dependent gene expression by live cell luciferase reporters reveals fundamental insights into stress signaling. We make the following discoveries applying this non-invasive reporter technology. (1) Signal transduction sensitivities can be compared and we apply this here to salt, oxidative and xenobiotic stress responsive transcription factors. (2) Stress signaling depends on where and how the damage is generated within the cell. Specifically we show that two ROS-generating agents, menadione and hydrogen peroxide, differ in their dependence on mitochondrial respiration. (3) Stress signaling is conditioned by the cells history. We demonstrate here that positive memory or an acquired resistance towards oxidative stress is induced dependent on the nature of the previous stress experience. (4) The metabolic state of the cell impinges on the sensitivity of stress signaling. This is shown here for the shift towards higher stress doses of the response profile for yeast cells moved from complex to synthetic medium. (5) The age of the cell conditions its transcriptional response capacity, which is demonstrated by the changes of the dose response to oxidative stress during both replicative and chronological aging. We conclude that capturing dose dependent gene expression in real time will be of invaluable help to understand stress signaling and its dynamic modulation.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Saccharomyces cerevisiae/genética , Transcripción Genética , Genes Reporteros , Luciferasas/genética , Presión Osmótica , Estrés Oxidativo/genética , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
Repair and removal of damaged mitochondria is a key process for eukaryotic cell homeostasis. Here we investigate in the yeast model how different protein complexes of the mitochondrial electron transport chain are subject to specific degradation upon high respiration load and organelle damage. We find that the turnover of subunits of the electron transport complex I equivalent and complex III is preferentially stimulated upon high respiration rates. Particular mitochondrial proteases, but not mitophagy, are involved in this activated degradation. Further mitochondrial damage by valinomycin treatment of yeast cells triggers the mitophagic removal of the same respiratory complexes. This selective protein degradation depends on the mitochondrial fusion and fission apparatus and the autophagy adaptor protein Atg11, but not on the mitochondrial mitophagy receptor Atg32. Loss of autophagosomal protein function leads to valinomycin sensitivity and an overproduction of reactive oxygen species upon mitochondrial damage. A specific event in this selective turnover of electron transport chain complexes seems to be the association of Atg11 with the mitochondrial network, which can be achieved by overexpression of the Atg11 protein even in the absence of Atg32. Furthermore, the interaction of various Atg11 molecules via the C-terminal coil domain is specifically and rapidly stimulated upon mitochondrial damage and could therefore be an early trigger of selective mitophagy in response to the organelles dysfunction. Our work indicates that autophagic quality control upon mitochondrial damage operates in a selective manner.
RESUMEN
Sphingolipids are regulators of mitochondria-mediated cell death in higher eukaryotes. Here, we investigate how changes in sphingolipid metabolism and downstream intermediates of sphingosine impinge on mitochondrial function. We found in yeast that within the sphingolipid degradation pathway, the production via Dpl1p and degradation via Hfd1p of hexadecenal are critical for mitochondrial function and cell death. Genetic interventions, which favor hexadecenal accumulation, diminish oxygen consumption rates and increase reactive oxygen species production and mitochondrial fragmentation and vice versa. The location of the hexadecenal-degrading enzyme Hfd1p in punctuate structures all along the mitochondrial network depends on a functional ERMES (endoplasmic reticulum-mitochondria encounter structure) complex, indicating that modulation of hexadecenal levels at specific ER-mitochondria contact sites might be an important trigger of cell death. This is further supported by the finding that externally added hexadecenal or the absence of Hfd1p enhances cell death caused by ectopic expression of the human Bax protein. Finally, the induction of the sphingolipid degradation pathway upon stress is controlled by the Hog1p MAP kinase. Therefore, the stress-regulated modulation of sphingolipid degradation might be a conserved way to induce cell death in eukaryotic organisms.
Asunto(s)
Mitocondrias/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Esfingolípidos/metabolismo , Estrés Fisiológico , Aldehídos/farmacología , Muerte Celular/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estrés Fisiológico/efectos de los fármacosRESUMEN
Peroxisomes and mitochondria are the main intracellular sources for reactive oxygen species. At the same time, both organelles are critical for the maintenance of a healthy redox balance in the cell. Consequently, failure in the function of both organelles is causally linked to oxidative stress and accelerated aging. However, it has become clear that peroxisomes and mitochondria are much more intimately connected both physiologically and structurally. Both organelles share common fission components to dynamically respond to environmental cues, and the autophagic turnover of both peroxisomes and mitochondria is decisive for cellular homeostasis. Moreover, peroxisomes can physically associate with mitochondria via specific protein complexes. Therefore, the structural and functional connection of both organelles is a critical and dynamic feature in the regulation of oxidative metabolism, whose dynamic nature will be revealed in the future. In this review, we will focus on fundamental aspects of the peroxisome-mitochondria interplay derived from simple models such as yeast and move onto discussing the impact of an impaired peroxisomal and mitochondrial homeostasis on ROS production, aging, and disease in humans.
Asunto(s)
Envejecimiento/metabolismo , Antioxidantes/metabolismo , Mitocondrias/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Peroxisomas/metabolismo , Animales , Autofagia , Humanos , Enfermedades Neurodegenerativas/patología , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Levaduras/metabolismoRESUMEN
Citrinin (CIT) and ochratoxin A (OTA) are important mycotoxins, which frequently co-contaminate foodstuff. In order to assess the toxicologic threat posed by the two mycotoxins separately or in combination, their biological effects were studied here using genomic transcription profiling and specific live cell gene expression reporters in yeast cells. Both CIT and OTA cause highly transient transcriptional activation of different stress genes, which is greatly enhanced by the disruption of the multidrug exporter Pdr5. Therefore, we performed genome-wide transcription profiling experiments with the pdr5 mutant in response to acute CIT, OTA, or combined CIT/OTA exposure. We found that CIT and OTA activate divergent and largely nonoverlapping gene sets in yeast. CIT mainly caused the rapid induction of antioxidant and drug extrusion-related gene functions, while OTA mainly deregulated developmental genes related with yeast sporulation and sexual reproduction, having only a minor effect on the antioxidant response. The simultaneous exposure to CIT and OTA gave rise to a genomic response, which combined the specific features of the separated mycotoxin treatments. The application of stress-specific mutants and reporter gene fusions further confirmed that both mycotoxins have divergent biological effects in cells. Our results indicate that CIT exposure causes a strong oxidative stress, which triggers a massive transcriptional antioxidant and drug extrusion response, while OTA mainly deregulates developmental genes and only marginally induces the antioxidant defense.