RESUMEN
Glutathione is the most abundant low molecular weight thiol in the eukaryotic cytosol. The compartment-specific ratio and absolute concentrations of reduced and oxidized glutathione (GSH and GSSG, respectively) are, however, not easily determined. Here, we present a glutathione-specific green fluorescent protein-based redox probe termed redox sensitive YFP (rxYFP). Using yeast with genetically manipulated GSSG levels, we find that rxYFP equilibrates with the cytosolic glutathione redox buffer. Furthermore, in vivo and in vitro data show the equilibration to be catalyzed by glutaredoxins and that conditions of high intracellular GSSG confer to these a new role as dithiol oxidases. For the first time a genetically encoded probe is used to determine the redox potential specifically of cytosolic glutathione. We find it to be -289 mV, indicating that the glutathione redox status is highly reducing and corresponds to a cytosolic GSSG level in the low micromolar range. Even under these conditions a significant fraction of rxYFP is oxidized.
Asunto(s)
Cistina/biosíntesis , Citosol/metabolismo , Disulfuros/metabolismo , Proteínas/metabolismo , Genes Reporteros , Glutarredoxinas , Glutatión/metabolismo , Oxidación-Reducción , Oxidorreductasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
In Saccharomyces cerevisiae, glucose depletion causes a profound alteration in metabolism, mediated in part by global transcriptional changes. Many of the transcription factors that regulate these changes act combinatorially. We have analyzed combinatorial regulation by Adr1 and Cat8, two transcription factors that act during glucose depletion, by combining genome-wide expression and genome-wide binding data. We identified 32 genes that are directly activated by Adr1, 28 genes that are directly activated by Cat8, and 14 genes that are directly regulated by both. Our analysis also uncovered promoters that Adr1 binds but does not regulate and promoters that are indirectly regulated by Cat8, stressing the advantage of combining global expression and global localization analysis to find directly regulated targets. At most of the coregulated promoters, the in vivo binding of one factor is independent of the other, but Adr1 is required for optimal Cat8 binding at two promoters with a poor match to the Cat8 binding consensus. In addition, Cat8 is required for Adr1 binding at promoters where Adr1 is not required for transcription. These data provide a comprehensive analysis of the direct, indirect, and combinatorial requirements for these two global transcription factors.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/genética , Transactivadores/fisiología , Factores de Transcripción/fisiología , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Genes Fúngicos , Genoma Fúngico , Glucosa/fisiología , Regiones Promotoras Genéticas/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transactivadores/genética , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
The transcription factor Adr1 directly activates the expression of genes encoding enzymes in numerous pathways that are upregulated after the exhaustion of glucose in the yeast Saccharomyces cerevisiae. ADH2, encoding the alcohol dehydrogenase isozyme required for ethanol oxidation, is a highly glucose-repressed, Adr1-dependent gene. Using a genetic screen we isolated >100 mutants in 12 complementation groups that exhibit ADR1-dependent constitutive ADH2 expression on glucose. Temperature-sensitive alleles are present among the new constitutive mutants, indicating that essential genes play a role in ADH2 repression. Among the genes we cloned is MOT1, encoding a repressor that inhibits TBP binding to the promoter, thus linking glucose repression with TBP access to chromatin. Two genes encoding proteins involved in vacuolar function, FAB1 and VPS35, and CDC10, encoding a nonessential septin, were also uncovered in the search, suggesting that vacuolar function and the cytoskeleton have previously unknown roles in regulating gene expression. Constitutive activation of ADH2 expression by Adr1 is SNF1-dependent in a strain with a defective MOT1 gene, whereas deletion of SNF1 did not affect constitutive ADH2 expression in the mutants affecting vacuolar or septin function. Thus, the mutant search revealed previously unknown Snf1-dependent and -independent pathways of ADH2 expression.
Asunto(s)
Alcohol Deshidrogenasa/genética , Regulación Fúngica de la Expresión Génica , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Alcohol Deshidrogenasa/química , Alelos , Cromatina/metabolismo , Eliminación de Gen , Prueba de Complementación Genética , Glucosa/metabolismo , Modelos Genéticos , Mutación , Plásmidos/metabolismo , Regulación hacia ArribaRESUMEN
Glutathione is present in millimolar concentrations in the cell, but its relative distribution among cellular compartments remains elusive. We have chosen the endoplasmic reticulum (ER) as an example organelle to study compartment-specific glutathione levels. Using a glutaredoxin sensor (sCGrx1pER), which rapidly and specifically equilibrates with the reduced glutathione (GSH)-glutathione disulfide (GSSG) redox couple with known equilibrium constant, we showed that the [GSH]:[GSSG] ratio in the ER of intact HeLa cells is less than 7:1. Taking into consideration the previously determined value for [GSH](2):[GSSG] in the ER of 83 mM, this translates into a total glutathione concentration in the ER ([GStot]=[GSH]+2[GSSG]) of greater than 15 mM. Since the integrated, intracellular [GStot] was measured as ~7 mM, we conclude the existence of a [GStot] gradient across the ER membrane. A possible homeostatic mechanism by which cytosol-derived glutathione is trapped in the ER is discussed. We propose a high [GStot] as a distinguishing feature of the ER environment compared to the extracellular space.
Asunto(s)
Retículo Endoplásmico/metabolismo , Glutarredoxinas/metabolismo , Glutatión/metabolismo , Citosol/metabolismo , Glutarredoxinas/genética , Células HeLa , Homeostasis , HumanosRESUMEN
Trisulfides and other oligosulfides are widely distributed in the biological world. In plants, for example, garlic, trisulfides are associated with potentially beneficial properties. However, an extra neutral sulfur atom covalently bound between the two sulfur atoms of a pair of cysteines is not a common post-translational modification, and the number of proteins in which a trisulfide has been unambiguously identified is small. Nevertheless, we believe that its prevalence may be underestimated, particularly with the increasing evidence for significant pools of sulfides in living tissues and their possible roles in cellular metabolism. This review focuses on examples of proteins that are known to contain a trisulfide bridge, and gives an overview of the chemistry of trisulfide formation, and the methods by which it is detected in proteins.
Asunto(s)
Proteínas/química , Proteínas/metabolismo , Sulfuros/química , Animales , HumanosRESUMEN
BACKGROUND: Post-translational modification regulates promoter-binding by Adr1, a Zn-finger transcriptional activator of glucose-regulated genes. Support for this model includes the activation of an Adr1-dependent gene in the absence of Adr1 protein synthesis, and a requirement for the kinase Snf1 for Adr1 DNA-binding. A fusion protein with the Adr1 DNA-binding domain and a heterologous activation domain is glucose-regulated, suggesting that the DNA binding region is the target of regulation. METHODOLOGY/PRINCIPAL FINDINGS: Peptide mapping identified serine 98 adjacent to the Zn-fingers as a phosphorylation site. An antibody specific for phosphorylated serine 98 on Adr1 showed that the level of phosphorylated Adr1 relative to the level of total Adr1 decreased with glucose derepression, in a Snf1-dependent manner. Relative phosphorylation decreased in a PHO85 mutant, and this mutant constitutively expressed an Adr1-dependent reporter. Pho85 did not phosphorylate Adr1 in vitro, suggesting that it affects Adr1 indirectly. Mutation of serine 98 to the phosphomimetic amino acid aspartate reduced in vitro DNA-binding of the recombinant Adr1 DNA-binding domain. Mutation to aspartate or alanine affected activation of a reporter by full-length Adr1, and in vivo promoter binding. CONCLUSIONS/SIGNIFICANCE: Mutation of Adr1 serine 98 affects in vitro and in vivo DNA binding, and phosphorylation of serine 98 in vivo correlates with glucose availability, suggesting that Adr1 promoter-binding is regulated in part by serine 98 phosphorylation.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , ADN/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Activación Transcripcional , Análisis Mutacional de ADN , Glucosa/metabolismo , Modelos Moleculares , Mutación , Mapeo Peptídico , Fosforilación , Regiones Promotoras Genéticas , Unión Proteica , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Serina/química , Dedos de ZincRESUMEN
The transcription factor Adr1 activates numerous genes in nonfermentable carbon source metabolism. An unknown mechanism prevents Adr1 from stably binding to the promoters of these genes in glucose-grown cells. Glucose depletion leads to Snf1-dependent binding. Chromatin immunoprecipitation showed that the Adr1 DNA-binding domain could not be detected at the ADH2 promoter under conditions in which the binding of the full-length protein occurred. This suggested that an activation domain is required for stable binding, and coactivators may stabilize the interaction with the promoter. Artificial recruitment of Mediator tail subunits by fusion to the Adr1 DNA-binding domain overcame both the inhibition of promoter binding and glucose repression of ADH2 expression. In contrast, an Adr1 DNA-binding domain-Tbp fusion did not overcome glucose repression, although it was an efficient activator of ADH2 expression under derepressing conditions. When Mediator was artificially recruited, ADH2 expression was independent of SNF1, SAGA, and Swi/Snf, whereas ADH2 expression was dependent on these factors with wild-type Adr1. These results suggest that in the presence of glucose, the ADH2 promoter is accessible to Adr1 but that other interactions that occur when glucose is depleted do not take place. Artificial recruitment of Mediator appears to overcome this requirement and to allow stable binding and transcription under normally inhibitory conditions.
Asunto(s)
Alcohol Deshidrogenasa/metabolismo , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica , Glucosa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Alcohol Deshidrogenasa/genética , Sitios de Unión , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Transcripción Genética/genéticaRESUMEN
Snf1, the yeast AMP kinase homolog, is essential for derepression of glucose-repressed genes that are activated by Adr1. Although required for Adr1 DNA binding, the precise role of Snf1 is unknown. Deletion of histone deacetylase genes allowed constitutive promoter binding of Adr1 and Cat8, another activator of glucose-repressed genes. In repressed conditions, at the Adr1-and Cat8-dependent ADH2 promoter, partial chromatin remodeling had occurred, and the activators recruited a partial preinitiation complex that included RNA polymerase II. Transcription did not occur, however, unless Snf1 was activated, suggesting a Snf1-dependent event that occurs after RNA polymerase II recruitment. Glucose regulation persisted because shifting to low glucose increased expression. Glucose repression could be completely relieved by combining the three elements of 1) chromatin perturbation by mutation of histone deacetylases, 2) activation of Snf1, and 3) the addition of an Adr1 mutant that by itself confers only weak constitutive activity.
Asunto(s)
Proteínas Serina-Treonina Quinasas/fisiología , Cromatina/química , ADN/química , Cartilla de ADN/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/fisiología , Eliminación de Gen , Glucosa/metabolismo , Histona Desacetilasas/metabolismo , Modelos Biológicos , Mutación , Nucleosomas/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Transactivadores/metabolismo , Factores de Transcripción/química , Factores de Transcripción/fisiologíaRESUMEN
Endoplasmic reticulum-associated degradation (ERAD) mediates the turnover of short-lived and misfolded proteins in the ER membrane or lumen. In spite of its important role, only subtle growth phenotypes have been associated with defects in ERAD. We have discovered that the ERAD proteins Ubc7 (Qri8), Cue1, and Doa10 (Ssm4) are required for growth of yeast that express high levels of the sterol biosynthetic enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Interestingly, the observed growth defect was exacerbated at low temperatures, producing an HMGR-dependent cold sensitivity. Yeast strains lacking UBC7, CUE1, or DOA10 also assembled aberrant karmellae (ordered arrays of membranes surrounding the nucleus that assemble when HMGR is expressed at high levels). However, rather than reflecting the accumulation of abnormal karmellae, the cold sensitivity of these ERAD mutants was due to increased HMGR catalytic activity. Mutations that compromise proteasomal function also resulted in cold-sensitive growth of yeast with elevated HMGR, suggesting that improper degradation of ERAD targets might be responsible for the observed cold-sensitive phenotype. However, the essential ERAD targets were not the yeast HMGR enzymes themselves. The sterol metabolite profile of ubc7Delta cells was altered relative to that of wild-type cells. Since sterol levels are known to regulate membrane fluidity, the viability of ERAD mutants expressing normal levels of HMGR was examined at low temperatures. Cells lacking UBC7, CUE1, or DOA10 were cold sensitive, suggesting that these ERAD proteins have a role in cold adaptation, perhaps through effects on sterol biosynthesis.
Asunto(s)
Aclimatación/fisiología , Proteínas Portadoras/fisiología , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/fisiología , Fosfoproteínas Fosfatasas/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/fisiología , Esteroles/biosíntesis , Enzimas Ubiquitina-Conjugadoras/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Proteínas Portadoras/genética , Frío , Eliminación de Gen , Proteínas de la Membrana/genética , Fosfoproteínas Fosfatasas/genética , Complejo de la Endopetidasa Proteasomal/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Esteroles/análisis , Enzimas Activadoras de Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Ubiquitina-Proteína Ligasas/genética , Regulación hacia ArribaRESUMEN
We have examined the activities of promoters of a number of yeast genes encoding resident endoplasmic reticulum proteins, and found increased expression in a strain with severe protein disulphide isomerase deficiency. Serial deletion in the promoter of the MPD1 gene, which encodes a PDI1-homologue, revealed a cis-acting element responding to deficiency of protein disulphide isomerase activity (designated CERP). The presence of the sequence element is necessary and sufficient for the upregulation in response to disulphide isomerase deficiency, as measured by a minimal promoter containing the CERP element. The sequence (GACACG) does not resemble the unfolded protein response element. It is present in the upstream regions of the MPD1, MPD2, KAR2, PDI1 and ERO1 genes.