Zinc Cluster Proteins Research Articles

Novel Botrytis cinerea Zn(II)2Cys6 Transcription Factor BcFtg1 Enhances the Virulence of the Gray Mold Fungus by Promoting Organic Acid Secretion and Carbon Source Utilization.

The Zn(II)2Cys6 zinc cluster protein family comprises a subclass of zinc-finger proteins that serve as transcriptional regulators involved in a diverse array of fugal biological processes. However, the roles and mechanisms of the Zn(II)2Cys6 transcription factors in mediating Botrytis cinerea, a necrotrophic fungus that causes gray mold in over 1000 plant species, development and virulence remain obscure. Here, we demonstrate that a novel B. cinerea pathogenicity-associated factor BcFTG1 (fungal transcription factor containing the GAL4 domain), identified from a virulence-attenuated mutant M20162 from a B. cinerea T-DNA insertion mutant library, plays an important role in oxalic acid (OA) secretion, carbon source absorption and cell wall integrity. Loss of BcFTG1 compromises the ability of the pathogen to secrete OA, absorb carbon sources, maintain cell wall integrity, and promote virulence. Our findings provide novel insights into fungal factors mediating the pathogenesis of the gray mold fungus via regulation of OA secretion, carbon source utilization and cell wall integrity.

Transcription factor AacmrA mediated melanin synthesis regulates the growth, appressorium formation, stress response and pathogenicity of pear fungal Alternaria alternata.

CmrA, as transcription factor for regulating DHN-melanin synthesis, controls melanin synthesis gene expression, and also regulate growth, development, stress response and virulence of plant fungi. However, little is known about the roles of CmrA on infection structure formation, penetration and pathogenicity of pear fungal Alternaria alternata. Here, we identified cmrA gene in A.alternata and assigned as AacmrA, sequence alignment and phylogenetic analysis revealed that AacmrA is highly conserved among fungi and encoded protein contain two Cys2His2 zinc finger motifs and one Zn(II)2Cys6 zinc cluster protein motif. ΔAacmrA severely decreased melanin production and DHN melanin synthesis related genes expression. Deletion of AacmrA impaired the morphology of spore and hyphae. Spore germination and appressorium formation induced by hydrophobicity surfaces and fruit wax significantly decreased in ΔAacmrA mutant. ΔAacmrA mutants were more sensitive than the wild type to osmotic stress and cell wall inhibitors, especially more sensitive to oxidative stress. In addition, lesion diameter of pear fruit wound inoculated with the ΔAacmrA mutant was reduced by 40.8% compared with the wild type 12d after inoculation. All findings of this study suggested that AacmrA is required for melanin biosynthesis, infection structure formation, and pathogenicity in A.alternata.

Genome-wide characterization of the Zn(II)2Cys6 zinc cluster-encoding gene family in Pleurotus ostreatus and expression analyses of this family during developmental stages and under heat stress.

Pleurotus ostreatus is one of the most widely cultivated mushrooms in China. The regulatory mechanisms of fruiting body formation and the response to heat stress in P. ostreatus are main research focuses. The Zn(II)2Cys6 family is one of the largest families of transcriptional factors and plays important roles in multiple biological processes in fungi. In this study, we identified 66 zinc cluster proteins in P. ostreatus (PoZCPs) through a genome-wide search. The PoZCPs were classified into 15 types according to their zinc cluster domain. Physical and chemical property analyses showed a huge diversity among the PoZCPs. Phylogenetic analysis of PoZCPs classified these proteins into six groups and conserved motif combinations and similar gene structures were observed in each group. The expression profiles of these PoZCP genes during different developmental stages and under heat stress were further investigated by RNA-sequencing (RNA-seq), revealing diverse expression patterns. A total of 13 PoZCPs that may participate in development or the heat stress response were selected for validation of their expression levels through real-time quantitative PCR (RT-qPCR) analysis, and some developmental stage-specific and heat stress-responsive candidates were identified. The findings contribute to our understanding of the roles and regulatory mechanisms of ZCPs in P. ostreatus.

From Lipid Homeostasis to Differentiation: Old and New Functions of the Zinc Cluster Proteins Ecm22, Upc2, Sut1 and Sut2.

Zinc cluster proteins are a large family of transcriptional regulators with a wide range of biological functions. The zinc cluster proteins Ecm22, Upc2, Sut1 and Sut2 have initially been identified as regulators of sterol import in the budding yeast Saccharomyces cerevisiae. These proteins also control adaptations to anaerobic growth, sterol biosynthesis as well as filamentation and mating. Orthologs of these zinc cluster proteins have been identified in several species of Candida. Upc2 plays a critical role in antifungal resistance in these important human fungal pathogens. Upc2 is therefore an interesting potential target for novel antifungals. In this review we discuss the functions, mode of actions and regulation of Ecm22, Upc2, Sut1 and Sut2 in budding yeast and Candida.

Phytohormone sensing in the biotrophic fungus Ustilago maydis - the dual role of the transcription factor Rss1.

SummaryThe phenolic compound salicylic acid (SA) is a key signalling molecule regulating local and systemic plant defense responses, mainly against biotrophs. Many microbial organisms, including pathogens, share the ability to degrade SA. However, the mechanism by which they perceive SA is unknown. Here we show that Ustilago maydis, the causal agent of corn smut disease, employs a so far uncharacterized SA sensing mechanism. We identified and characterized the novel SA sensing regulator, Rss1, a binuclear zinc cluster protein with dual functions as putative SA receptor and transcriptional activator regulating genes important for SA and tryptophan degradation. Rss1 represents a major component in the identified SA sensing pathway during the fungus’ saprophytic stage. However, Rss1 does not have a detectable impact on virulence. The data presented in this work indicate that alternative or redundant sensing cascades exist that regulate the expression of SA‐responsive genes in U. maydis during its pathogenic development.

Mutations in transcription factor Mrr2p contribute to fluconazole resistance in clinical isolates of Candida albicans

The Candida albicans zinc cluster proteins are a family of transcription factors (TFs) that play essential roles in the development of antifungal drug resistance. Gain-of-function mutations in several TFs, such as Tac1p, Mrr1p and Upc2p, have been previously well documented in azole-resistant clinical C. albicans isolates. Mrr2p (multidrug resistance regulator 2) is a novel TF controlling expression of the ABC transporter gene CDR1 and mediating fluconazole resistance. In this study, the relationship between naturally occurring mutations in MRR2 and fluconazole resistance in clinical C. albicans isolates was investigated. Among a group of 20 fluconazole-resistant clinical C. albicans and 10 fluconazole-susceptible C. albicans, 12 fluconazole-resistant isolates overexpressed CDR1 by at least two-fold compared with the fluconazole-susceptible isolates. Of these 12 resistant isolates, three (C7, C9, C15) contained 11 identical missense mutations, 6 of which occurred only in the azole-resistant isolates. The contribution of these mutations to CDR1 overexpression and therefore to fluconazole resistance was further verified by generating recombinant strains containing the mutated MRR2 gene. The mutated MRR2 alleles from isolate C9 contributed to an almost six-fold increase in CDR1 expression and an eight-fold increase in fluconazole resistance; the missense mutations S466L and T470N resulted in an increase in CDR1 expression of more than two-fold and a four-fold increase in fluconazole resistance. In contrast, the other four missense mutations conferred only two- to four-fold increases in fluconazole resistance, with no significant increase in CDR1 expression. These findings provide some insight into the mechanism by which MRR2 regulates C. albicans multidrug resistance.

Zinc cluster protein Znf1, a novel transcription factor of non-fermentative metabolism in Saccharomyces cerevisiae.

The ability to rapidly respond to nutrient changes is a fundamental requirement for cell survival. Here, we show that the zinc cluster regulator Znf1 responds to altered nutrient signals following glucose starvation through the direct control of genes involved in non-fermentative metabolism, including those belonged to the central pathways of gluconeogenesis (PCK1, FBP1 and MDH2), glyoxylate shunt (MLS1 and ICL1) and the tricarboxylic acid cycle (ACO1), which is demonstrated by Znf1-binding enrichment at these promoters during the glucose-ethanol shift. Additionally, reduced Pck1 and Fbp1 enzymatic activities correlate well with the data obtained from gene transcription analysis. Cells deleted for ZNF1 also display defective mitochondrial morphology with unclear structures of the inner membrane cristae when grown in ethanol, in agreement with the substantial reduction in the ATP content, suggesting for roles of Znf1 in maintaining mitochondrial morphology and function. Furthermore, Znf1 also plays a role in tolerance to pH and osmotic stress, especially during the oxidative metabolism. Taken together, our results clearly suggest that Znf1 is a critical transcriptional regulator for stress adaptation during non-fermentative growth with some partial overlapping targets with previously reported regulators in Saccharomyces cerevisiae.

The switch from fermentation to respiration in Saccharomyces cerevisiae is regulated by the Ert1 transcriptional activator/repressor.

In the yeast Saccharomyces cerevisiae, fermentation is the major pathway for energy production, even under aerobic conditions. However, when glucose becomes scarce, ethanol produced during fermentation is used as a carbon source, requiring a shift to respiration. This adaptation results in massive reprogramming of gene expression. Increased expression of genes for gluconeogenesis and the glyoxylate cycle is observed upon a shift to ethanol and, conversely, expression of some fermentation genes is reduced. The zinc cluster proteins Cat8, Sip4, and Rds2, as well as Adr1, have been shown to mediate this reprogramming of gene expression. In this study, we have characterized the gene YBR239C encoding a putative zinc cluster protein and it was named ERT1 (ethanol regulated transcription factor 1). ChIP-chip analysis showed that Ert1 binds to a limited number of targets in the presence of glucose. The strongest enrichment was observed at the promoter of PCK1 encoding an important gluconeogenic enzyme. With ethanol as the carbon source, enrichment was observed with many additional genes involved in gluconeogenesis and mitochondrial function. Use of lacZ reporters and quantitative RT-PCR analyses demonstrated that Ert1 regulates expression of its target genes in a manner that is highly redundant with other regulators of gluconeogenesis. Interestingly, in the presence of ethanol, Ert1 is a repressor of PDC1 encoding an important enzyme for fermentation. We also show that Ert1 binds directly to the PCK1 and PDC1 promoters. In summary, Ert1 is a novel factor involved in the regulation of gluconeogenesis as well as a key fermentation gene.

The novel zinc cluster regulator Tog1 plays important roles in oleate utilization and oxidative stress response in Saccharomyces cerevisiae

Many zinc cluster proteins have been shown to play a role in the transcriptional regulation of glucose-repressible genes during glucose exhaustion and diauxic shift. Here, we studied an additional member of this family called Yer184c (herein called Tog1) for transcriptional regulator of oleate. Our results showed that a Δtog1 strain displays impaired growth with several non-fermentable carbons. Tog1 is also implicated in oxidative stress tolerance. Importantly, during the glucose–oleate shift, combined results from quantitative real time-PCR and chromatin immunoprecipitation (ChIP) experiments showed that Tog1 acts as a direct activator of oleate utilizing genes, encoded key enzymes in β-Oxidation and NADPH regeneration (POX1, FOX2, POT1 and IDP2), the glyoxylate shunt (MLS1 and ICL1), and gluconeogenesis (PCK1 and FBP1). A transmission electron microscopy (TEM) analysis of the Δtog1 strain assayed with oleate also revealed a substantial decrease in peroxisome abundance that is vital for fatty acid oxidation. Overall, our results clearly demonstrated that Tog1 is a newly characterized zinc cluster regulator that functions in the complex network of non-fermentable carbon metabolism in Saccharomycescerevisiae.

Phenotypic Analysis of a Family of Transcriptional Regulators, the Zinc Cluster Proteins, in the Human Fungal Pathogen Candida glabrata

Candida glabrata is the second most important human fungal pathogen. Despite its formal name, C. glabrata is in fact more closely related to the nonpathogenic budding yeast Saccharomyces cerevisiae. However, less is known about the biology of this pathogen. Zinc cluster proteins form a large family of transcriptional regulators involved in the regulation of numerous processes such as the control of the metabolism of sugars, amino acids, fatty acids, as well as drug resistance. The C. glabrata genome encodes 41 known or putative zinc cluster proteins, and the majority of them are uncharacterized. We have generated a panel of strains carrying individual deletions of zinc cluster genes. Using a novel approach relying on tetracycline for conditional expression in C. glabrata at the translational level, we show that only two zinc cluster genes are essential. We have performed phenotypic analysis of nonessential zinc cluster genes. Our results show that two deletion strains are thermosensitive whereas two strains are sensitive to caffeine, an inhibitor of the target of rapamycin pathway. Increased salt tolerance has been observed for eight deletion strains, whereas one strain showed reduced tolerance to salt. We have also identified a number of strains with increased susceptibility to the antifungal drugs fluconazole and ketoconazole. Interestingly, one deletion strain showed decreased susceptibility to the antifungal micafungin. In summary, we have assigned phenotypes to more than half of the zinc cluster genes in C. glabrata. Our study provides a resource that will be useful to better understand the biological role of these transcription factors.

Insights into Dynamic Mitotic Chromatin Organization Through the NIMA Kinase Suppressor SonC, a Chromatin-Associated Protein Involved in the DNA Damage Response

The nuclear pore complex proteins SonA and SonB, the orthologs of mammalian RAE1 and NUP98, respectively, were identified in Aspergillus nidulans as cold-sensitive suppressors of a temperature-sensitive allele of the essential mitotic NIMA kinase (nimA1). Subsequent analyses found that sonB1 mutants exhibit temperature-dependent DNA damage sensitivity. To understand this pathway further, we performed a genetic screen to isolate additional conditional DNA damage-sensitive suppressors of nimA1. We identified two new alleles of SonA and four intragenic nimA mutations that suppress the temperature sensitivity of the nimA1 mutant. In addition, we identified SonC, a previously unstudied binuclear zinc cluster protein involved with NIMA and the DNA damage response. Like sonA and sonB, sonC is an essential gene. SonC localizes to nuclei and partially disperses during mitosis. When the nucleolar organizer region (NOR) undergoes mitotic condensation and removal from the nucleolus, nuclear SonC and histone H1 localize in a mutually exclusive manner with H1 being removed from the NOR region and SonC being absent from the end of the chromosome beyond the NOR. This region of chromatin is adjacent to a cluster of nuclear pore complexes to which NIMA localizes last during its progression around the nuclear envelope during initiation of mitosis. The results genetically extend the NIMA regulatory system to include a protein with selective large-scale chromatin location observed during mitosis. The data suggest a model in which NIMA and SonC, its new chromatin-associated suppressor, might help to orchestrate global chromatin states during mitosis and the DNA damage response.

Genetic and Functional Investigation of Zn2Cys6Transcription Factors RSE2 and RSE3 in Podospora anserina

In Podospora anserina, the two zinc cluster proteins RSE2 and RSE3 are essential for the expression of the gene encoding the alternative oxidase (aox) when the mitochondrial electron transport chain is impaired. In parallel, they activated the expression of gluconeogenic genes encoding phosphoenolpyruvate carboxykinase (pck) and fructose-1,6-biphosphatase (fbp). Orthologues of these transcription factors are present in a wide range of filamentous fungi, and no other role than the regulation of these three genes has been evidenced so far. In order to better understand the function and the organization of RSE2 and RSE3, we conducted a saturated genetic screen based on the constitutive expression of the aox gene. We identified 10 independent mutations in 9 positions in rse2 and 11 mutations in 5 positions in rse3. Deletions were generated at some of these positions and the effects analyzed. This analysis suggests the presence of central regulatory domains and a C-terminal activation domain in both proteins. Microarray analysis revealed 598 genes that were differentially expressed in the strains containing gain- or loss-of-function mutations in rse2 or rse3. It showed that in addition to aox, fbp, and pck, RSE2 and RSE3 regulate the expression of genes encoding the alternative NADH dehydrogenase, a Zn2Cys6 transcription factor, a flavohemoglobin, and various hydrolases. As a complement to expression data, a metabolome profiling approach revealed that both an rse2 gain-of-function mutation and growth on antimycin result in similar metabolic alterations in amino acids, fatty acids, and α-ketoglutarate pools.

Analysis of a fungus‐specific transcription factor family, the Candida albicans zinc cluster proteins, by artificial activation

The zinc cluster proteins are a family of transcription factors that are unique to the fungal kingdom. In the pathogenic yeast Candida albicans, zinc cluster transcription factors control the expression of virulence-associated traits and play key roles in the development of antifungal drug resistance. Gain-of-function mutations in several zinc cluster transcription factors, which result in constitutive overexpression of their target genes, are a frequent cause of azole resistance in clinical C. albicans isolates. We found that zinc cluster proteins can also be artificially activated by C-terminal fusion with the heterologous Gal4 activation domain. We used this strategy to create a comprehensive library of C. albicans strains expressing all 82 zinc cluster transcription factors of this fungus in a potentially hyperactive form. Screening of this library identified regulators of invasive filamentous growth and other phenotypes that are important during an infection. In addition, the approach uncovered several novel mediators of fluconazole resistance, including the multidrug resistance regulator Mrr2, which controls the expression of the major C. albicans multidrug efflux pump CDR1. Artificial activation therefore is a highly useful method to study the role of zinc cluster transcription factors in C. albicans and other fungi of medical, agricultural and biotechnological importance.

Regulation of mating in the budding yeast Saccharomyces cerevisiae by the zinc cluster proteins Sut1 and Sut2

The zinc cluster proteins Sut1 and Sut2 play a role in sterol uptake and filamentous growth in the budding yeast Saccharomyces cerevisiae. In this study, we show that they are also involved in mating. Cells that lack both SUT1 and SUT2 were defective in mating. The expression of the genes NCE102 and PRR2 was increased in the sut1 sut2 double deletion mutant suggesting that Sut1 and Sut2 both repress the expression of NCE102 and PRR2. Consistent with these data, overexpression of either SUT1 or SUT2 led to lower expression of NCE102 and PRR2. Furthermore, expression levels of NCE102, PRR2 and RHO5, another target gene of Sut1 and Sut2, decreased in response to pheromone. Prr2 has been identified as a mating inhibitor before. Here we show that overexpression of NCE102 and RHO5 also reduced mating. Our results suggest that Sut1 and Sut2 positively regulate mating by repressing the expression of the mating inhibitors NCE102, PRR2 and RHO5 in response to pheromone.

Controlling Drug Resistance in Fungal Systems by Zinc Cluster Proteins

Drug resistance is a serious complication for patients with an infection, and unfortunately, bacteria and fungi are quickly rendering many therapeutic drugs ineffective. In Saccharomyces cerevisiae, this mechanism is defined as pleiotropic drug-resistance (PDR). Overexpression of drug efflux pumps, which transport drugs out of the cell, is controlled by two major transcriptional regulators, Pdr1p and Pdr3p. Homologous PDR pathways have been identified in other pathogenic species, such as Canadida albicans. Pdr1p and Pdr3p are zinc cluster transcription factors that recognize a pair of CGG repeats in the promoter region of target PDR genes. Interestingly, these transcriptional regulators tolerate sequence variation within the binding site and recognize a small group of variant sites. Pdr1p and Pdr3p have been shown to selectively regulate PDR genes through canonical and variant binding sites. This is observed in the differential regulation of two PDR genes: PDR5, the prominent ABC transporter and HXT11, a hexose transporter. Pdr1p and Pdr3p recognize a variant binding site in the promoter region of PDR5; however, the transcriptional regulators do not recognize the same variant site in the promoter of HXT11. The regulatory mechanism for how canonical and variant binding sites lead to differential PDR gene expression has yet to be explored. These protein/DNA interactions have been studied using two approaches, binding affinity and specificity studies. Fluorescence anisotropy and surface plasmon resonance were used to determine the individual binding affinities of the DNA-binding domain (DBD) for canonical and variant binding sites, and x-ray crystallography was utilized to resolve the molecular interactions between the DBD complex with canonical and variant binding sites. Differences in the binding motifs and in the DBDs of Pdr1p and Pdr3p must allow for differential regulation, making this a unique system for investigation and a possible drug target.

Yeast Zinc Cluster Proteins Dal81 and Uga3 Cooperate by Targeting Common Coactivators for Transcriptional Activation of Γ-Aminobutyrate Responsive Genes

In Saccharomyces cerevisiae, optimal utilization of various compounds as a nitrogen source is mediated by a complex transcriptional network. The zinc cluster protein Dal81 is a general activator of nitrogen metabolic genes, including those for γ-aminobutyrate (GABA). In contrast, Uga3 (another zinc cluster protein) is an activator restricted to the control of genes involved in utilization of GABA. Uga3 binds to DNA elements found in the promoters of target genes and increases their expression in the presence of GABA. Dal81 appears to act as a coactivator since the DNA-binding activity of this factor is dispensable but its mode of action is not known. In this study, we have mapped a regulatory, as well as an activating, region for Uga3. A LexA-Uga3 chimeric protein activates a lexA reporter in a GABA- and Dal81-dependent manner. Activation by Uga3 requires the SAGA complex as well as Gal11, a component of mediator. ChIP analysis revealed that Uga3 is weakly bound to target promoters. The presence of GABA enhances binding of Uga3 and allows recruitment of Dal81 and Gal11 to target genes. Recruitment of Gal11 is prevented in the absence of Dal81. Importantly, Dal81 by itself is a potent activator when tethered to DNA and its activity depends on SAGA and Gal11 but not Uga3. Overexpression of Uga3 bypasses the requirement for Dal81 but not for SAGA or Gal11. Thus, under artificial conditions, both Dal81 and Uga3 can activate transcription independently of each other. However, under physiological conditions, both factors cooperate by targeting common coactivators.

The Saccharomyces cerevisiae zinc factor protein Stb5p is required as a basal regulator of the pentose phosphate pathway

In Saccharomyces cerevisiae, the oxidative stress-activated zinc cluster protein Stb5p activates genes involved in NADPH production and most genes of the pentose phosphate (PP) pathway. To gain insight into the role of Stb5p, we studied the behaviour of stb5 deletion mutants during aerobic and anaerobic growth on glucose. stb5 mutants were auxotrophic for methionine and pyrimidine nucleotides. The methionine auxotrophy phenotype was air dependent, suggesting an impaired aerobic NADPH status. Consistent with this, the acetate level was reduced and the α-ketoglutarate level was increased in the stb5 mutant. stb5 cells also required pyrimidine nucleotides for aerobic and anaerobic growth, consistent with a reduction in 5-phosphoribosyl-1-pyrophosphate production caused by a reduced flux through the PP pathway. Strains overexpressing STB5 could not grow on glucose. This growth defect was restored by overproduction of an NADPH-butanediol dehydrogenase, which reoxidizes the excess NADPH in the oxidative PP pathway. These findings suggest a major role for the transcription factor Stb5p in maintaining a basal flux through the PP pathway to meet the NADPH requirements for aerobic growth, and to provide the nucleotide precursors. Our data also demonstrate the potential use of a system based on overproduction of this transcription factor to increase flux through the PP pathway.

Transcriptional regulation of nonfermentable carbon utilization in budding yeast

Saccharomyces cerevisiae preferentially uses glucose as a carbon source, but following its depletion, it can utilize a wide variety of other carbons including nonfermentable compounds such as ethanol. A shift to a nonfermentable carbon source results in massive reprogramming of gene expression including genes involved in gluconeogenesis, the glyoxylate cycle, and the tricarboxylic acid cycle. This review is aimed at describing the recent progress made toward understanding the mechanism of transcriptional regulation of genes responsible for utilization of nonfermentable carbon sources. A central player for the use of nonfermentable carbons is the Snf1 kinase, which becomes activated under low glucose levels. Snf1 phosphorylates various targets including the transcriptional repressor Mig1, resulting in its inactivation allowing derepression of gene expression. For example, the expression of CAT8, encoding a member of the zinc cluster family of transcriptional regulators, is then no longer repressed by Mig1. Cat8 becomes activated through phosphorylation by Snf1, allowing upregulation of the zinc cluster gene SIP4. These regulators control the expression of various genes including those involved in gluconeogenesis. Recent data show that another zinc cluster protein, Rds2, plays a key role in regulating genes involved in gluconeogenesis and the glyoxylate pathway. Finally, the role of additional regulators such as Adr1, Ert1, Oaf1, and Pip2 is also discussed.

Weak Organic Acids Trigger Conformational Changes of the Yeast Transcription Factor War1 in Vivo to Elicit Stress Adaptation

The Saccharomyces cerevisiae zinc cluster regulator War1 mediates an essential transcriptional and adaptive response to weak organic acid stress. Here we investigate the mechanism of War1 activation upon weak acid stress. We identified several gain-of-function WAR1 alleles mapping to the central War1 region. These mutations constitutively increase levels of the plasma membrane ABC transporter Pdr12, the main War1 target mediating stress adaptation. Functional analysis of War1 reveals that the central region and its C-terminal activation domain are required for function. Notably, the native DNA-binding and dimerization domains appear dispensable for War1 activity, because they can be replaced by a LexA DNA-binding domain. Chromatin immunoprecipitation demonstrates elevated promoter affinity of activated War1, because its PDR12 promoter association increases upon stress. Hyperactive WAR1 alleles have constitutively high PDR12 promoter association. Furthermore, fluorescence resonance energy transfer of functional CFP-War1-YFP proteins also demonstrates conformational changes of stress-activated War1 in vivo. Our results suggest a mechanism whereby War1 activation is accompanied by conformational changes enhancing promoter association, thus initiating the adaptation process.

Saccharomyces cerevisiae Rds2 transcription factor involvement in cell wall composition and architecture.

Although the cell wall is very important in yeasts, relatively little is known about the relationship between its structure and function. In Saccharomyces cerevisiae, a family of 55 transcription factor proteins unique to fungi, so-called zinc cluster proteins, has been described. Of these, Rds2 has been identified as an activator/inhibitor of gluconeogenesis. However, previous studies have pointed out additional roles for this protein, specifically, in the modulation of cell-wall architecture and drug sensitivity. In this work, evidence regarding the role of Rds2 as a regulator of cell-wall architecture and composition is presented based on phenotypical analysis of the cell walls prepared from a S. cerevisiae Rds2 mutant strain. Analyses of the sensitivity of this rds2Delta mutant to different drugs and to osmotic stress showed that Rds2 is indeed involved in the drug-sensitivity response and plays a role in determining osmotic sensitivity.