Positive regulation of gene expression by the yeast Saccharomyces cerevisiae transcription factor Yap1p is required for normal tolerance of oxidative stress elicited by the redox-active agents diamide and H 2 O 2 . Several groups have provided evidence that a cluster of cysteine residues in the extreme C terminus of the factor are required for normal modulation of Yap1p by oxidant challenge. Deletion of this C-terminal cysteine-rich domain (c-CRD) produces a protein that is highly active under both stressed and nonstressed conditions and is constitutively located in the nucleus. We have found that a variety of different c-CRD mutant proteins are hyperactive in terms of their ability to confer diamide tolerance to cells but fail to provide even normal levels of H 2 O 2 resistance. Although the c-CRD mutant forms of Yap1p activate an artificial Yap1p-responsive gene to the same high level in the presence of either diamide or H 2 O 2 , these mutant factors confer hyperresistance to diamide but hypersensitivity to H 2 O 2 . To address this discrepancy, we have examined the ability of c-CRD mutant forms of Yap1p to activate expression of an authentic target gene required for H 2 O 2 tolerance, TRX2. When assayed in the presence of c-CRD mutant forms of Yap1p, a TRX2-lacZ fusion gene fails to induce in response to H 2 O 2 . We have also identified a second cysteine-rich domain, in the N terminus (n-CRD), that is required for H 2 O 2 but not diamide resistance and influences the localization of the protein. These data are consistent with the idea that the function of Yap1p is different at promoters of loci involved in H 2 O 2 tolerance from promoters of genes involved in diamide resistance.To grow in the presence of oxygen, cells must be able to deal with reactive oxygen species (ROS) that are produced during metabolism. Aerobes have the ability both to detoxify ROS and to repair macromolecules that are damaged by these highly reactive compounds (26). Owing to the potentially lethal action of ROS, cells maintain constant surveillance of intracellular ROS levels and rapidly activate the expression of loci involved in oxidative stress tolerance (7).The yeast Saccharomyces cerevisiae has been a useful model for studies of the eukaryotic response to oxidant challenge (15). S. cerevisiae produces a variety of enzymes, such as catalase, superoxide dismutase, and glutathione peroxidase, and small molecules and peptides (glutathione and thioredoxins) that detoxify ROS (reviewed in reference 10). Recent data have provided insight into the regulation of the biosynthesis of these enzymatic activities.One of the key regulators of oxidative stress tolerance in S. cerevisiae is the Yap1p transcription factor. The YAP1 gene is required for normal tolerance to a wide variety of oxidants and is essential for normal synthesis of a variety of antioxidant activities, including glutathione and glutathione reductase (4,5,19,27). Later studies established that Yap1p-dependent transactivation was markedly enhanced when cells were challenged w...

The action of ␥-aminobutyrate (GABA) as an intercellular signaling molecule has been intensively studied, but the role of this amino acid metabolite in intracellular metabolism is poorly understood. In this work, we identify a Saccharomyces cerevisiae homologue of the GABA-producing enzyme glutamate decarboxylase (GAD) that is required for normal oxidative stress tolerance. A high copy number plasmid bearing the glutamate decarboxylase gene (GAD1) increases resistance to two different oxidants, H 2 O 2 and diamide, in cells that contain an intact glutamate catabolic pathway. Structural similarity of the S. cerevisiae GAD to previously studied plant enzymes was demonstrated by the crossreaction of the yeast enzyme to a antiserum directed against the plant GAD. The yeast GAD also bound to calmodulin as did the plant enzyme, suggesting a conservation of calcium regulation of this protein. Loss of either gene encoding the downstream steps in the conversion of glutamate to succinate reduced oxidative stress tolerance in normal cells and was epistatic to high copy number GAD1. The gene encoding succinate semialdehyde dehydrogenase (UGA5) was identified and found to be induced by H 2 O 2 exposure. Together, these data strongly suggest that increases in activity of the glutamate catabolic pathway can act to buffer redox changes in the cell.

CAP1 encodes a basic region‐leucine zipper (bZip) transcriptional regulatory protein that is required for oxidative stress tolerance in Candida albicans. Cap1p is a homologue of a Saccharomyces cerevisiae bZip transcription factor designated Yap1p that is both required for oxidative stress tolerance and localized to the nucleus in response to the presence of oxidants. Oxidant‐regulated localization of Yap1p to the nucleus requires the presence of a carboxy‐terminal cysteine residue (C629) that is conserved in Cap1p as C477. To examine the role of this conserved cysteine residue, C477 was replaced with an alanine residue. This mutant protein, C477A Cap1p, was analysed for its behaviour both in S. cerevisiae and C. albicans. Wild type and C477A Cap1p were able to complement the oxidant hypersensitivity of a Δyap1 S. cerevisiae strain. Whereas a Yap1p‐responsive lacZ fusion gene was oxidant inducible in the presence of YAP1, the C. albicans Cap1p derivatives were not oxidant responsive in S. cerevisiae. Introduction of wild type and C477A Cap1p‐expressing plasmids into C. albicans produced differential resistance to oxidants. Glutathione reductase activity was found to be inducible by oxidants in the presence of Cap1p but was constitutively elevated in the presence of C477A Cap1p. Western blot assays indicate Cap1p is post‐translationally regulated by oxidants. Green fluorescent protein fusions to CAP1 showed that this protein is localized to the nucleus only in the presence of oxidants while C477A Cap1p is constitutively nuclear localized. Directly analogous to S. cerevisiae Yap1p, regulated nuclear localization of C. albicans Cap1p is crucial for its normal function.

The yeast transcriptional regulator Yap1p is a key determinant in oxidative stress resistance. This protein is found in the cytoplasm under non-stressed conditions but rapidly accumulates in the nucleus following oxidant exposure. There it activates transcription of genes encoding antioxidants that return the redox balance of the cell to an acceptable range. Yap1p localization to the nucleus requires the oxidant-specific formation of disulfide bonds in the N-terminal cysteine-rich domain (N-CRD) and/or the C-terminal cysteine-rich domain (C-CRD). H 2 O 2 exposure triggers the formation of two interdomain disulfide bonds between the N-and C-CRDs. This dually disulfide-bonded structure has been argued to mask the nuclear export signal in the C-CRD that would otherwise prevent Yap1p nuclear accumulation. The C-CRD is required for wild-type H 2 O 2 tolerance but dispensable for resistance to diamide. The Saccharomyces cerevisiae TRX2 gene, encoding a thioredoxin protein, cannot be induced by H 2 O 2 in the presence of various mutant forms of Yap1p lacking the normally functioning C-CRD. In this work, we demonstrate that the proper folding of Yap1p in the presence of H 2 O 2 is required for recruitment of the mediator component Rox3p to the TRX2 promoter in addition to the nuclear accumulation of Yap1p during stress by this oxidant. These data demonstrate that the dually disulfide-bonded Yap1p N-and C-CRDs form a bifunctional protein domain controlling both nuclear localization and transcriptional activation.

Saccharomyces cerevisiae cells express a family of transcription factors belonging to the basic regionleucine zipper family. Two of these proteins, yAP-1 and Gcn4p, are known to be involved in oxidative stress tolerance and general control of amino acid biosynthesis, respectively. Strains lacking the YAP1 or GCN4 structural gene have very different phenotypes, which have been taken as evidence that these transcriptional regulatory proteins control separate batteries of target genes. In this study, we provide evidence that both yAP-1 and Gcn4p control the expression of a putative integral membrane protein, Atr1p. Both yAP-1 and Gcn4p can elevate resistance to 3-amino-1,2,4-triazole and 4-nitroquinoline-N-oxide but only if the ATR1 gene is intact. Expression of ATR1 is enhanced in the presence of constitutively active alleles of YAP1 and GCN4. Regulation of ATR1 transcription by yAP-1 and Gcn4p occurs through a common DNA element related to the yAP-1 recognition element found upstream of other yAP-1-regulated genes. These data provide the first indication of overlap between the regulatory networks defined by yAP-1 and Gcn4p.

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We generated transgenic mice containing a chimeric construct consisting of the alpha-cardiac myosin heavy chain (alpha cMHC) promoter and the human renin (hRen) gene in order to target hRen synthesis specifically to the heart. The construct consisted of three segments: (i) an alpha cMHC DNA segment including 4.5 kb of 5' flanking DNA and an additional 1.1 kb of genomic DNA encompassing exons I-III (non-coding) and the first two introns; (ii) a partial hRen cDNA consisting of exons I-VI; and (iii) a hRen genomic segment containing exons VII through IX, their intervening introns, and 400 bp of 3' flanking DNA. This results in the formation of a 909 bp internal fusion exon consisting of alpha cMHC, polylinker, and hRen sequences. Despite the presence of splice acceptor and donor sites bracketing this exon, transcription of this transgene resulted in a major alternatively spliced mRNA lacking the exon and therefore a majority of the hRen coding sequence. Cloning and sequencing of RT-PCR products from several heart samples from two independent transgenic lines confirmed accurate and faithful splicing of alpha cMHC exon II to hRen exon VII thus bypassing the internal fusion exon. All other exons (alpha cMHC exons I and II and hRen exons VII, VIII and IX) were appropriately spliced. These results are consistent with the hypothesis on exon definition which states that internal exons have a size limitation. Moreover, the results demonstrate that transgenes present in the genome at independent insertion sites and in either a single copy or multiple copies can be subject to exon skipping. The implications for transgene design will be discussed.

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