We originally identified the Yap1p protein as an activity in nuclear extracts from S. cerevisiae that recognized the AP-1 binding site in the SV40 early enhancer region. The protein was biochemically purified using this DNA binding assay and monoclonal antibodies raised against the factor. These monoclonal antibodies allowed the cloning of theYAP1 structural gene. Using the cloned gene, we demonstrated that Yap1p was capable of activating a heterologous reporter gene in S. cerevisiae through its action at the SV40 AP-1 binding site. Cells lacking YAP1 had no obvious phenotype.
Later work from the lab of Karl-Dieter Entian in Germany demonstrated that YAP1 was required for normal resistance to oxidative stress. We found that yap1 mutant cells were hypersensitive to the heavy metal cadmium and normal function of Yap1p required that this protein be able to act as a positive regulator of transcription. Shusuke Kuge and Nic Jones demonstrated that oxidant exposure acted to enhance Yap1p-dependent activation of gene expression. More recently, this same group has provided evidence that Yap1p is localized to the nucleus only after oxidative challenge.
Our group has focused on the dissection of functional domains within the Yap1p protein. We demonstrated that this protein possessed two separable and independently acting transactivation domains. More recently, we have found that the activation of yAP-1 function by oxidative stress occurs at a post-translational level. A key experiment supporting this conclusion is shown below.
Figure 1. Regulation of Yap1p occurs at a post-translational level. A strain lacking the YAP1 structural gene was transformed with a low-copy vector plasmid (vector) or the same plasmid carrying a copy of the wild-type YAP1 gene (wild-type). Transformants were then grown under non-stressed (control) or oxidatively-stressed conditions by challenging with diamide, hydrogen peroxide or diethylmaleate (DEM). Protein extracts were prepared from the cells and analyzed by western blotting with a rabbit antiserum directed against Yap1p. NS indicates the position of a non-specific protein that cross-reacts with the anti-Yap1p antibody. This experiment demonstrates that while genes under Yap1p-mediated transactivation is much higher in the presence of oxidants, the steady-state levels of the protein do not change.
Our goal is to understand the molecular basis underlying the post-translational activation of Yap1p function by oxidative stress. We have recently found that a cluster of cysteine residues at the carboxy-terminus of Yap1p is critical for normal redox regulation of the factor. There are six cysteine residues in the protein present in two clusters: three between residues and three between residues 598 and 629. The three cysteine residues located between positions 598 and 629 are present as tripeptide repeat sequences of cysteine-serine-glutamate (CSE). To address the role of these C-terminal CSE repeats, we prepared alanine scanning mutations in each repeat. The CSE repeat containing cysteine 629 produced the most striking phenotypic change and the analysis of this mutant form of Yap1p is shown below.
Figure 2. Loss of the CSE629 repeat produces a factor with oxidant-specific function. A cell lacking the YAP1 gene was transformed with an empty low-copy vector plasmid (vector) or the same plasmid expressing either wild-type (wild-type) or the CSE629AAA form of Yap1p. Transformants carrying the indicated plasmids were then grown in minimal media and assayed for beta-galactosidase activity (A) or the ability to tolerate oxidant challenge (B). A. Beta-galactosidase levels were determined in minimal media in the absence (no addition) or the presence (H2O2 or diamide) of oxidative stress. Enzyme activities were reported in units/OD600. B. Transformants from part A were grown to mid-log phase and placed on rich media (YPD) alone (no addition) or supplemented with the indicated oxidants.
These data indicate the importance of CSE629 in regulation of Yap1p function. Loss of this tripeptide causes the factor to behave as a constitutive activator of transcription and eliminates the influence of oxidants on its function. Surprisingly, the CSE629AAA Yap1p only confers hyper-resistance to diamide but not H2O2. We hypothesize that this is due to a differential action of Yap1p in response to the two different oxidative stresses produced by H2O2 and diamide. The synthetic Yap1p-responsive reporter ARE-TRP5-lacZ correctly predicts the phenotype of every mutant form of Yap1p that we have analyzed. However, this same reporter fails to predict the H2O2 phenotype of CSE629AAA and a number of other C-terminal mutant forms of Yap1p. We believe this reflects differential functional requirements for the Yap1p C-terminus to induce genes that are expressed in response to H2O2 challenge. Current work seeks to identify these genes.
Our intent is to use Yap1p as both a model for how cells respond to oxidative stress and to understand the network of genes that are activated in response to a given oxidative challenge. We believe that while Yap1p is responsive to many different oxidants the behavior of the protein in response to these different oxidants is not identical. Use of the powerful genetics of yeast will allow the detailed description of how this model eukaryote responds at the molecular level to different oxidative stress agents. Since oxidative stress is a challenge faced by all cells, our studies of yeast will provide important new insight into these pathways in human cells.