TheSaccharomyces cerevisiaeAP-1 Protein Discriminates between Oxidative Stress Elicited by the Oxidants H2O2and Diamide
John A. Wemmie, Susanne M. Steggerda, W. Scott Moye-Rowley
1997
Journal of Biological Chemistry
The Saccharomyces cerevisiae AP-1 protein (yAP-1) is a key mediator of oxidative stress tolerance. Transcriptional activation by yAP-1 has been shown to be inducible by exposure of cells to H 2 O 2 and diamide, among other oxidative stress eliciting compounds. Here we define the segments of the yAP-1 protein that are required to respond to this environmental challenge. Western blotting analyses indicated that levels of yAP-1 do not change during oxidative stress. Deletion mutagenesis and gene
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... sion experiments indicate that two different segments of yAP-1 are required for oxidative stress inducibility. These two domains function differentially depending on the type of oxidant used to generate oxidative stress. Three repeated cysteine-serine-glutamate sequences located in the carboxyl terminus are required for normal regulation of yAP-1 function during oxidative stress. Replacement of these cysteine-serine-glutamate repeats by alanine residues does not similarly affect H 2 O 2 and diamide regulation of yAP-1 function. While yAP-1 transactivation is enhanced by exposure to either H 2 O 2 or diamide, the protein responds to the oxidative stress produced by these compounds in nonidentical ways. Oxidative stress is a challenge faced by all cells that grow in an aerobic environment. Cellular damage resulting from oxidative stress has been implicated in a wide variety of pathological conditions including Down's Syndrome (1), familial amyotrophic lateral sclerosis (2), and cancer (3) as well as normal cellular processes like aging (4) and apoptosis (5). Not surprisingly then, cells possess a robust ability to detoxify the reactive oxygen species that mediate cellular damage by oxidative stress. Regulation of the production of this defense network has been the focus of much recent work. Studies in animal cells have shown that the activity of the transcription factors c-Jun and NF-B is modulated in response to the intracellular redox environment (6 -8). These regulatory proteins are likely to act to link oxidative stress to a downstream response to this challenge. The yeast Saccharomyces cerevisiae serves as an invaluable model system for fundamental eukaryotic processes such as oxidative stress tolerance. As first shown by Schnell et al. (9), the YAP1 locus is a key determinant of oxidative stress tolerance. YAP1 encodes a basic region-leucine zipper (bZip) transcription factor of 650 amino acids that is a positive regulator of gene expression (9 -12). Domain mapping experiments indicate the presence of two separable transactivation domains in the protein, located between amino acids 220 -379 and 430 -537 (13). We have found that transcriptional control of GSH1 and production of normal levels of glutathione require the action of yAP-1 (14). Kuge and Jones (15) demonstrated that both an artificial yAP-1-responsive reporter and TRX2 gene expression were inducible by a variety of oxidative stress agents in a yAP-1-dependent fashion. The induction of yAP-1-dependent transactivation correlated with an increase in yAP-1 DNA binding activity. In this work, we have explored the yAP-1 protein sequences required for oxidative stress induction of yAP-1 transactivation. Through the use of deletion mutagenesis and lexA fusion proteins, two different regions of yAP-1 have been identified that mediate the response of this protein to H 2 O 2 and diamideinduced oxidative stress. Intriguingly, while both of these two regions affect the response of yAP-1 to H 2 O 2 and diamide, the consequences of mutating either region varies with the type of oxidant used to generate stress. These data strongly suggest that oxidative stress conditions generated by H 2 O 2 and diamide are differentially sensed by yAP-1. MATERIALS AND METHODS Yeast Methods-The yeast strains used in this study are: SEY6210 (MAT␣,
doi:10.1074/jbc.272.12.7908
pmid:9065458
fatcat:oakgumt3prfhtip73c3oabyiu4