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M008377200v1
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Papers In Press, published online ahead of print October 30, 2000
J. Biol. Chem, 10.1074/jbc.M008377200
Submitted on September 13, 2000
Revised on October 24, 2000
Accepted on October 28, 2000

Multiple Yap1p-binding sites mediate induction of the yeast major facilitator FLR1 gene in response to drugs, oxidants, and alkylating agents

Duc-Thang Nguyen, Anne-Marie Alarco, and Martine Raymond

Institut de recherches cliniques de Montréal, Montréal, Québec H2W 1R7

Corresponding Author: raymonm{at}ircm.qc.ca

The bZip transcription factor Yap1p plays an important role in oxidative stress response and multidrug resistance in Saccharomyces cerevisiae. We have previously demonstrated that the FLR1 gene, encoding a multidrug transporter of the major facilitator superfamily, is a transcriptional target of Yap1p. The FLR1 promoter contains three potential Yap1p response elements (YREs) at positions -148 (YRE1), -167 (YRE2) and -364 (YRE3). To address the function of these YREs, the three sites have been individually mutated and tested in transactivation assays. Our results show that i) each of the three YREs is functional and important for the optimal transactivation of FLR1 by Yap1p; and that ii) the three YREs are not functionally equivalent, mutation of YRE3 being the most deleterious, followed by YRE2 and YRE1. Simultaneous mutation of the three YREs abolished transactivation of the promoter by Yap1p, demonstrating that the three sites are essential for the regulation of FLR1 by Yap1p. Gel retardation assays confirmed that Yap1p differentially binds to the three YREs (YRE3>YRE2>YRE1). We show that the transcription of FLR1 is induced upon cell treatment with the oxidizing agents diamide, diethylmaleate, hydrogen peroxide, and tert-butyl hydroperoxide, the antimitotic drug benomyl and the alkylating agent methylmethane sulfonate, and that this induction is mediated by Yap1p through the three YREs. Finally, we show that FLR1 overexpression confers resistance to diamide, diethylmaleate and menadione but hypersensitivity to H2O2, demonstrating that the Flr1p transporter participates in Yap1p-mediated oxidative stress response in S. cerevisiae.


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