Stimulation of Yeast 3-Phosphoglycerate Kinase Gene Promoter by Paraquat

Abstract

Yeast cells exposed to adverse conditions employ a number of defense mechanisms in order to respond effectively to the stress and sustain a high proliferation rate. It has been shown that several glycolytic enzymes are induced upon heat treatment of yeast. In this work, we used a reporter plasmid construct to study the effects of oxidative stress, induced by the 0;--generating compound paraquat (PQ), on the yeast a-phosphoglycerate kinase gene (PGK) promoter. Our results show that (i) moderate, as opposed to excessive, doses of PQ induce increased stimulation of the PGK promoter, at midlogarithmic phase of growth; and (ii) the thiol antioxidant N-acetylcysteine cancels this stimulatory effect. These observations may represent one aspect of a more general role for glycolysis in maintaining the energy pools of yeast cells under stress. ID 2000 Academic Press Key Words: Saccharomyces cerevisiae; a-phosphoglycerate kinase gene promoter; glycolysis; oxidative stress response.The unicellular eukaryotic organism Saccharomyces cerevisiae has been extensively used as a model experimental system for the study of oxidative stress responses (1). Oxidative stress can be induced using a variety of redox cycling compounds that can act intracellulary to generate the superoxide radical (0;-), hydroxyl (HO') radical, and hydrogen peroxide (H 2 0 2 ). One such compound is paraquat (1,l'-dimethyl-4,4'-bipyridinium dichloride; PQ) which generates 0;-(2).Vlhile investigating the response of yeast cells to oxidative stress induced by PQ, we have recently observed a possible stimulatory effect of PQ on the promoter of the glycolytic gene 3-phosphoglycerate kinase (PGl{) in plasmid constructs of the promoter and the Escherichia coli iron superoxide dismutase (FeSOD) gene (3). TheAbbreviations used: PQ, paraquat; PGK, 3-phosphoglycerate kinase; ROS, reactive oxygen species; NAC, N-acetylcysteine.1 To whom correspondence should be addressed. Fax: +356-310577. E-mail: rbal1@Um.edu.mt. PGK enzyme is highly expressed in S. cerevisiae, contributing to approximately 5% of the total cellular protein (4). In view of the knowledge that oxidative stress can compromise a cell's ability to generate ATP (5), particularly by interfering with normal mitochondrial energy metabolism, we wanted to examine whether generation of reactive oxygen species (ROS), by paraquat, stimulates the PGK promoter in yeast. Using a reporter gene construct with the yeast PGK promoter sequence, we show that sublethal, as opposed to lethal, doses ofPQ result in increased stimulation of the PGK promoter at midlogarithmic phase of growth. Addition of the antioxidant N-acetylcysteine (NAC) abolishes this stimulatory effect. Furthermore, brief (1 h) exposure to PQ does not elicit increased transcription from the PGK promoter, indicating that the biological nmction of this glycolytic response is probably not intended for immediate protection against stress. MATERIALS AND METHODSBacterial strain and culture conditions. The E. coli strain used in the standard cloning procedures ...

PGK1, the gene encoding the glycolitic enzyme phosphoglycerate kinase, acts as a multicopy suppressor of apoptotic phenotypes in S. cerevisiae

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Cu,Zn‐superoxide dismutase of Saccharomyces cerevisiae is required for resistance to hyperosmosis

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Microarray based analysis of temperature and oxidative stress induced messenger RNA in Schistosoma mansoni

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