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J. Biol. Chem. 275 (38): 29207-29216

© 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

A Novel Plant Glutathione S-Transferase/Peroxidase Suppresses Bax Lethality in Yeast*

Sotirios C. KampranisDagger §, Radostina DamianovaDagger §, Mirna AtallahDagger §, Garabet Toby, Greta KondiDagger , Philip N. Tsichlis||, and Antonios M. MakrisDagger **

From the Dagger  Mediterranean Agronomic Institute of Chania, Chania 73100, Greece,  Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, and || Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

The mammalian inducer of apoptosis Bax is lethal when expressed in yeast and plant cells. To identify potential inhibitors of Bax in plants we transformed yeast cells expressing Bax with a tomato cDNA library and we selected for cells surviving after the induction of Bax. This genetic screen allows for the identification of plant genes, which inhibit either directly or indirectly the lethal phenotype of Bax. Using this method a number of cDNA clones were isolated, the more potent of which encodes a protein homologous to the class theta  glutathione S-transferases. This Bax-inhibiting (BI) protein was expressed in Escherichia coli and found to possess glutathione S-transferase (GST) and weak glutathione peroxidase (GPX) activity. Expression of Bax in yeast decreases the intracellular levels of total glutathione, causes a substantial reduction of total cellular phospholipids, diminishes the mitochondrial membrane potential, and alters the intracellular redox potential. Co-expression of the BI-GST/GPX protein brought the total glutathione levels back to normal and re-established the mitochondrial membrane potential but had no effect on the phospholipid alterations. Moreover, expression of BI-GST/GPX in yeast was found to significantly enhance resistance to H2O2-induced stress. These results underline the relationship between oxidative stress and Bax-induced death in yeast cells and demonstrate that the yeast-based genetic strategy described here is a powerful tool for the isolation of novel antioxidant and antiapoptotic genes.

* This work was supported by Grant PENED 99 ED404 from the General Secretariat of Science and Technology of Greece (to S. C. K. and A. M. M.) and National Institutes of Health Grant R01CA57436 (to P. N. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF193439.

§ These authors contributed equally to this work.

** To whom correspondence should be addressed: Mediterranean Agronomic Inst. of Chania, Alsyllion agrokepion, P.O. Box 85, 73100 Chania, Greece. Tel.: 30-821-81151; Fax: 30-821-81154; E-mail: antonios@maich.gr.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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