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

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 θ glutathioneS-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. 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 θ glutathioneS-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. reactive oxygen species mitochondrial permeability transition glutathioneS-transferase glutathione peroxidase hemagglutinin galactose and raffinose complete medium γ-glutamylcystinilglycine glutathione disulfide 2′,7′-dichlorohydrofluorescein 3,3′dihexylocarbocyanine iodide activation domain B42 Bax-inhibiting glucose 2′,7′-dichlorodihydrofluorescein diacetate Apoptosis, or programmed cell death, is a crucial component of normal development and plays an important role in organogenesis, tissue homeostasis, and the editing of the immune system to remove autoreactive clones. Apoptosis is a morphologically recognizable form of cell death that is implemented by a mechanism conserved through evolution from nematode to man. Homologues of the genes that implement cell death in nematodes do likewise in mammals, although in mammals the process is considerably more complex (1Ellis R.E. Yuan J.Y. Horvitz H.R. Annu. Rev. Cell Biol. 1991; 7: 663-698Crossref PubMed Scopus (1945) Google Scholar, 2Ucker D.S. New Biol. 1991; 3: 103-109PubMed Google Scholar, 3Korsmeyer S.J. Blood. 1992; 80: 879-886Crossref PubMed Google Scholar, 4Reed J.C. J. Cell Biol. 1994; 124: 1-6Crossref PubMed Scopus (2390) Google Scholar). In plants, programmed cell death is involved in the terminal differentiation of xylem vessels and perhaps in developmental processes (5Mittler R. Lam E. Plant Physiol. 1995; 108: 489-493Crossref PubMed Scopus (141) Google Scholar, 6Dietrich R.A. Richberg M.H. Schmidt R. Dean C. Dangl J.L. Cell. 1997; 88: 685-694Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 7Richberg M.H. Aviv D.H. Dangl J.L. Curr. Opin. Plant Biol. 1998; 1: 480-485Crossref PubMed Scopus (96) Google Scholar). Moreover, inhibition of pathogen growth in plants also involves a rapid process of programmed cell death localized at the site of invasion. The death of plant cells during this “hypersensitive response” creates a zone of dead cells, called the “hypersensitive response lesion,” which limits the spread of the pathogen, playing an important role in the plant's defense mechanism (8Godiard L. Grant M.R. Dietrich R.A. Kiedrowski S. Dangl J.L. Curr. Opin. Genet. Dev. 1994; 4: 662-671Crossref PubMed Scopus (35) Google Scholar, 9Dangl J.L. Dietrich R.A. Richberg M.H. Plant Cell. 1996; 8: 1793-1807Crossref PubMed Scopus (723) Google Scholar, 10Mittler R. Lam E. Trends Microbiol. 1996; 4: 10-15Abstract Full Text PDF PubMed Scopus (152) Google Scholar). The process of apoptosis can be divided into three distinct components. The first component consists of the apoptotic stimulus and the signal it triggers. This signal may be delivered through surface receptors or may originate inside the cell from a secondary action of a drug, toxin, or radiation. The second component consists of the detection and the transduction of the signal, whereas the third component includes the activation and regulation of the effectors of apoptosis (11Vaux D.L. Strasser A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2239-2244Crossref PubMed Scopus (909) Google Scholar). A highly conserved group of genes that are involved in this third component, i.e. the regulation of the apoptotic process, is the bcl-2 family of genes. The proteins encoded by these genes interact with each other and either promote (e.g. Bax, Bcl-xS, Bak, Bid, Bik, and Hrk) or inhibit (e.g.Bcl-2, Bcl-xL, Bcl-w, Bfl-1, Brag-1, Mcl-1, and A1) apoptosis (12Kroemer G. Nat. Med. 1997; 3: 614-620Crossref PubMed Scopus (1713) Google Scholar). 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