108 Abstract The glutathione-mediated pathway for the detox- ification of endogenously and exogenously derived toxic compounds was investigated using a pigment that accu- mulates in certain adenine biosynthetic mutants of yeasts. The ade1 / ade2 mutants of Saccharomyces cerevisiae, when grown on adenine-limiting medium, accumulate a characteristic red pigment (ade pigment) in their vacuoles. The precursors of the ade pigments are toxic intermedi- ates that form conjugates with glutathione, followed by their transport inside the vacuole. In this study, this red pigment was used as a phenotypic screen to obtain insight regarding new genes involved in the three phases of this de- toxification pathway: the activation phase (phase I), the con- jugation phase (phase II), and the efflux phase (phase III). Components of the phase III (efflux) pathway which in- cludes, in addition to the previously characterized Ycf1p and Bpt1p, another member of the ‘Ycf1p family’, Bat1p, as well as a vacuolar H + -ATPase-dependent transport were identified. In the investigation of phase II (conjugation), it was found that glutathione S-transferases, encoded by GTT1 and GTT2,do not appear to play a role in this pro- cess. By contrast, two other previously characterized genes, the oxidative stress transcription factor gene, SKN7, and the yeast caesin protein kinase gene, YCK1, of S. cere- visiae do participate in this pathway. Keywords Glutathione · Detoxification · Adenine · Vacuole · Glutathione conjugate · Pump/ Introduction All organisms use different strategies to combat various toxic substances generated either endogenously by vari- ous metabolic activities or acquired exogenuosly from the surrounding environment. One of the mechanism of de- toxification is to conjugate the toxins with glutathione, glucuronic acid, or glucose and then transport the conju- gates outside the cell through the plasma membrane (in animal cells) or in a vacuole (in plants and yeast). In gen- eral, this detoxification pathway involves three sequential steps (Ishikawa 1992). In phase Ι, the toxic compounds are prepared for conjugation by oxidation/hydroxylation reactions with cytochrome P450 enzymes which expose reactive sites. In phase ΙΙ, conjugates are formed enzymat- ically, or non-enzymatically, with either glutathione, glu- curonic acid, or sulfate. In phase ΙΙΙ, these conjugates are removed from the cytosol with the help of specific efflux pumps. Earlier studies clearly demonstrated that these pathways also exist in yeast, e.g. Saccharomyces cerevisiae and Schizosaccharomyces pombe, serving to remove both endo- genously derived and exogenously derived toxic com- pounds (Li. et al. 1996; Chaudhuri et al. 1997; Sharma et al. 2002), While not all components of the pathway have been completely identified, yeast are nonetheless excel- lent models to genetically investigate these events. Limited information is available on the direct involve- ment of cytochrome P450s or other mixed-function oxi- dases in phase Ι in yeasts, although in vitro studies have shown that many xenobiotic compounds are known to be acted upon by the yeast cytochrome P450 systems (King et al. 1985; Kelly et al. 1997). Regarding phase ΙΙ, al- though two glutathione-S-transferases, Gtt1p and Gtt2p, have been identified in S. cerevisiae, they have not been shown to play any role in drug resistance; interestingly, the glutathione-S-transferase activity of these enzymes Kailash Gulshan Sharma · Rupinder Kaur · Anand K. Bachhawat The glutathione-mediated detoxification pathway in yeast: an analysis using the red pigment that accumulates in certain adenine biosynthetic mutants of yeasts reveals the involvement of novel genes Arch Microbiol (2003) 180 : 108–117 DOI 10.1007/s00203-003-0566-z Received: 2 January 2003 / Revised: 12 May 2003 / Accepted: 15 May 2003 / Published online: 19 June 2003 ORIGINAL PAPER K. G. Sharma · R. Kaur · A. K. Bachhawat (✉) Sector 39-A, Insitute of Microbial Technology, 160 036 Chandigarh, India Tel.: +91-172-690908, Fax: +91-172-690632/+91-172-690585, e-mail: akbachhawat@hotmail.com Present address: K. G. Sharma Department of Physiology and Biophysics, University of Iowa, Iowa, USA Present address: R. Kaur Deptartment of Molecular Biology and Genetics, Johns Hopkins School of Medicine, 725 North Wolfe Street, PCTB 504, Baltimore, MD, USA © Springer-Verlag 2003