Reverse genetic analysis of the glutathione metabolic pathway suggests a novel role of PHGPX and URE2 genes in aluminum resistance in Saccharomyces cerevisiae

We have taken a systematic genetic approach to study the potential role of glutathione metabolism in aluminum (A1) toxicity and resistance, using disruption mutants available in Saccharomyces cerevisiae. Yeast disruption mutants defective in phospholipid hydroperoxide glutathione peroxidases (PHGPX; phgpx1 A, phgpx2 Delta, and phgpx3Delta), were tested for their sensitivity to A1. The triple mutant, phgpx1 Delta/2Delta/3Delta, was more sensitive to A1 (55% reduction in growth at 300 muM A1) than any single phgpx mutant, indicating that the PHGPX genes may collectively contribute to A1 resistance. The hypersensitivity of phgpx3Delta to A1 was overcome by complementation with PHGPX3, and all PHGPX genes showed increased expression in response to A1 in the wild-type strain (YPH250), with maximum induction of approximately 2.5-fold for PHGPX3. Both phgpx3Delta and phgpx1Delta/2Delta/3Delta mutants were sensitive to oxidative stress (exposure to H2O2 or diamide). Lipid peroxidation was also increased in the phgpx1Delta/2Delta/3Delta mutant compared to the parental strain. Disruption mutants defective in genes for glutathione S-transferases (GSTs) (gtt1Delta and gtt2Delta), glutathione biosynthesis (gsh1Delta and gsh2Delta), glutathione reductase (glr1Delta) and a glutathione transporter (opt1Delta) did not show hypersensitivity to A1 relative to the parental strain BY4741. Interestingly, a strain deleted for URE2, a gene which encodes a prion precursor with homology to GSTs, also showed hypersensitivity to A1. The hypersensitivity of the ure2Delta mutant could be overcome by complementation with URE2. Expression of URE2 in the parental strain increased approximately 2-fold in response to exposure to 100 muM A1. Intracellular oxidation levels in the ure2A mutant showed a 2-fold (non-stressed) and 3-fold (when exposed-to 2 mM H2O2) increase compared to BY4741; however, the ure2Delta mutant showed no change in lipid peroxidation compared to the control. The phgpx1Delta/2Delta/3Delta and ure2Delta mutants both showed increased accumulation of A1. These findings suggest the involvement of PHGPX genes and a novel role of URE2 in A1 toxicity/resistance in S. cerevisiae.