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

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Abstract
We have taken a systematic genetic approach to study the potential role of glutathione metabolism in aluminum (Al) toxicity and resistance, using disruption mutants available in Saccharomyces cerevisiae. Yeast disruption mutants defective in phospholipid hydroperoxide glutathione peroxidases (PHGPX; phgpx1 Δ, phgpx2 Δ, and phgpx3Δ), were tested for their sensitivity to Al. The triple mutant, phgpx1 Δ/2Δ/3Δ, was more sensitive to Al (55% reduction in growth at 300 μM Al) than any single phgpx mutant, indicating that the PHGPX genes may collectively contribute to Al resistance. The hypersensitivity of phgpx3Δ to Al was overcome by complementation with PHGPX3, and all PHGPX genes showed increased expression in response to Al in the wild-type strain (YPH250), with maximum induction of approximately 2.5-fold for PHGPX3. Both phgpx3Δ and phgpx1Δ/2Δ/3Δ mutants were sensitive to oxidative stress (exposure to H2O2 or diamide). Lipid peroxidation was also increased in the phgpx1Δ/2Δ/3Δ mutant compared to the parental strain. Disruption mutants defective in genes for glutathione S-transferases (GSTs) (gtt1Δ and gtt2Δ), glutathione biosynthesis (gsh1Δ and gsh2Δ), glutathione reductase (glr1Δ) and a glutathione transporter (opt1Δ) did not show hypersensitivity to Al 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 Al. The hypersensitivity of the ure2Δ 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 μM Al. Intracellular oxidation levels in the ure2Δ mutant showed a 2-fold (non-stressed) and 3-fold (when exposed-to 2 mM H2O2) increase compared to BY4741; however, the ure2Δ mutant showed no change in lipid peroxidation compared to the control. The phgpx1Δ/2Δ/3Δ and ure2Δ mutants both showed increased accumulation of Al. These findings suggest the involvement of PHGPX genes and a novel role of URE2 in Al toxicity/resistance in S. cerevisiae.