Delineation of the molecular mechanism for disulfide stress-induced aluminium toxicity

Following our previous finding that the sulfhydryl-oxidising chemical diamide induced a marked elevation of cellular Al3+ (Wu et al., Int J Mol Sci, 12:8119–8132, 2011), a further investigation into the underlying molecular mechanism was carried out, using the eukaryotic model organism Saccharomyces cerevisiae. The effects of non-toxic dose of diamide (0.8 mM) and a mild dose of aluminium sulphate (Al3+) (0.4 mM) were determined prior to the screening of gene deletion mutants. A total of 81 deletion mutants were selected for this study according to the available screening data against Al3+ only (Kakimoto et al., BioMetals, 18: 467–474, 2005) and diamide only (Thorpe et al., Proc Natl Acad Sci USA, 101: 6564–6569, 2004). On the basis of our screening data and the cluster analysis, a cluster containing the gene deletions (rpe1∆, sec72∆, pdr5∆ and ric1∆) was found to be specifically sensitive to the mixture of diamide and Al3+. However gnp1∆, mch5∆ and ccc1∆ mutants were resistant. Dithiothreitol (DTT) and ascorbate markedly reversed the diamide-induced Al3+ toxicity. Inductively-coupled plasma optical emission spectrometry demonstrated that DTT reduced the intracellular Al3+ content in diamide/Al3+-treated yeast cells six-fold compared to the non-DTT controls. These data together revealed that the pleiotropic drug resistance transporter (Pdr5p) and vacuolar/vesicular transport-related proteins (Ric1p and Sec72p) are the targets of diamide. A dysfunctional membrane-bound Pdr5p terminates the detoxification pathway for Al3+ at the final step, leading to intracellular Al3+ accumulation and hence toxicity. As Al3+ toxicity has been a problem in agriculture and human health, this study has provided a significant step forward in understanding Al3+ toxicity.