Aluminium toxicity and resistance in Saccharomyces cerevisiae

Abstract

The conditions required for maximal toxicity of aluminium (Al) to the yeast Saccharomyces cerevisiae were investigated, with the aim of developing a medium for the selection of novel yeast strains with increased Al resistance. In synthetic medium with a low pH and a low inorganic phosphate (Pi) content, yeast strains were found to be relatively tolerant to ionic Al. The major modification required to increase Al toxicity was a reduction in the concentration of magnesium (Mg2+) ions. Alterations to the phosphate, calcium or potassium concentration had little effect on Al toxicity. Organic acids known to chelate Al3+ reduced Al toxicity, suggesting that Al3+ was the toxic Al species. The unique ability of Mg2+ ions to ameliorate Al toxicity suggested that Al3+ inhibited Mg2+ uptake by yeast. Mg2+, Co2+Zn2+, Ni2+ and Mn2+ ions are thought to enter yeast cells via the same low affinity, low specificity uptake system (Fuhrmann and Rothstein 1968). Al3+ was found to inhibit the accumulation of57Co2+ by yeast cells more effectively than gallium (Ga3+), Ianthanum (La3+) or Mg2+ ions. In addition, a cot7 mutant strain with a defect in divalent cation uptake proved to be more sensitive to Al3+ than a wild-type strain. The results suggested that Al blocked Mg2+. Uptake by yeast, causing Mg2 deficiency. Supporting this hypothesis, light microscopy showed that Al-treated and Mg2+ -deficient yeast cells showed similar morphological abnormalities. Genetic analysis of Al toxicity was performed by screening for yeast genes which conferred Al tolerance when overexpressed. Two yeast strains were transformed with a yeast genomic library constructed in a high copy vector, and the transformants plated on Al-containing medium to select tolerant recipients. Several tolerant strains were isolated, and three different genomic clones were found to be responsible for the increased tolerance. Restriction digests and Southern analysis indicated that two of the clones overlapped the same locus. By deletion mapping and partial sequence analysis of the clones, the two genes which conferred Al3+-resistance were identified (designated ALR1 and ALR2). The ALR genes are closely related, and encode large hydrophilic proteins with two predicted membrane-spanning domains. Database homology searches indicated the ALR proteins had similarities to the S.typhimurium CorA protein, which was known to mediate Mg2+ and Co2+ transport across the periplasmic membrane. Overexpression of the ALR genes from the strong GALI promoter increased tolerance to Al3+ and Ga3+ ions in LPM, but induced sensitivity to Zn2+, Mn2+, Ni2+, Cu2+, Ca2+ and La3+. Many of these latter ions were previously identified as substrates for a yeast low affinity divalent cation uptake system, and the results of the genetic analysis suggested the ALR genes encoded this system. The function of the two ALR genes was investigated using gene disruption. Inactivation of the ALR1 gene resulted in a lethal phenotype in normal medium, but alr1 strains could be grown in medium supplemented with a high Mg2+ concentration. Disruption of ALR2 had no apparent effect on growth. Double alr1 alr2 mutant strains resembled single alr1 mutant strains in phenotype. Northern analysis and genetic evidence indicated ALR2 was not expressed in the genetic background used. However, the Mg2+-dependent phenotype of the alr1 mutation could be corrected by increased expression of ALR2, indicating the ALR genes were functionally redundant. The phenotype of the alr1 mutant suggested that the main physiological role of the Alr1 (and Alr2) proteins was to mediate Mg3+ uptake by yeast. The effect of altered ALR gene expression on divalent cation uptake was investigated, using 57Co2+ as an analogue for Mg2+. Deletion of atr1 was associated with decreased Co2+ uptake, and overexpression of either of the ALR genes resulted in higher rates of Co2+ uptake. Overexpression of either ALR gene could counteract the inhibition of Co2+ uptake by Al3+. The results suggested a model for Al toxicity to yeast, and for the Al3+ and Ga3+ tolerance conferred by the ALR genes. The similarity of the hydrated Al3+ and Mg2+ ions in size and charge suggested Al3+ blocked the Mg2+ transport systems encoded by the ALR genes, leading to decreased Mg2+ uptake and Mg2+ deficiency. Increased expression of the ALR genes was able to counteract this inhibition by an increased capacity for Mg2+ uptake. YKLO64w (MNR2), a gene of unknown function located on chromosome XI, was found to be related in sequence to the ALR genes. Overexpression of MNR2 did not confer Al tolerance, and could not effectively complement the growth defect induced by the alr1 mutation, suggesting the MNR2 gene product did not normally mediate Mg2+. Uptake. In contrast, deletion of MNR2 induced sensitivity to high concentrations of Mn2+, Ca2+ and Zn2+ ions. Co2+ uptake assays indicated mnr2 mutants accumulated more Co2+, and strains overexpressing MNR2 accumulated less. The increased Co2+ uptake in mnr2 mutants was dependent on a functional ALR1 gene. The results indicated the MNR2 gene product contributes to the regulation of intracellular cation accumulation, and probably mediates efflux of cations such as Ca2+ and Mn2* from the cell. In conclusion, the work described in this thesis has established yeast as a model system for the molecular genetic study of Al toxicity, and may enable further progress in cloning Al tolerance genes from diverse organisms. In addition, the identification of genes encoding the yeast magnesium transport system may enable the cloning and physiological characterisation of other eukaryotic membrane proteins capable of mediating magnesium transport.