Mutagenesis of the Yeast ALR1 Mg Transport Gene

Abstract

Magnesium is an essential element and the most abundant divalent cation in eukaryotic and prokaryotic cells, but its transport is not well understood. Mutagenesis was used to study the function of the ALR1 (aluminium resistance) gene, which encodes the major Mg2+ uptake system in Saccharomyces cerevisiae. Random PCR mutagenesis was undertaken of the C-terminal part of ALR1 that is homologous to the bacterial CorA magnesium transport family. The mutants with the most severe phenotype all had amino acid changes in a small region of Alr1 containing the putative transmembrane (TM) domains. Eighteen single amino acid mutants in this critical region were classified into three categories: no, low and moderate activity. One ‘no activity’ mutation, M762L, affected the GMN motif that is a characteristic of the CorA super-family genes. Two other conservative mutations that reduced or inactivated uptake led me to identify Ser729 and Ile746 as critical amino acid residues in Alr1. High expression of inactive mutants inhibited the capability of the wild-type Alr1 protein to transport magnesium, consistent with the idea that Alr1 may form homo-oligomers. The results confirm the classification of ALR1 as a member of the CorA family of magnesium transport genes Random mutagenesis was also undertaken of the critical region of Alr1 containing the TM domains, in order to find important residues for Al3+ toxicity. Two types of Altolerant mutants were obtained: one with increased sensitivity to Co2+ and a second with no change in sensitivity to Co2+ ions. The former class was shown to have an increased rate of Mg2+ uptake, consistent with the hypothesis that Al3+ toxicity results from Mg2+ deficiency via inhibition of Alr1 activity. The latter class of mutants was shown to have normal rates of Mg2+ uptake but with a reduced sensitivity to inhibition by Al3+ ions. The three individual mutants in the latter class were combined in all combinations and the results indicated that their Al3+ tolerance was likely to be additive and that the mutants operate independently. The most tolerant mutant in this class, I746L, involved a conservative change (alteration of the relative location of methyl groups on the amino acid side chain), to a residue that is located within a TM and that was shown above to be critical for Mg2+ uptake. Therefore, Ile746 plays a very important role in both Mg2+ uptake and Al3+ tolerance in Alr1. These results indicate that Al3+ may inhibit Mg2+ uptake by directly competing for binding sites within the pore of the Alr1 protein. Truncation of N-terminal extension of Alr1 showed that the N-terminal 239 amino acids and the C-terminal 53 amino acids are not essential for magnesium uptake. They might be serving some other functions such as protein regulation. In conclusion, these mutagenesis results firmly establish ALR1 as a magnesium transport gene belonging to the CorA super-family and provide direct experimental support for the hypothesis that Al3+ toxicity in yeast occurs by direct inhibition of Mg2+ uptake via the Alr1 protein.