Residues of the yeast ALR1 protein that are critical for Magnesium uptake
Mutagenesis was used to study the function by the ALR1 (aluminium resistance) gene, which encodes the major Mg2+ uptake system in yeast. Truncation of Alr1 showed that the N-terminal 239 amino acids and the C-terminal 53 amino acids are not essential for magnesium uptake. 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 containing the putative transmembrane domains. Eighteen single amino acid mutants in this critical region were classified into three categories for magnesium uptake: no, low and moderate activity. Seventeen of the 18 mutants expressed a cross-reacting band of similar size and intensity as wild-type Alr1. Conservative mutations that reduced or inactivated uptake led us to identify Ser729, Ile746 and Met762 (part of the conserved GMN motif) as critical amino acid residues in Alr1. High expression of inactive mutants inhibited the capability of wild-type Alr1 to transport magnesium, consistent with Alr1 forming homo-oligomers. The results confirm the classification of ALR1 as a member of the CorA family of magnesium transport genes.
KeywordsSaccharomyces cerevisiaePCR mutagenesisMagnesiumCorA gene familyTransmembrane domainsDominant negativeAtomic absorption spectrophotometry
yeast extract peptone dextrose
YPD plus high magnesium
low pH low magnesium
Polymerase chain reaction