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 Mg(2+) 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 Ser(729), Ile(746) and Met(762) (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.
Lee
Jong-Min
JM
School of Biological Sciences, University of Auckland, 92019 Auckland, New Zealand.
Gardner
Richard C
RC
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D027682:Cation Transport Proteins; D008274:Magnesium / Q000493:pharmacokinetics*; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000254:growth & development* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D017385:Sequence Homology
C110206:ALR1 protein, S cerevisiae; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D008274:Magnesium