Current Genetics

, Volume 49, Issue 1, pp 7–20

Residues of the yeast ALR1 protein that are critical for Magnesium uptake

Authors

  • Jong-min Lee
    • School of Biological SciencesUniversity of Auckland
    • School of Biological SciencesUniversity of Auckland
Research Article

DOI: 10.1007/s00294-005-0037-y

Cite this article as:
Lee, J. & Gardner, R.C. Curr Genet (2006) 49: 7. doi:10.1007/s00294-005-0037-y

Abstract

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.

Keywords

Saccharomyces cerevisiaePCR mutagenesisMagnesiumCorA gene familyTransmembrane domainsDominant negativeAtomic absorption spectrophotometry

Abbreviations

TM

transmembrane

YPD

yeast extract peptone dextrose

YPDM

YPD plus high magnesium

LPM

low pH low magnesium

SC

synthetic complete

OD

Optical density

HA

Haemaglutenin

ER

endoplasmic reticulum

PCR

Polymerase chain reaction

Copyright information

© Springer-Verlag 2005