Mutational analysis of NHAoc/NHA2 in Saccharomyces cerevisiae.
BACKGROUND
NHAoc/NHA2 is highly and selectively expressed in osteoclasts and plays a role(s) in normal osteoclast differentiation, apoptosis and bone resorptive function in vitro. Extensive mutational analysis of a bacterial homologue, NhaA, has revealed a number of amino acid residues essential for its activity. Some of these residues are evolutionarily conserved and have been shown to be essential not only for activity of NhaA in bacteria, but also of NHAoc/NHA2 in eukaryotes.


METHODS
The salt-sensitive Saccharomyces cerevisiae strain BW31a was used for heterologous expression of mutants of NHAoc/NHA2. Membrane expression of NHAoc/NHA2 was confirmed by confocal microscopy. Intracellular concentration of Na+ (a measure of Na+ antiporter activity) was estimated by atomic absorption spectroscopy. The growth phenotypes of cells expressing NHAoc/NHA2 mutants were studied on YNB agar supplemented with NaCl and by growth curves in YNB broth.


RESULTS
Mutations in amino acid residues V161 and F357 reduced the ability of transfected BW31a cells to remove intracellular sodium and to grow in NaCl-containing medium. Yeast expressing the double mutant F357 F437 cannot grow in 0.4M NaCl, suggesting that these residues are also essential for antiporter activity.


CONCLUSIONS
Evolutionarily conserved amino acids are required for full antiporter function.


CONCLUSIONS
Mutations in these amino acid residues may impact NHAoc activity and therefore osteoclast function in vitro and in vivo.
Huang
Xiaobin
X
Institute of Radiation Medicine, Chinese Academy of Medical Sciences, Peking Union, Medical College, Tsinghua University, China.
Morse
Leslie R
LR
Xu
Yan
Y
Zahradka
Jaromir
J
Sychrová
Hana
H
Stashenko
Phil
P
Fan
Feiyue
F
Battaglino
Ricardo A
RA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017920:Antiporters / Q000235:genetics* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017136:Ion Transport; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic
D017920:Antiporters; C492183:NHX1 protein, S cerevisiae; C527589:SLC9B2 protein, human; D029701:Saccharomyces cerevisiae Proteins; D017923:Sodium-Hydrogen Exchangers; D049452:Green Fluorescent Proteins; D012965:Sodium Chloride; D012964:Sodium