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Mutational analysis of NHAoc/NHA2 in Saccharomyces cerevisiae.
- Source :
-
Biochimica et biophysica acta [Biochim Biophys Acta] 2010 Dec; Vol. 1800 (12), pp. 1241-7. Date of Electronic Publication: 2010 Aug 14. - Publication Year :
- 2010
-
Abstract
- 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.<br />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.<br />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.<br />Conclusions: Evolutionarily conserved amino acids are required for full antiporter function.<br />General Significance: Mutations in these amino acid residues may impact NHAoc activity and therefore osteoclast function in vitro and in vivo.<br /> (Copyright © 2010 Elsevier B.V. All rights reserved.)
- Subjects :
- Amino Acid Sequence
Amino Acid Substitution
Cell Membrane metabolism
Genetic Complementation Test
Green Fluorescent Proteins genetics
Green Fluorescent Proteins metabolism
Humans
Ion Transport
Microscopy, Confocal
Molecular Sequence Data
Phenotype
Saccharomyces cerevisiae drug effects
Saccharomyces cerevisiae growth & development
Saccharomyces cerevisiae Proteins metabolism
Sequence Homology, Amino Acid
Sodium metabolism
Sodium Chloride pharmacology
Sodium-Hydrogen Exchangers metabolism
Transformation, Genetic
Antiporters genetics
Antiporters metabolism
Mutation
Saccharomyces cerevisiae genetics
Saccharomyces cerevisiae Proteins genetics
Sodium-Hydrogen Exchangers genetics
Subjects
Details
- Language :
- English
- ISSN :
- 0006-3002
- Volume :
- 1800
- Issue :
- 12
- Database :
- MEDLINE
- Journal :
- Biochimica et biophysica acta
- Publication Type :
- Academic Journal
- Accession number :
- 20713131
- Full Text :
- https://doi.org/10.1016/j.bbagen.2010.08.001