Physiological consequences of expression of the Na+/H+ antiporter sod2 in Escherichia coli (original) (raw)
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Journal of Bacteriology, 2001
Environmental DNA libraries prepared from three different soils were screened for genes conferring Na ؉ (Li ؉ )/H ؉ antiporter activity on the antiporter-deficient Escherichia coli strain KNabc. The presence of those genes was verified on selective LK agar containing 7.5 mM LiCl. Two positive E. coli clones were obtained during the initial screening of 1,480,000 recombinant E. coli strains. Both clones harbored a plasmid (pAM1 and pAM3) that conferred a stable Li ؉ -resistant phenotype. The insert of pAM2 (1,886 bp) derived from pAM1 contained a gene (1,185 bp) which encodes a novel Na ؉ /H ؉ antiporter belonging to the NhaA family. The insert of pAM3 harbored the DNA region of E. coli K-12 containing nhaA, nhaR, and gef. This region is flanked by highly conserved insertion elements. The sequence identity with E. coli decreased significantly outside of the insertion sequence elements, indicating that the unknown organism from which the insert of pAM3 was cloned is different from E. coli. The products of the antiporter genes located on pAM2 and pAM3 revealed functional homology to NhaA of E. coli and enabled the antiporter-deficient E. coli mutant to grow on solid media in the presence of up to 450 mM NaCl or 250 mM LiCl at pH 8.0. The Na ؉ /H ؉ antiporter activity in everted membrane vesicles that were derived from the E. coli strains KNabc/pAM2 and KNabc/pAM3 showed a substantial increase between pHs 7 and 8.5. The maximal activity was observed at pHs 8.3 and 8.6, respectively. The K m values of both antiporters for Na ؉ were approximately 10-fold higher than the values for Li ؉ .
Biochemistry, 1998
We identified amino acid residues important for activity of sod2, the Na + /H + antiporter of Schizosaccharomyces pombe. We mutated all eight His residues of sod2 into Arg. Only His367fArg affected function and resulted in complete inability of sod2 to allow growth of S. pombe in LiCl-containing medium. Mutant S. pombe (H367R) could not expel sodium in acidic (pH 4.0) medium and were defective in their ability to alkalinize external medium. When His367 was replaced by Asp, sodium export of S. pombe was suppressed at acidic pH while the sodium-dependent proton influx at pH 6.1 was increased compared to wild type. We also mutated three residues conserved in putative membrane regions of various eukaryotic and prokaryotic Na + /H + exchangers. S. pombe containing Asp241fAsn and Asp266,267fAsn mutations had greatly impaired growth in LiCl-containing medium. In addition, sodium-dependent proton influx at external pH 6.1 was impaired. Sodium export from S. pombe cells at external pH 4.0 was also almost completely abolished by the D266,267N mutation; however, the D241N mutant protein retained almost normal Na + export. The results demonstrate that His367, Asp241, and Asp266,267 are important in the function of the eukaryotic Na + /H + exchanger sod2.
Characterization of the NHA1 gene encoding a Na+/H+-antiporter of the yeast Saccharomyces cerevisiae
FEBS Letters, 1996
The AFL41 gene (2958 nt) encoding a putative Na+/ H+ antiporter (986 aa) in Saccharomyces cerevisiae was cloned-by selection based on increased NaCl tolerance. The putative protein is highly similar to sodium/proton antiporters from Schizosaccharomyces pombe (gene sod2), and Zygosaccharomyces rouxii (gene Z-SODI). Overexpression of the NHAl gene results in higher and partially pH-dependent tolerance to sodium and lithium; its disruption leads to an increased sensitivity towards these ions.
Heterologous expression of mammalian Na/H antiporters in Saccharomyces cerevisiae
Biochimica et Biophysica Acta (BBA) - General Subjects, 2006
Na + /H + antiporters, integral membrane proteins that exchange protons for alkali metal cations, play multiple roles in probably all living organisms (preventing cells from excessive amounts of alkali metal cations, regulating intracellular pH and cell volume). In this work, we studied the functionality of rat plasma membrane NHE1-3 exchangers upon their heterologous expression in alkali-metal-cation sensitive Saccharomyces cerevisiae, and searched for conditions that would increase their level in the plasma membrane and improve their functionality. Though three tested exchangers were partially localized to the plasma membrane (and two of them (NHE2 and NHE3) in an active form), the bulk of the synthesized proteins were arrested along the secretory pathway, mainly in the ER. To increase the level of exchangers in the yeast plasma membrane several approaches (truncation of C-terminal regulatory sequences, expression in mutant yeast strains, construction of rat/yeast protein chimeras, various growth conditions and chemical chaperones) were tested. The only increase in the amount of NHE exchangers in the plasma membrane was obtained upon expression in a strain with the npi1 mutation, which significantly lowers the level of Rsp5 ubiquitin ligase in cells. This mutation helped to stabilize proteins in the plasma membrane.
Canadian Journal of Physiology and Pharmacology, 2005
In the fission yeast Schizosaccharomyces pombe, the Na+/H+ exchanger, Sod2, plays a major role in the removal of excess intracellular sodium, and its disruption results in a sodium-sensitive phenotype. We examined the subcellular distribution and dynamics of Sod2 expression in S. pombe using a sod2-GFP fusion protein under the control of an attenuated version of the inducible nmt promoter. Sod2 was localized throughout the plasma membrane, the nuclear envelope, and some internal membrane systems. In exponentially growing cells, in which sod2-GFP was expressed and then the promoter turned-off, previously synthesized sod2-GFP was stable for long periods and found localized to the plasma membrane in the medial regions of the cell. It was not present at the actively growing cell ends. This suggests that these regions of the cell contain old plasma membrane protein vs. newly synthesized plasma membrane without Sod2 at the growing ends. Sod2 localization was not affected by salt stress. T...
The Journal of biological chemistry, 1998
pH controls the activity of the NhaA Na ؉ /H ؉ antiporter of Escherichia coli. In the present work we show that replacement of glycine 338 of NhaA with serine (G338S) alleviates the pH control of the antiporter. Monitoring Na ؉ -dependent collapse of ⌬pH, to assess antiporter activity in isolated membrane vesicles, shows that the mutant protein is practically independent of pH, between pH 7 and 9, and even at pH 6 is 70% active. Similarly the purified reconstituted mutant protein catalyzes pH-independent passive efflux of 22 Na from proteoliposomes as well as ⌬pH-driven influx. Whereas the native NhaA in isolated membrane vesicles is exposed to digestion by trypsin only above pH 7, the mutated protein is degraded already at pH 6.5. ⌬nhaA⌬nhaB cells transformed with a plasmid encoding the pH-independent antiporter are sensitive to Na ؉ but not to K ؉ at alkaline pH, while growing in the presence of both ions at neutral pH. Several possibilities that could explain the Na ؉ sensitivity of the mutant at alkaline pH were excluded; Western analysis and measurement of Na ؉ /H ؉ antiporter activity in membrane vesicles, isolated from cells shifted to the non-permissive growth conditions, showed neither reduced expression of G338S-NhaA nor defective activity. The finding that the mutated protein is electrogenic led to the retraction of the idea that the protein is active in vitro but not in vivo at alkaline pH, when only ⌬ exists in the cells. The Na ؉ concentration needed for half-maximal activity of G338S in isolated everted membrane vesicles is similar to that of the wild type. Therefore an increase in intracellular Na ؉ due to a reduced antiporter affinity could not explain the results. It is suggested that the loss of growth at alkaline pH in the presence of Na ؉ is due to the loss of the pH control of the mutated NhaA. Indeed, in the four mutations suppressing G338S phenotype, growth at alkaline pH was restored together with the pH regulation of NhaA. Three of the four suppressor mutations cluster in helix IV, whereas the original mutation is in helix XI, suggesting that the two helixes interact.
Journal of Biological Chemistry
A gene has been cloned from a DNA library from alkaliphilic Bacillus firmus OF4 that functionally complements a mutant strain of Escherichia coli, NM81, that carries a deletion for one of that strain's Na+/H+ antiporter genes (delta nhaA). The cloned alkaliphile gene restored to NM81 the ability to grow at pH 7.5 in the presence of 0.6 M NaCl and on 100 mM Li+ in the presence of melibiose, and concomitantly led to an increase in the membrane associated Na+/H+ antiport activity. The biologically active alkaliphile DNA was identified as an incomplete open reading frame, the sequence of which would encode a hydrophobic protein. The insert was used to isolate clones containing the complete open reading frame, which would be predicted to encode a protein with a molecular weight of 42,960 and multiple membrane spanning regions. When the open reading frame was expressed under the control of the T7 promoter, the gene product was localized in the membrane. Southern analysis indicated no ...