Functional Analysis of Amino Acid Residues Essential for Activity in the Na+/H+ Exchanger of Fission Yeast (original) (raw)
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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...
Scientific Reports, 2019
The Na + /H + exchanger of the plasma membrane of S. pombe (SpNHE1) removes excess intracellular sodium in exchange for an extracellular proton. We examined the functional role of acidic amino acids of a yeast specific periplasmic extracellular loop 6 (EL6) and of Glu 74 and Arg 77 of transmembrane segment 3. Glu 74 and Arg 77 are conserved in yeast species while Glu 74 is conserved throughout various phyla. the mutation E74A caused a minor effect, while mutation R77A had a larger effect on the ability of SpNHE1 to confer salt tolerance. Mutation of both residues to Ala or Glu also eliminated the ability to confer salt tolerance. Arg 341 and Arg 342 were also necessary for SpNHE1 transport in S. pombe. Deletion of 3 out of 4 acidic residues (Asp 389 , Glu 390 , Glu 392 , Glu 397) of EL6 did not greatly affect SpNHE1 function while deletion of all did. Replacement of EL6 with a segment from the plant Na + /H + exchanger SOS1 also did not affect function. We suggest that EL6 forms part of a cation coordination sphere, attracting cations for transport but that the region is not highly specific for the location of acidic charges. Overall, we identified a number of polar amino acids important in SpNHE1 function. Na + /H + exchangers are integral membrane proteins that exist in all known plants, yeast and mammalian cells 1. In mammals, they remove excess intracellular protons in exchange for external sodium and are important in ischemic heart disease and breast cancer 2,3. In contrast, in plants and yeast, plasma membrane members of this group of proteins catalyze the reverse process: exchanging intracellular sodium for extracellular protons. They therefore function in salt tolerance extruding sodium ions through these plasma membrane transporters. The energy of transport comes from the proton gradient generated by the plasma membrane H +-ATPase 4. In plants, salt tolerance is significant in agriculture and in this regard, overexpression of plasma membrane salt tolerance proteins has been shown to improve salt tolerance in plants 5. In the fission yeast Schizosaccharomyces pombe, the Na + /H + exchanger SpNHE1 (previously known as sod2) plays the major role in salt removal from the cytosol and in salt tolerance. It is an excellent model system with which to study plasma membrane salt tolerance proteins 6. S. pombe has limited other salt tolerance mechanisms in the plasma membrane, so the deletion of SpNHE1 causes this yeast to exhibit a salt sensitive phenotype 7. By reintroducing the protein, salt tolerance is restored and this allows assay of defective protein's activity. We have used this assay in earlier works 6,8,9. SpNHE1 can also enhance salt tolerance in plants 10 and in phylogenetic analysis SpNHE1 clusters with plant plasma membrane salt tolerance proteins 11. The mechanism of transport of plasma membrane salt tolerance proteins and Na + /H + exchangers in general, is of interest both as a fundamental scientific problem and from the economic view of modification of plants to improve agricultural output. Crystal structures of some forms of Na + /H + exchanger are known such the E. coli sodium transporter NhaA 12 , Thermos thermophilus (NapA) 13 , Pyrococcus abyssi (PaNhaP) 14 and Methanococcus
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.
Structure and function of yeast and fungal Na+ /H+ antiporters
IUBMB Life, 2018
Sodium proton antiporters (or sodium proton exchangers [NHEs]) are a critical family of membrane proteins that exchange sodium for protons across cell membranes. In yeast and plants, their primary function is to keep the sodium concentration low inside the cytoplasm. One class of NHE constitutively expressed in yeast is the plasma membrane Na+ /H+ antiporter, and another class is expressed on the endosomal/vacuolar membrane. At present, four bacterial plasma membrane antiporter structures are known and nuclear magnetic resonance structures are available for the membrane spanning transmembrane helices of mammalian and yeast NHEs. Additionally, a vast amount of mutational data are available on the role of individual amino acids and critical motifs involved in transport. We combine this information to obtain a more detailed picture of the yeast NHE plasma membrane protein and review mechanisms of transport, conserved motifs, unique residues important in function, and regulation of these proteins. The Na+ /H+ antiporter of Schizosaccharomyces pombe, SpNHE1, is an interesting model protein in an easy to study system and is representative of fungal Na+ /H+ antiporters.
Physiological consequences of expression of the Na+/H+ antiporter sod2 in Escherichia coli
Molecular and cellular biochemistry, 1998
Sod2 is the sodium-proton antiporter on the plasma membrane of the fission yeast Schizosaccharomyces pombe. It is vitally important for sodium export and pH homeostasis in this organism. Recently, the sod2 gene has been cloned and sequenced. However, initial attempts to express sod2 in Escherichia coli using the T7 promoter failed. In the present work we examined physiological consequences of expression of sod2 in E. coli. To alleviate problems caused by expression of sod2 we: (i) used sodium-free media at all steps; (ii) used the moderate tac promoter for expression and; (iii) used E. coli strain MH1 which has impaired sodium exchange. The effect of sod2 expression on E. coli varied depending on the E. coli genotype. When sod2 was expressed in BL21 cells which have normal Na+/H+ antiporters, the result was a Li+ sensitive phenotype. LiCl completely arrested or prevented growth of BL21 E. coli transformed with the sod2 gene. The effect on growth was pronounced in media of low extern...
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.
European Journal of Biochemistry, 1999
We have previously shown that fission yeast encodes a PPZ-like phosphatase, designated Pzh1, which is an important determinant of cation homeostasis. pzh1D mutants display increased tolerance to Na + ions, but they are hypersensitive to KCl [Balcells, L., Go Âmez, N., Casamayor, A., Clotet, J. & Arin Äo, J. (1997) Eur. J. Biochem. 250, 476±483]. We have immunodetected Pzh1 in yeast extracts and found that this phosphatase is largely associated with particulate fractions. Cells defective in Pzh1 do not show altered efflux of Na + or Li + ions, but they accumulate these cations more slowly than wild-type cells. K + ion content of pzh1D cells is about twice that of wild-type cells, and this can be explained by decreased efflux of K + . Therefore, Pzh1 may regulate both Na + influx and K + efflux in fission yeast. To test the possible relationship between K + uptake, Na + tolerance and Pzh1 function, we deleted the trk1 + gene, which encodes a putative high-affinity transporter of K + ions. trk1D mutants grew well even at relatively low concentrations of KCl and did not show significantly altered content or influx of K + ions. However, they showed a Na + -sensitive phenotype which was greatly intensified by deletion of the sod2 + gene (which encodes the major determinant for efflux of Na + ions), and clearly ameliorated by deletion of the pzh1 phosphatase, as well as by moderate concentrations of KCl in the medium. These results suggest that Trk1 does not mediate the effect of Pzh1 on NaCl tolerance and that fission yeast contains efficient systems, other than Trk1, for uptake of K + ions.
Molecular and cellular biochemistry, 2003
The Na+/H+ exchanger is an integral membrane protein found in the plasma membrane of eukaryotic and prokaryotic cells. In eukaryotes it functions to exchange one proton for a sodium ion. In mammals it removes intracellular protons while in plants and fungal cells the plasma membrane form removes intracellular sodium in exchange for extracellular protons. In this study we used the Na+/H+ exchanger of Schizosaccharomyces pombe (Sod2) as a model system to study amino acids critical for activity of the protein. Twelve mutant forms of the Na+/H+ exchanger were examined for their ability to translocate protons as assessed by a Cytosensor microphysiometer. Mutation of the amino acid Histidine 367 resulted in defective proton translocation. The acidic residues Asp145, Asp178, Asp266 and Asp267 were important in the proton translocation activity of the Na+/H+ exchanger. Mutation of amino acids His98, His233 and Asp241 did not significantly impair proton translocation by the Na+/H+ exchanger....