A conserved domain in the tail region of the Saccharomyces cerevisiae Na+/H+ antiporter (Nha1p) plays important roles in localization and salinity-resistant cell-growth

The Saccharomyces cerevisiae Na(+)/H(+) antiporter Nha1p has a two-domain structure consisting of an N-terminal integral membrane region and a C-terminal cytoplasmic region. We previously identified six distinct cytoplasmic domains (C1-C6) conserved among yeast species and here we performed detailed structure-function analysis of the C1 domain (16 residues). Deletion of the C1 domain causes extensive inhibition of cell-growth under high salinity conditions. Mutants with single residue deletions or various amino acid substitutions affecting the C1 domain were analyzed with respect to salinity-dependent growth and Nha1p localization. The C1 domain was found to consist of two subdomains: (i) The first three N-proximal residues, which in conjunction with the integral membrane region play a crucial role in the targeting of Nha1p to the cytoplasmic membrane, and (ii) the portion between Leu-439 and Thr-449, which is not required for localization, but in which four residues (Gly-440, Arg-441, His-442, and Ile-446) affect salinity-sensitive cell-growth by possibly influencing the antiporter activity. Based on the overall similarity of the two-domain structure of Nha1p to that of mammalian Na(+)/H(+) antiporters, the functional importance of domains proximal to the membrane region is discussed.     

C5 conserved region of hydrophilic C‐terminal part of Saccharomyces cerevisiae Nha1 antiporter determines its requirement of Erv14 COPII cargo receptor for plasma‐membrane targeting

Erv14, a conserved cargo receptor of COPII vesicles, helps the proper trafficking of many but not all transporters to the yeast plasma membrane, for example, three out of five alkali‐metal‐cation transporters in Saccharomyces cerevisiae. Among them, the Nha1 cation/proton antiporter, which participates in cell cation and pH homeostasis, is a large membrane protein (985 aa) possessing a long hydrophilic C‐terminus (552 aa) containing six conserved regions (C1–C6) with unknown function. A short Nha1 version, lacking almost the entire C‐terminus, still binds to Erv14 but does not need it to be targeted to the plasma membrane. Comparing the localization and function of ScNha1 variants shortened at its C‐terminus in cells with or without Erv14 reveals that only ScNha1 versions possessing the complete C5 region are dependent on Erv14. In addition, our broad evolutionary conservation analysis of fungal Na⁺/H⁺ antiporters identified new conserved regions in their C‐termini, and our experiments newly show C5 and other, so far unknown, regions of the C‐terminus, to be involved in the functionality and substrate specificity of ScNha1. Taken together, our results reveal that also relatively small hydrophilic parts of some yeast membrane proteins underlie their need to interact with the Erv14 cargo receptor.     

A novel membrane protein capable of binding the Na+/H+ antiporter (Nha1p) enhances the salinity-resistant cell growth of Saccharomyces cerevisiae

The Na+/H+ antiporter Nha1p of Saccharomyces cerevisiae plays an important role in maintaining intracellular pH and Na+ homeostasis. Nha1p has a two-domain structure composed of integral membrane and hydrophilic tail regions. Overexpression of a peptide of approximately 40 residues (C1+C2 domains) that is localized in the juxtamembrane area of its cytoplasmic tail caused cell growth retardation in highly saline conditions, possibly by decreasing Na+/H+ antiporter activity. A multicopy suppressor gene of this growth retardation was identified from a yeast genome library. The clone encodes a novel membrane protein denoted as COS3 in the genome data base. Overexpression or deletion of COS3 increases or decreases salinity-resistant cell growth, respectively. However, in nha1Delta cells, overexpression of COS3 alone did not suppress the growth retardation. Cos3p and a hydrophilic portion of Cos3p interact with the C1+C2 peptide in vitro, and Cos3p is co-precipitated with Nha1p from yeast cell extracts. Cos3p-GFP mainly resides at the vacuole, but overexpression of Nha1p caused a portion of the Cos3p-GFP proteins to shift to the cytoplasmic membrane. These observations suggest that Cos3p is a novel membrane protein that can enhance salinity-resistant cell growth by interacting with the C1+C2 domain of Nha1p and thereby possibly activating the antiporter activity of this protein.     

Mutagenesis analysis of the yeast Nha1 Na+/H+ antiporter carboxy-terminal tail reveals residues required for function in cell cycle

The yeast Nha1 Na(+),K(+)/H(+) antiporter may play an important role in regulation of cell cycle, as high-copy expression of the NHA1 gene is able to rescue the blockage at the G(1)/S transition of cells lacking Sit4 protein phosphatase and Hal3 activities. Interestingly, this function was independent of the role of the antiporter in improving tolerance to sodium cations, it required the integrity of a relatively large region (from residues 800 to 948) of its carboxy-terminal moiety, and was not performed by the fission yeast homolog antiporter Sod2, which lacks a carboxy-terminal tail. Here we show that a hybrid protein composed of the Sod2 antiporter fused to the carboxy-terminal half of Nha1 strongly increased sodium tolerance, but did not allow growth at high potassium nor did rescue growth of the sit4 hal3 conditional mutant strain. Deletion of Nha1 residues from 800 to 849, 900 to 925 or 926 to 954 abolished the function of Nha1 in cell cycle without affecting sodium tolerance. A screening for loss-of-function mutations at the 775-980 carboxy-terminal tail of Nha1 has revealed a number of residues required for function in cell cycle, most of them clustering in two regions, from residues 869 to 876 (cluster A) and 918 to 927 (cluster B). The later is rather conserved in other related antiporters, while the former is not.     

Oligomerization of the Saccharomyces cerevisiae Na+/H+ antiporter Nha1p: implications for its antiporter activity

The Na(+)/H(+) antiporter (Nha1p) from the budding yeast Saccharomyces cerevisiae plays an important role in intracellular pH and Na(+) homeostasis. Here, we show by co-precipitation of differently tagged Nha1p proteins expressed in the same cell that the yeast Nha1p l forms an oligomer. In vitro cross-linking experiments then revealed that Nha1p-FLAG is present in the membranes as a dimer. Differently tagged Nha1p proteins were also co-precipitated from sec18-1 mutant cells in which ER-to-Golgi traffic is blocked under non-permissive temperatures, suggesting that Nha1p may already dimerize in the ER membrane. When we over-expressed a mutant Nha1p with defective antiporter activity in cells that also express the wild-type Nha1p-EGFP fusion protein, we found impaired cell growth in highly saline conditions, even though the wild-type protein was appropriately expressed and localized correctly. Co-immunoprecipitation assays then showed the inactive Nha1p-FLAG mutant interacted with the wild-type Nha1p-EGFP protein. These results support the notion that Nha1p exists in membranes as a dimer and that the interaction of its monomers is important for its antiporter activity.     

A screening for high copy suppressors of the sit4 hal3 synthetically lethal phenotype reveals a role for the yeast Nha1 antiporter in cell cycle regulation

A screening for multicopy suppressors of the G(1)/S blockage of a conditional sit4 hal3 mutant yielded the NHA1 gene, encoding a Na(+),K(+)/H(+) antiporter, composed of a transmembrane domain and a large carboxyl-terminal tail, which has been related to cation detoxification processes. Expression of either the powerful Saccharomyces cerevisiae Ena1 Na(+)/H(+)-ATPase or the Schizosaccharomyces pombe Sod2 Na(+)/H(+) antiporter, although increasing tolerance to sodium, was unable to mimic the Nha1 function in the cell cycle. Mutation of the conserved Asp residues Asp(266)-Asp(267) selectively abolished Na(+) efflux without modifying K(+) efflux and did not affect the capacity of Nha1 to relieve the G(1) blockage. Mutagenesis analysis revealed that the region near the carboxyl-terminal end of Nha1 comprising residues 800-948 is dispensable for sodium detoxification but necessary for transport of K(+) cations. Therefore, this portion of the protein contains structural elements that selectively modulate Nha1 antiporter functions. This region is also required for Nha1 to function in the cell cycle. However, expression of the closely related Cnh1 antiporter from Candida albicans, which also contains a long carboxyl-terminal extension, although allowing efficient K(+) transport does not relieve cell cycle blockage. This indicates that although the determinants for Nha1-mediated regulation of potassium transport and the cell cycle map very closely in the protein, most probably the function of Nha1 on cell cycle is independent of its ability to extrude potassium cations.     

Cnh1 Na(+) /H(+) antiporter and Ena1 Na(+) -ATPase play different roles in cation homeostasis and cell physiology of Candida glabrata

Yeasts tightly regulate their intracellular concentrations of alkali metal cations. In Saccharomyces cerevisiae, the Nha1 Na(+) /H(+) -antiporter and Ena1 Na(+) -ATPase, mediate the efflux of toxic sodium and surplus potassium. We report the characterization of Candida glabrata CgCnh1 and CgEna1 homologues. Their substrate specificity and transport properties were compared upon expression in S. cerevisiae, and their function characterized directly in C. glabrata. The CgCnh1 antiporter and the CgEna1 ATPase transport both potassium and sodium when expressed in S. cerevisiae. CgEna1p fully complements the lack of S. cerevisiae own Na(+) -ATPases but the activity of the CgCnh1 antiporter is lower than that of ScNha1p. Candida glabrata deletion mutants and analyses of their phenotypes revealed that though both transporters have a broad substrate specificity, their function in C. glabrata cells is not the same. Their differing physiological roles are also reflected in their regulation of expression, CgENA1 is highly upregulated by an increased osmotic pressure or sodium concentration, whereas CgCNH1 is expressed constitutively. The Cnh1 antiporter is involved in the regulation of potassium content and the Ena1 ATPase in sodium detoxification of C. glabrata cells. This situation differs from S. cerevisiae, where the Nha1 antiporter and Ena ATPases both participate together in Na(+) detoxification and in the regulation of K(+) homeostasis.     

Identification of conserved prolyl residue important for transport activity and the substrate specificity range of yeast plasma membrane Na+/H+ antiporters

Yeast plasma membrane Na+/H+ antiporters are divided according to their substrate specificity in two distinct subfamilies. To identify amino acid residues responsible for substrate specificity determination (recognition of K+), the Zygosaccharomyces rouxii Sod2-22 antiporter (non-transporting K+) was mutagenized and a collection of ZrSod2-22 mutants that improved the KCl tolerance of a salt-sensitive Saccharomyces cerevisiae strain was isolated. Several independent ZrSod2-22 mutated alleles contained the replacement of a highly conserved proline 145 with a residue containing a hydroxyl group (Ser, Thr). Site-directed mutagenesis of Pro145 proved that an amino acid with a hydroxyl group at this position is enough to enable ZrSod2-22p to transport K+. Simultaneously, the P145(S/T) mutation decreased the antiporter transport activity for both Na+ and Li+. Replacement of Pro145 with glycine resulted in a ZrSod2-22p with extremely low activity only for Na+, and the exchange of a charged residue (Asp, Lys) for Pro145 completely stopped the activity. Mutagenesis of the corresponding proline in the S. cerevisiae Nha1 antiporter (Pro146) confirmed that this proline of the fifth transmembrane domain is a critical residue for antiporter function. This is the first evidence that a non-polar amino acid residue is important for the substrate specificity and activity of yeast Nha antiporters.     

The Zygosaccharomyces rouxii strain CBS732 contains only one copy of the HOG1 and the SOD2 genes

The osmotolerant yeast Zygosaccharomyces rouxii CBS732 contains only one copy of the ZrHOG1 and ZrSOD2-22 genes. Both genes were cloned and sequenced (Acc. Nos. AJ132606 and AJ252273, respectively) and their sequences were compared to homologous pairs of genes from Z. rouxii ATCC42981 (genes Z-HOG1, Z-HOG2, Z-SOD2, Z-SOD22). The CBS732 ZrHog1p is shorter than its ATCC42981 counterparts (380 aa residues vs. 407 and 420 aa, respectively) and is more similar to ATCC42981 Z-Hog2p than to Z-Hog1p. Also its promoter region corresponds to that one of Z-HOG2. The CBS732 ZrHOG1 promoter region is recognised by Saccharomyces cerevisiae, and the gene product (MAP kinase ZrHog1p) presence fully complements the osmosensitivity of a S. cerevisiae hog1 mutant strain. The CBS ZrSOD2-22 gene is highly similar to ATCC42981 Z-SOD2 but it contains also a segment of 15 aa residues specific for Z-SOD22. Z. rouxii ZrSod2-22 Na(+)/H(+) antiporter expressed in S. cerevisiae shows better activity toward toxic Na(+) and Li(+) cations than does S. cerevisiae's own Nha1 antiporter, and is efficient in improving the halotolerance of some S. cerevisiae wild types.     

Functional study of the Saccharomyces cerevisiae Nha1p C-terminus

Saccharomyces cerevisiae cells possess an alkali metal cation antiporter encoded by the NHA1 gene. Nha1p is unique in the family of yeast Na+/H+ antiporters on account of its broad substrate specificity (Na+, Li+, K+) and its long C-terminus (56% of the whole protein). In order to study the role of the C-terminus in Nha1p function, we constructed a series of 13 truncated NHA1 versions ranging from the complete one (2958 nucleotides, 985 amino acids) down to the shortest version (1416 nucleotides, 472 amino acids), with only 41 amino acid residues after the last putative transmembrane domain. Truncated NHA1 versions were expressed in an S. cerevisiae alkali metal cation-sensitive strain (B31; ena1-4Delta nha1Delta). We found that the entire Nha1p C-terminus domain is not necessary for either the proper localization of the antiporter in the plasma membrane or the transport of all four substrates (we identified rubidium as the fourth Nha1p substrate). Partial truncation of the C-terminus of about 70 terminal amino acids improves the tolerance of cells to Na+, Li+ and Rb+ compared with cells expressing the complete Nha1p. The presence of the neighbouring part of the C-terminus (amino acids 883-928), rich in aspartate and glutamate residues, is necessary for the maintenance of maximum Nha1p activity towards sodium and lithium. In the case of potassium, the participation of the long C-terminus in the regulation of intracellular potassium content is demonstrated. We also present evidence that the Nha1p C-terminus is involved in the cell response to sudden changes in environmental osmolarity.     

Ability of Sit4p to promote K+ efflux via Nha1p is modulated by Sap155p and Sap185p

We demonstrate here that SAP155 encodes a negative modulator of K+ efflux in the yeast Saccharomyces cerevisiae. Overexpression of SAP155 decreases efflux, whereas deletion increases efflux. In contrast, a homolog of SAP155, called SAP185, encodes a positive modulator of K+ efflux: overexpression of SAP185 increases efflux, whereas deletion decreases efflux. Two other homologs, SAP4 and SAP190, are without effect on K+ homeostasis. Both SAP155 and SAP185 require the presence of SIT4 for function, which encodes a PP2A-like phosphatase important for the G1-S transition through the cell cycle. Overexpression of either the outwardly rectifying K+ channel, Tok1p, or the putative plasma membrane K+/H+ antiporter, Kha1p, increases efflux in both wild-type and sit4Delta strains. However, overexpression of the Na+-K+/H+ antiporter, Nha1p, is without effect in a sit4Delta strain, suggesting that Sit4p signals to Nha1p. In summary, the combined activities of Sap155p and Sap185p appear to control the function of Nha1p in K+ homeostasis via Sit4p.     

Protein stabilization through phage display

RNase S consists of two proteolytic fragments of RNase A, residues 1-20 (S20) and residues 21-124 (S pro). A 15-mer peptide (S15p) with high affinity for S pro was selected from a phage display library. Peptide residues that are buried in the structure of the wild type complex are conserved in S15p though there are several changes at other positions. Isothermal titration calorimetry studies show that the affinity of S15p is comparable to that of the wild type peptide at 25 degrees C. However, the magnitudes of DeltaH(o) and DeltaC(p) are lower for S15p, suggesting that the thermal stability of the complex is enhanced. In agreement with this prediction, at pH 6, the T(m) of the S15p complex was found to be 10 degrees C higher than that of the wild type complex. This suggests that for proteins where fragment complementation systems exist, phage display can be used to find mutations that increase protein thermal stability.     

Characterization of the ion transport activity of the budding yeast Na+/H+ antiporter, Nha1p, using isolated secretory vesicles

The Saccharomyces cerevisiae Nha1p, a plasma membrane protein belonging to the monovalent cation/proton antiporter family, plays a key role in the salt tolerance and pH regulation of cells. We examined the molecular function of Nha1p by using secretory vesicles isolated from a temperature sensitive secretory mutant, sec4-2, in vitro. The isolated secretory vesicles contained newly synthesized Nha1p en route to the plasma membrane and showed antiporter activity exchanging H+ for monovalent alkali metal cations. An amino acid substitution in Nha1p (D266N, Asp-266 to Asn) almost completely abolished the Na+/H+ but not K+/H+ antiport activity, confirming the validity of this assay system as well as the functional importance of Asp-266, especially for selectivity of substrate cations. Nha1p catalyzes transport of Na+ and K+ with similar affinity (12.7 mM and 12.4 mM), and with lower affinity for Rb+ and Li+. Nha1p activity is associated with a net charge movement across the membrane, transporting more protons per single sodium ion (i.e., electrogenic). This feature is similar to the bacterial Na+/H+ antiporters, whereas other known eukaryotic Na+/H+ antiporters are electroneutral. The ion selectivity and the stoichiometry suggest a unique physiological role of Nha1p which is distinct from that of other known Na+/H+ antiporters.     

Involvement of Nha1 antiporter in regulation of intracellular pH in Saccharomyces cerevisiae

The Nha1 antiporter is involved in regulation of intracellular pH in Saccharomyces cerevisiae. We report that deletion of the NHA1 gene resulted in an increase of cytoplasmic pH in cells suspended in water or acidic buffers. Addition of KCl or NaCl to exponentially growing cells lowered the internal pH but the difference between cells with or without NHA1 was maintained. Addition of KCl to starved cells resulted in much higher alkalinization of cytoplasmic pH in a strain lacking Nha1p compared to the wild-type or Nha1p-overexpressing strains. The H+/K+(Na+) exchange mechanism of Nha1p was confirmed in reconstituted plasma membrane vesicles.     

Characterization of the ion transport activity of the budding yeast Na/H antiporter, Nha1p, using isolated secretory vesicles

The Saccharomyces cerevisiae Nha1p, a plasma membrane protein belonging to the monovalent cation/proton antiporter family, plays a key role in the salt tolerance and pH regulation of cells. We examined the molecular function of Nha1p by using secretory vesicles isolated from a temperature sensitive secretory mutant, sec4-2, in vitro. The isolated secretory vesicles contained newly synthesized Nha1p en route to the plasma membrane and showed antiporter activity exchanging H⁺ for monovalent alkali metal cations. An amino acid substitution in Nha1p (D266N, Asp-266 to Asn) almost completely abolished the Na⁺/H⁺ but not K⁺/H⁺ antiport activity, confirming the validity of this assay system as well as the functional importance of Asp-266, especially for selectivity of substrate cations. Nha1p catalyzes transport of Na⁺ and K⁺ with similar affinity (12.7 mM and 12.4 mM), and with lower affinity for Rb⁺ and Li⁺. Nha1p activity is associated with a net charge movement across the membrane, transporting more protons per single sodium ion (i.e., electrogenic). This feature is similar to the bacterial Na⁺/H⁺ antiporters, whereas other known eukaryotic Na⁺/H⁺ antiporters are electroneutral. The ion selectivity and the stoichiometry suggest a unique physiological role of Nha1p which is distinct from that of other known Na⁺/H⁺ antiporters.     

Importance of the seryl and threonyl residues of the fifth transmembrane domain to the substrate specificity of yeast plasma membrane Na+/H+ antiporters

The Zygosaccharomyces rouxii Na+/H+ antiporter Sod2-22p is a member of the subfamily of yeast plasma membrane Nha/Sod antiporters that do not recognize potassium as their substrate. A functional study of two ZrSod2-22p mutated versions that improved the tolerance of a S. cerevisiae alkali-metal-cation sensitive strain to high extracellular concentration of KCl identified two polar non-charged amino-acid residues in the fifth transmembrane domain, Thr141 and Ser150, as being involved in substrate recognition and transport in yeast Nha/Sod antiporters. A reciprocal substitution of amino-acid residues with a hydroxyl group at these positions, T141S or S150T, produced a broadened cation selectivity of the antiporter for K+, in addition to Na+ and Li+. Site-directed mutagenesis of Ser150 showed that while the replacement of Ser150 with a small hydrophobic (valine) or negatively charged (aspartate) amino acid did not produce a significant change in ZrSod2-22p substrate specificity, the introduction of a positive charge at this position stopped the activity of the antiporter. This data demonstrates that the amino-acid composition of the fifth transmembrane domain, mainly the presence of amino acids containing hydroxyl groups in this part of the protein, is critical for the recognition and transport of substrates and could participate in conformational movements during the binding and/or cation transport cycle in yeast plasma membrane Na+/H+ antiporters.     

跨膜逆向转运蛋白NHXFS1多克隆抗体的制备及应用

NHXFS1基因是通过DNA家族改组（DNA family shuffling）技术,以拟南芥、水稻和菊花的液泡膜Na＋/H＋逆向转运蛋白基因（NHX1）为亲本获得的活性显著增强的新基因。为制备该蛋白的多克隆抗体,对该蛋白进行跨膜结构分析,选取跨膜蛋白的C末端为靶标,并将其克隆到原核表达载体pET32a中,成功构建了原核融合蛋白pET32a-NHXFS1-抗原表达载体,转化大肠杆菌BL21（DE3）并诱导表达。通过镍柱亲和层析纯化该融合表达蛋白,获得了纯度约为80%的纯化蛋白,用于免疫新西兰大白兔制备多克隆抗体。ELISA实验表明,该抗体的效价达到1：128 000,提取表达NHXFS1蛋白的酵母液泡经该多克隆抗体Western blot检测,证明该抗体具有较好的NHXFS1蛋白特异性。NHXFS1多克隆抗体的制备为进一步认识NHXFS1新蛋白结构与功能以及植物耐盐分子生物学的研究奠定了基础。     

The Na+,K+/H+ -antiporter Nha1 influences the plasma membrane potential of Saccharomyces cerevisiae

There are three different sodium transport systems (Ena1-4p, Nha1p, Nhx1p) in Saccharomyces cerevisiae. The effect of their absence on the tolerance to alkali-metal cations and on the membrane potential was studied. All three sodium transporters were found to participate in the maintenance of Na+, Li+, K+ and Cs+ homeostasis. Measurements of the distribution of a fluorescent potentiometric probe (diS-C3(3) assay) in cell suspensions showed that the lack of all three transporters depolarizes the plasma membrane. The overexpression of the Na+,K+/H+ antiporter Nha1 resulted in the hyperpolarization of the plasma membrane and consequently increased the sensitivity to Cs+, Tl+ and hygromycin B. This is the first evidence that the activity of a Na+,K+/H+ antiporter could play a role in the homeostatic regulation of the plasma membrane potential in yeast cells.     

C lambda 2 and C lambda 4 immunoglobulin light chain genes in a wild-derived inbred mouse strain

A genomic clone containing the lambda constant (C) region genes (C lambda 2S and C lambda 4S) has been isolated from a genomic library from the mouse strain SPE. SPE is an inbred strain derived from progenitors trapped near Grenada in Spain and has been classified as mus 3 or mus spretus. The sequence of the C lambda 2S gene is virtually identical to that of BALB/c both in the coding region and in flanking sequences, suggesting that it is an expressed gene in the SPE strain. By contrast, the C lambda 4S gene on the same cluster has diverged in sequence from that of BALB/c and contains a large deletion that precludes its normal expression. Whereas BALB/c J lambda 4 region contains substitutions that probably preclude its usage, the SPE J lambda 4 gene includes all sequences required for a functional J gene. Comparison of the C lambda 2S and C lambda 4S gene sequences with those available for BALB/c C lambda 3 and C lambda 1 confirms the close relationship between the C lambda 1-C lambda 4 and C lambda 2-C lambda 3 gene pairs. The C lambda 3 gene of BALB/c is more closely related to C lambda 2S than is C lambda 1 of BALB/c to C lambda 4S. If it is assumed that C lambda 1 and C lambda 2 are respective duplicates of C lambda 4 and C lambda 3 and that these duplications occurred at the same time, then the C lambda 2 gene has been under stronger selective pressure than C lambda 4.