Article

A Hydrophobic Filter Confers the Cation Selectivity of Zygosaccharomyces rouxii Plasma-Membrane Na + /H + Antiporter Olga Kinclova-Zimmermannova 1 , Pierre Falson 2 , Denis Cmunt 1 and Hana Sychrova 1 1 - Department of Membrane Transport, Institute of Physiology, Academy of Sciences of the Czech Republic, v.v.i., Videnska 1083, Prague 4, Czech Republic 142 20 2 - Drug Resistance Mechanism and Modulation Group, Molecular and Structural Basis of Infectious Systems, National Centre for Scientific Research and Lyon I University Laboratory No. 5086, Institute of Biology and Chemistry of Proteins, Lyon 69 367, France

Correspondence to Olga Kinclova-Zimmermannova: [email protected] http://dx.doi.org/10.1016/j.jmb.2015.02.012 Edited by B. Poolman

Abstract Na +/H + antiporters may recognize all alkali-metal cations as substrates but may transport them selectively. Plasma-membrane Zygosaccharomyces rouxii Sod2-22 antiporter exports Na + and Li +, but not K +. The molecular basis of this selectivity is unknown. We combined protein structure modeling, site-directed mutagenesis, phenotype analysis and cation efflux measurements to localize and characterize the cation selectivity region. A three-dimensional model of the Zr Sod2-22 transmembrane domain was generated based on the X-ray structure of the Escherichia coli NhaA antiporter and primary sequence alignments with homologous yeast antiporters. The model suggested a close proximity of Thr141, Ala179 and Val375 from transmembrane segments 4, 5 and 11, respectively, forming a hydrophobic hole in the putative cation pathway's core. A series of mutagenesis experiments verified the model and showed that structural modifications of the hole resulted in altered cation selectivity and transport activity. The triple Zr Sod2-22 mutant T141S-A179T-V375I gained K + transport capacity. The point mutation A179T restricted the antiporter substrate specificity to Li + and reduced its transport activity, while serine at this position preserved the native cation selectivity. The negative effect of the A179T mutation can be eliminated by introducing a second mutation, T141S or T141A, in the preceding transmembrane domain. Our experimental results confirm that the three residues found through modeling play a central role in the determination of cation selectivity and transport activity in Z. rouxii Na +/H + antiporter and that the cation selectivity can be modulated by repositioning a single local methyl group. © 2015 Elsevier Ltd. All rights reserved.

Introduction Na +/H + antiporters participate in ensuring the optimal intracellular level of alkali-metal cations and protons (pH) in the cells of most organisms [1]. As typical secondary active transporters, they harness the electrochemical gradient of one ion to energize the uphill transport of the other [2]. They are vital for the adjustment of cellular pH, cell volume or membrane potential [3–6], and their dysfunction is associated with a variety of human diseases [7]. Due to their multiplicity of physiological roles, the structure, function and transport mechanism of various Na +/H + antiporters are studied intensively. 0022-2836/© 2015 Elsevier Ltd. All rights reserved.

Genes encoding Na +/H + antiporters have been classified into the monovalent cation/proton antiporters (CPA) superfamily [8]. Our study is focused on the characterization of yeast plasma-membrane Na +/H + antiporters (Nha/Sod family) that belong to a clade distantly related to bacterial NhaA [1,9]. The three-dimensional (3D) structure of yeast or any other eukaryotic Na +/H + antiporter has not been resolved yet. The crystal structure of proteins from the CPA superfamily was only resolved for the prokaryotic Escherichia coli NhaA antiporter [10] and Thermus thermophilus NapA [11]. Several important (negatively charged) residues located within the transmembrane part of Na + /H + antiporters that J Mol Biol (2015) 427, 1681–1694

Cation Selectivity of the Na+/H + Antiporter

1682 participate in the transfer of Na + and H + have been identified [12,13]. These residues are conserved in antiporters from various types of organisms and their significance for protein functionality has been confirmed experimentally [14–18]. Genes encoding plasma-membrane Nha/Sod antiporters have been found in all sequenced fungi genomes, and several members of the Nha/Sod family have been characterized (in terms of their substrate specificity and transport activity) upon their heterologous expression in Saccharomyces cerevisiae [19]. Most of the yeast plasma-membrane Na +/H + antiporters mediate the efflux of toxic cations (Na +, Li + and Rb +), as well as K +. On the other hand, the substrate specificity of a few homologues (e.g., from Zygosaccharomyces rouxii, Schizosaccharomyces pombe, Candida versatilis) is limited to Na + and Li + (see Ref. [19] and its references and Ref. [20]). In contrast to most other organisms, the capability of yeast Na +/H + antiporters to export K + from cells and their physiological role related to this K + efflux capacity (such as the regulation of intracellular pH, membrane potential, cell cycle and response to osmotic shock) is rather unique [4]. Members of the yeast Nha/Sod family consist of a short hydrophilic N terminus, 12 transmembrane segments (TMS) and a hydrophilic C terminus. The membrane domains of yeast Na +/H + antiporters are highly conserved and are sufficient for ion exchange [21]. The C-terminal cytoplasmic parts are more variable in terms of their length and amino acid composition [22,23] and have a regulatory function rather than being directly involved in ion translocation [21,24–26]. In our previous studies, we identified amino acid residues determining the substrate specificity (ability to recognize K +) of yeast Na +/H + antiporters. The Z. rouxii Sod2-22 antiporter (non-transporting K +) was mutagenized and a collection of Zr Sod2-22 mutants that improved the KCl tolerance of a salt-sensitive S. cerevisiae strain (i.e., K +-transporting Zr Sod2-22 antiporters) was isolated. Three residues in close proximity to each other (Thr141, Pro145 and Ser150; see Fig. 1) that are involved in substrate recognition and transport in yeast Nha/Sod antiporters were identified [27,28]. The single mutation of any of these residues (T141S, P145S/T and S150T) was sufficient to enable Zr Sod2-22 to transport K +. The importance of a conserved proline (Pro146) for the determination of an antiporter transport properties was also confirmed for S. cerevisiae Nha1 [27] and S. pombe Sod2 [29]. Recently, the implication of the hydroxyl groups in the region containing amino acid residues 140–150 for this function was also confirmed for the S. pombe Sod2 antiporter [30]. In this work, we identified other residues that form a hydrophobic filter and determine the substrate specificity of Z. rouxii Sod2-22 antiporter. Based on a 3D model of the Zr Sod2-22 transmembrane domain,

we experimentally proved the importance of three residues in TMS 4, 5 and 11, respectively, that are required for the binding and transport of the substrate and/or activity regulation of yeast Nha/ Sod antiporters. Our results indicate an interaction among TMS 4, 5 and 11 and show that the position of a single methyl group within this part of the protein structure determines its ability to transport a particular alkali-metal cation.

Results A structural model of Zr Sod2-22 reveals a hydrophobic filter in the core of cation translocation pathway The Z. rouxii Sod2-22 antiporter is a protein that is 806 amino acids long [31]. According to Kyte-Doolittle hydropathy analysis [27], it consists of a transmembrane part (amino acids 1–431) and a long hydrophilic C terminus (~ 43% of the whole protein) [27]. Cation specificity and the binding site in yeast Na +/H + antiporters are believed to be determined by the composition of the transmembrane part [21,26]. To search for further residues involved in determining the substrate specificity of yeast plasma-membrane Na +/H + antiporters, we generated a topological (Fig. 1) and a 3D (Fig. 2 and Fig. S2) models of Zr Sod2-22. The 12 particular transmembrane domains were determined by aligning the primary sequence of Zr Sod2-22 with that of E. coli NhaA, S. cerevisiae Nha1 and S. pombe Sod2 using CLUSTAL W (Fig. S1). This led to the topological model of Zr Sod2-22 displayed in Fig. 1. According to the model, previously identified residues Thr141, Pro145 and Ser150, mutations of which modify the substrate specificity of Zr Sod2-22 antiporter [27,28], are located within TMS 4 (Fig. 1). Zr Sod2-22 may form a dimer in the membrane, as was shown for the E. coli NhaA or S. cerevisiae Nha1 antiporter [32,33], as there is a large extracellular loop 1 formed of two-stranded antiparallel β-sheets via which this intermolecular contact may occur. Based on our topological model and the X-ray structure of the E. coli NhaA antiporter, a 3D model of the Zr Sod2-22 transmembrane domain (residues 1–431) was created (Fig. 2a and Fig. S2, subscribed in the PMDB database as PM0079562). The long C-terminal tail of Zr Sod2-22 was not modeled because EcNhaA lacks this region. The model was built as described in Materials and Methods. It suggests that Zr Sod2-22 consists of 12 α-helices (N and C termini on the cytoplasmic side of the membrane) with the motif of unwound regions in two cross-helices (TMS 4 and 11) that seems to be shared by all CPAs [34]. In Zr Sod2-22, helices 4 and 11 are interrupted in their center similarly to the NhaA template, while helix 12

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Fig. 1. Topological model of Zr Sod2-22. Putative TMS are indicated by numbered boxes. TMS were determined on the basis of Zr Sod2-22 similarity with E. coli NhaA antiporter. The secondary structure of the hydrophilic C-terminal part was predicted previously [27]. Dark-gray residues show the positions of mutated amino acid residues found in the Zr Sod-94 mutant listed in Table 1; amino acid residues studied in this work are indicated with arrows with single amino acid letters and numbers. The previously characterized Pro145 and Ser150 in TMS 4 [27,28] are filled in with black.

appears more kinked than that in EcNhaA (Fig. 2a). This difference seems to be relevant, since the same was observed in the recent X-ray structure of the Na +/H + antiporter NapA from T. thermophilus [11]. The TMS 4/11 assembly in the middle of the membrane at the ion binding sites is thought to have a critical role in the cation exchange activity in EcNhaA [10]. Detailed investigation of the Zr Sod2-22 model of a putative core of the cation pathway found four residues in close proximity— Thr141 (TMS 4), Ala179 (TMS 5), Phe180 (TMS 5) and Val375 (TMS 11)—that together form a hydrophobic hole (Fig. 2b) with an acidic residue (Asp176) positioned just behind it (Fig. 2b). Asp176 may play a critical role in cation translocation and

binding in Zr Sod2-22, as was reported for the corresponding residue Asp164 in E. coli NhaA or Asp178 in S. pombe Sod2 [10,35]. We assumed that the hydrophobic hole can play the role of a filter through which cations can be selectively exchanged for protons. We have a collection of Zr Sod2-22 mutated versions that, in contrast to the native Sod2-22 antiporter, export K + [27]. The Zr Sod-94 mutated version from the collection contains eight amino acid substitutions, four of them located in the hydrophilic C terminus and four within the TMS (Table 1 and Ref. [28]). Three substitutions (T141S, A179T and V375I) involved residues that form a hydrophobic hole in our model (Fig. 2b). We have previously shown that the

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Fig. 2. 3D model of transmembrane part of Zr Sod2-22 based on E. coli NhaA antiporter structure. Drawn with PyMOL 1.6. (a) General side view of model built as described in Materials and Methods. The 3D model is shown in a rainbow of colors from blue for the N-terminal methionyl residue to red for the C-terminal seryl 431 residue of the membrane domain. TMS are numbered from 1 to 12 and their orientation is indicated with arrows. Residues Thr141, Ala179, Phe180 and Val375 are shown in a stick-and-surface representation. (b) Detailed view of cation filter. The 3D model is observed from the top among helices 2, 4, 5 and 11. Residues Thr141, Ala179, Phe180 and Val375 forming the cation filter are shown in a stick-and-surface representation; cation binding residue Asp176 is also represented in sticks.

conservative substitution of Thr141 for serine was enough to provide the mutated Zr Sod2-22 with a K + transport capacity, in addition to Li + and Na + [28]. Therefore, we focused on the other two residues (Ala179 and Val375) identified in our 3D model and in the Zr Sod-94 mutated version and their role in the antiporter cation selectivity was examined. We also tested the role of Met362 that is not clustered with the others according to 3D model but that was mutated in Zr Sod-94 (Table 1). We generated

Table 1. Mutations found in ZrSOD-94 allele and their position within the protein according to 3D Zr Sod2-22 model. Amino acid substitution (native → mutant)

Position of the mutationa

ACT → 141SerTCT 141Thr GCA Ala → 79ThrACA 179 ATG Met → 362IleATC 362 GTA → 375IleATA 375Val AGA → 509IleATA 509Arg TTC Phe → 618LeuCTC 618 GCA ACA Ala → 620 620Thr TGC → 703SerAGC 703Cys

T4 T5 T11 T11 C3 C C C

a T, TMS; C, C terminus; C3, third structurally conserved domain in the C terminus [22].

individual mutations of these residues and their combinations in ZrSOD2-22 (listed in Table 2) to characterize their contribution to the transporter functionality, together with their possible mutual dependency. Zr Sod2-22 mutated versions were expressed in the salt-sensitive S. cerevisiae BW31 strain, which lacks the main sodium and potassium extrusion systems, Ena Na +-ATPases and the Na +/H + antiporter Nha1 (ena1-4Δ nha1Δ; see Ref. [27]). The resulting transformants were tested for their tolerance to alkali-metal cation salts and the cation efflux activity from cells was compared to a strain expressing the wild-type Zr Sod2-22 antiporter. In this well-established heterologous expression system, improvements in the salt tolerance of cells expressing various Zr Sod2-22 antiporters observed in drop tests fully reflect the antiporter substrate specificity, and cation efflux measurements confirm the activity of a particular expressed antiporter [27,28,36]. Note that the low or absent transport activity shown below was not due to, most probably, the amount of the protein or its cell mis-localization since the overexpressed GFPlabeled native Zr Sod2-22 and the functional, low or non-functional mutated versions gave similar fluorescence signals and the proteins had an identical localization along the secretory pathway and in the cell periphery (Fig. S3).

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Table 2. Generated mutated versions of Zr Sod2-22 and their substrate specificities. Amino acid substitution (native → mutant)

Position of the mutationa

Substrate specificityb

T141A A179T A179S A179G A179D A179K M362I V375I T141A + T141S + T141S + T141S + T141S +

T4 T5 T5 T5 T5 T5 T11 T11 T4 + T4 + T4 + T4 + T4 +

(Li+), Na+, K+ (Li+) Li+, Na+ Li+, Na+ Li+, Na+ — Li+, Na+ Li+, Na+ Li+, Na+ Li+, Na+ Li+, Na+ Li+, Na+, K+ Li+, Na+, K+

a b

A179T A179T A179T + M362I A179T + V375I A179T + M362I + V375I

T5 T5 T5 + T11 T5 + T11 T5 + T11 + T11

T, TMS. Cations in parentheses are transported with a very low transport activity; “—” means no transport activity.

Mutation A179T is critical for the substrate specificity and transport activity of Zr Sod2-22 Figure 3a shows the salt tolerance of cells expressing Zr Sod2-22 antiporters with the point mutations of T141S, A179T, M362I or V375I, respectively, and the tolerance of cells expressing either the wild-type Zr Sod2-22 or no antiporter (empty vector) as controls. As expected, the native antiporter only increased cell tolerance to sodium and lithium; the T141S version improved cell growth in the presence of LiCl, NaCl and KCl, in agreement with its broader substrate specificity (Fig. 3a). Unexpectedly, the A179T mutated antiporter lost the ability to improve cell tolerance to NaCl, and the observed growth of cells on LiCl plates was only slightly better than cells with the empty vector (Fig. 3a). On the other hand, neither the M362I nor the V375I mutation in TMS 11 changed the substrate specificity of the protein (Fig. 3a). The transport capacity of mutated antiporters was estimated by measuring the sodium loss at pH 4.5 (Fig. 3b) from sodium-preloaded cells (cf. Materials and Methods). During the experiment, the intracellular concentration of Na + was stable in control cells without any antiporter (Fig. 3b, empty vector), indicating that the observed sodium effluxes were mediated exclusively by Zr Sod2-22 antiporters. According to the drop test results, no Na + efflux was observed from cells harboring Zr Sod2-22 with the A179T substitution (Fig. 3b and Table 3). The sodium efflux activity of the T141S and M362I mutants was slightly lower than that of the native Zr Sod2-22, while the sodium efflux via the V375I mutated transporter was slightly higher (Fig. 3b). The initial amount of Na + in the control strain lacking any antiporter was similar to those in cells expressing either the native ZrSod2-22 or T141S, A179T and M362I single-mutated antiporters (on average, 107 ± 9 nmol/mg dry weight). On the other hand,

we repeatedly observed a lower amount of Na + in sodium-preloaded cells expressing the V375I mutant (90 ± 6 nmol/mg dry weight) and the rate of sodium efflux from those cells was higher than that observed for the native antiporter (Fig. 3b). This indicates that the V375I mutation increases Na + efflux activity both at pH 4.5 and at neutral pH (i.e., during the sodium preloading of cells; cf. Materials and Methods). This observation was confirmed by a drop test on NaCl-containing plates with pH adjusted to 3.5 or 7.0. As shown in Fig. 3c, cells expressing the M362I or V375I versions tolerated the same concentration of NaCl as those expressing the native antiporter at pH 3.5. At pH 7.0, cells expressing Zr Sod2-22(V375I) grew slightly better than those with the native Zr Sod2-22. Taken together, these results suggest that Met362 (located outside of a putative catalytic center; Fig. 2) is not essential for the antiporter substrate specificity and/or activity, while Ala179 appears to play a critical role in the transport. The binding and/or transport of protons seem to be influenced by Val375, as the V375I mutation increased the activity of the antiporter at higher external pH, that is, under conditions where the proton gradient across the plasma membrane (which serves as a driving force to pump Na + against its concentration gradient) is lower. The size of a side chain at the position Ala179 influences the activity of Zr Sod2-22 According to the topological model shown in Fig. 1, Ala179 is located in TMS 5 within the highly conserved motif 171-ESGCNDGLAFPF-182 found in yeast plasma-membrane Na + /H + antiporters (Fig. 4a). To further investigate the importance of Ala179, we substituted a range of residues at this position. The drop test in Fig. 4b shows that, while Ser and Gly functioned similarly to Ala (i.e., the

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Fig. 3. Substrate specificity and transport activity of Zr Sod2-22 with point mutations found in transmembrane domain of Zr Sod-94. S. cerevisiae BW31 cells containing the empty vector, the native Zr Sod2-22 antiporter or Zr Sod2-22 versions with point mutations T141S, A179T, M362I and V375I as indicated were used in these experiments. All experiments were repeated three times and representative results are shown. (a) The substrate specificity of mutated Zr Sod2-22 was determined by the growth of cells on YNB plates supplemented with LiCl, NaCl and KCl as indicated; plates were incubated for 6 days at 30 °C. The position of the mutations within the protein (T = TMS) and the substrate specificities of the antiporters are indicated (cations in parentheses mean a very small improvement to cation tolerance). (b) Loss of Na + mediated by mutated Zr Sod2-22 versions. Cells were grown in YNB media and preloaded with Na +, and Na + efflux was estimated for 40 min as described in Materials and Methods. (c) Activity of mutated antiporters at various extracellular pH determined by growth of cells on pH 3.5 or 7.5 YNB plates and supplemented with NaCl as indicated; plates were incubated for 3 days at 30 °C.

antiporter was functional and had the same substrate specificity as the native protein), larger substitutions (e.g., negatively charged Asp or positively charged Lys) led to a decrease in or loss of function, respectively (Fig. 4b). These results suggest the importance of a small side chain at position 179 to the antiporter functionality.

Seryl and threonyl residues contain a hydroxyl group in their side chains. Though the replacement of Ala179 with Ser resulted in a functional antiporter, the incorporation of a larger threonyl residue with a methyl group in its β-branched side chain instead of Ala179 almost fully inactivated the antiporter (Figs. 3 and 4b). This indicates that an additional methyl group at

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Table 3. Loss of Na + and K + from S. cerevisiae BW31 cells containing empty vector or expressing native or mutated Zr Sod2-22 antiporters. Zr Sod2-22 antiporter in BW31 — Native T141A A179T T141A + A179T

Cation lossa +

Na (nmol/mg dry weight/40 min)

K+ (nmol/mg dry weight/120 min)

6±3 62 ± 6 41 ± 4 9±2 20 ± 5

48 46 66 50 45

± ± ± ± ±

8 7 5 13 18

a BW31 transformants were grown in YNB media and the Na+ and K+ loss was estimated as described in Materials and Methods. The values are the mean of at least three experiments plus or minus standard deviation.

position 179 in TMS 5 causes local conformational changes in Zr Sod2-22 or alters local hydrogen bonding, which disables the antiporter from transporting cations. We next studied the effect of particular side

chains in this part of the protein in more detail. We focused especially on the mutual importance of Thr141 and Ala179 and their relationship to Val375 located within the hydrophobic hole in the protein core (Fig. 2b).

Fig. 4. Alanine 179 is a crucial residue for Zr Sod2-22 functionality. (a) Protein sequence alignment showing the fifth transmembrane domain of yeast plasma-membrane Na +/H + antiporters. The highly conserved ESGCNDGLAFPF motif is boxed, alanine 179 is highlighted in dark gray and 100% conserved residues are in boldface. (b) The substrate specificity of Zr Sod2-22 with various substitutions of Ala179 was determined by the growth of cells harboring mutated Zr Sod2-22 on YNB plates supplemented with KCl, NaCl and LiCl as indicated; plates were incubated at 30 °C and pictures were taken after 2 or 5 days as indicated. The dilution series correspond to BW31 cells containing the empty vector, the native Zr Sod2-22 antiporter, Zr Sod2-22 versions with substitutions of Ala179 or K +-transporting Zr Sod-94 as indicated. The experiment was repeated three times and a representative result is shown.

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Fig. 5. Identification of interaction between the fourth and the fifth TMS via Thr141 and Ala179. The substrate specificity of mutated Zr Sod2-22 with point mutations T141A and A179T was determined by the growth of cells harboring mutated Zr Sod2-22 on YNB plates supplemented with LiCl, NaCl and KCl as indicated; plates were incubated for 3 days at 30 °C. The dilution series correspond to BW31 cells containing the empty vector, the native Zr Sod2-22 antiporter or Zr Sod2-22 versions with mutation T141A or A179T as indicated. The position of the mutations within the protein (T = TMS) and the substrate specificities of antiporters are indicated (cations in parentheses mean a very small improvement to cation tolerance). The experiment was repeated three times and a representative result is shown.

Swapping residues Thr141 and Ala179 result in a native substrate specificity of the antiporter though the two single mutations alter it The replacement of Thr141 with a smaller serine (i.e., the removal of a methyl group in the β-branched side chain) resulted in an antiporter with the new capacity to transport larger K + in addition to Li + and Na + (see Ref. [28]; Fig. 3). Figure 5 shows that the presence of another smaller amino acid (alanine) at position 141 resulted in an antiporter with broad substrate specificity for the three cations—Li +, Na + and K +—but with diminished ability to improve the tolerance to Li + and Na +. The Na + and K + efflux ability of T141A was confirmed by efflux measurements (Table 3). Compared to cells without any antiporter or with antiporters that did not transport K +, a pronounced efflux of K + was observed from cells containing the T141A mutated version (Table 3). The observed low efflux was sufficient to enable the long-term growth of cells in the presence of high concentrations of KCl (Fig. 5). As shown in the drop test in Fig. 5, the double-mutated T141A + A179T antiporter (i.e., in which the Thr141 and Ala179 were swapped) had the same substrate specificity as the native Zr Sod2-22 and only improved the tolerance of cells to sodium and lithium, but not to potassium. The sodium transport activities of the T141A and T141A + A179T antiporters were lower than that of the native Zr Sod2-22 (Table 3). Though in growth assays, the T141A + A179T antiporter provided cells with a higher NaCl tolerance than the T141A mutant (Fig. 5), the Na + efflux activity of the double-mutated T141A + A179T antiporter was lower than that of the T141A version. Two interconnected possibilities could be taken into account to explain this discrepancy: (i) the obtained ability to transport K + in T141A results in a decreased capacity to export Na + against its concentration gradient in the drop test assays, where both sodium and potassium cations are present in the cells and can compete for the binding

site; (ii) the efflux of K + is a disadvantage for cells when they need to cope with an excess of Na + in the cytoplasm, and it results in a higher yeast sensitivity to external NaCl. Nevertheless, our results show that the activity of the A179T mutated antiporter could be restored by introducing a second mutation (T141A) located in the neighboring TMS 4 proving that these two residues are in close proximity as shown in the 3D model (Fig. 2). Repositioning of a methyl group within the hydrophobic gate confers K + recognition and transport To further elucidate the relationship between the mutated residues found in the transmembrane domain of Zr Sod-94 and their role in its substrate selectivity, we consecutively introduced the mutations T141S, A179T, M362I and V375I into the ZrSOD2-22 gene (Fig. 6). Cells with an antiporter containing the single T141S mutation or the original Zr Sod-94, both of which recognize K + in addition to Li + and Na +, were used as controls (Fig. 6). The double-mutated antiporter T141S + A179T only recognized Li + and Na + (Fig. 6), which means that merely the removal of a methyl group in the β-branched side chain of Thr141 was enough to suppress the negative effect of the critical A179T substitution. This again proved a cross-relationship between these two residues. The introduction of the third mutation M362I to T141S + A179T did not change the substrate specificity of the antiporter (Fig. 6), confirming the non-essential role of Met362 in determining cation selectivity. On the other hand, the triple- or quadruple-mutated antiporters containing th e V 375 I m uta tio n i n TMS 11 (th e T141S + A179T + V375I and T141S + A179T + M362I + V375I versions, respectively) improved the tolerance of cells to all three cations (Fig. 6). This means that the addition of the V375I mutation to T141S + A179T restored (relative to the T141S substrate specificity) the ability to transport K +, in

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Fig. 6. Effect of combinations of T141S, A179T, M362I and V375I mutations for Zr Sod2-22 substrate specificity. The substrate specificity of mutated Zr Sod2-22 was determined by the growth of cells harboring mutated Zr Sod2-22 on YNB plates supplemented with LiCl, NaCl and KCl salts as indicated; plates were incubated at 30 °C and pictures were taken after 2 days (control), 4 days (LiCl and NaCl) or 6 days (KCl). The dilution series correspond to BW31 cells containing the empty vector; the native Zr Sod2-22 antiporter; Zr Sod2-22 versions with various combinations of point mutations T141S, A179T, M362I and V375I; or Zr Sod-94 as indicated. The position of the mutations within the protein (T, TMS; C, C terminus) and substrate specificities of the antiporters are indicated. The experiment was repeated three times and a representative result is shown.

addition to Li + and Na + . These observations demonstrated that the Val375 in TMS 11 is also in close proximity to Thr141 and Ala179. Taken together, our data confirmed an interaction predicted by the ZrSod2-22 3D model among TMS 4, 5 and 11 and showed that the substrate specificity of a Na +/H + antiporter can be modulated by slight changes in the size of the side chain of residues within the putative hydrophobic filter in the catalytic core of the transporter.

Discussion The family of yeast plasma-membrane Nha/Sod antiporters consists of transporters with broad substrate specificity that mediate the efflux of Li +, Na +, K + and Rb +, but there are also a few members that do not recognize and transport K + (Rb +). The distribution of yeast antiporters into subfamilies does not reflect their level of protein identity, since the non-K +-transporting Z. rouxii Sod2-22 is one of the closest homologues to K +-transporting Z. rouxii Nha1, S. cerevisiae Nha1 or Candida glabrata Cnh1 [23,37,38]. In this study, we have combined molecular modeling and experimental approaches to investigate the determinants of substrate specificity of yeast plasma-membrane Na +/H + antiporters. We found that there is a hydrophobic filter positioned in close proximity to the cation binding site in the core of the putative cationic pathway and that the position of a single methyl group within this part of the protein modulates the ability of the antiporter to recognize and transport particular cations. The only known 3D structures of the bacterial Na + /H + antiporters NhaA from E. coli and NapA from T. thermophilus [10,11] show that the cation pathway consists of two funnel-shaped domains that narrow toward the center of the membrane where a single cation binding site formed of

evolutionarily conserved negatively charged residues is positioned [10,11,39]. Only non-hydrated alkali-metal cations are able to access the end of the funnel [10]. Recently, it was proposed that the fast cation exchange via the Na + /H + antiporter is based on a large movement of the center of the molecule, resulting in the closure/opening of domains on either the periplasmic or the cytosolic side [11,40,41]. Since the protein structure and transport mechanism in Na + /H + antiporters seem to be evolutionarily preserved [42,43], the crystal structure of EcNhaA was used for modeling evolutionarily distant members of the CPA superfamily—human Na + /H + exchangers NHE1 and NHA2 [12,44]. Following the same rationale, we used the 3D structure of E. coli NhaA as a template and generated a 3D model of the plasma-membrane Sod2-22 antiporter from Z. rouxii (Fig. 2). According to this model, all residues that we identified previously (Thr141, Pro145 and Ser150) and in this work (Ala179 and Val375) as being involved in substrate specificity determination are located in the core of the putative cation passage. The crucial role of the region between amino acid 141 and amino acid 150 for yeast antiporter functionality was also confirmed by an alanine scanning experimental approach [29,30]. Our structural modeling of Zr Sod2-22 revealed that residues Thr141, Ala179 and Val375, although found in different transmembrane regions, are located close to each other and, together with Phe180, may form a hydrophobic filter through which cations can be translocated in a highly selective manner (Fig. 2b). In prokaryotic NhaA antiporters, a tandem of negatively charged aspartate residues in TMS 5 is thought to bind translocated cations. Dynamic simulation showed that Na + cations are mainly concentrated at the second aspartate, which is positioned at the base of the cytoplasmic cavity [11,41]. In fungi

1690 plasma-membrane Na + /H + antiporters, only the second aspartate (Asp176 in Zr Sod2-22) is evolutionarily conserved; the first aspartate is replaced with an asparagine (Fig. S1 and O. KinclovaZimmermannova, unpublished results). A detailed view of the hydrophobic cation filter from the periplasmic side in our 3D model shows that the corresponding aspartate 176 is ideally positioned behind the filter to play a role in cation binding (Figs. 2b and 7). Hence, subtle changes in the side-chain size of the residues forming the hydrophobic hole may influence the accessibility of this binding aspartate and affect the transport properties of the ZrSod2-22 antiporter as discussed below and shown in Fig. 7. Ion radii of dehydrated alkali-metal cations increase in the order Li + (0.76 Å) b Na + (1.02 Å) b K + (1.38 Å) [45]. As for Thr141, replacing it in Zr Sod2-22 with a smaller residue that does not contain a β-branched side chain (serine or alanine) resulted in the ability to transport large potassium cations. As shown in Fig. 7 for the T141A mutation, a removal of a β-branched methyl group and a hydroxyl group in the side chain at position 141 causes a void between TMS 4 and 5; consequently, the size of the hydrophobic hole increases compared to the native antiporter, and it enables large K + cations to pass through. Thus, the inability of Zr Sod2-22 to transport K + could arise from the capacity of Thr141 (in combination with other residues) to hamper the conformational changes required by the cation translocation cycle. In both Ala141 and Ser141 configurations, the gain of K + transport was accompanied by a decreased ability to transport small Li + ions. This phenomena was also observed previously for mutations of P145S/T [27] and is widely found in nature, where members of the K +-transporting subfamily of yeast plasma-membrane

Cation Selectivity of the Na+/H + Antiporter

Na +/H + antiporters transport Li + cations rather poorly compared to non-K +-transporting antiporters (e.g., S. cerevisiae Nha1 [21], C. glabrata Nha1 [38], Z. rouxii Nha1 [37] versus S. pombe Sod2 or Z. rouxii Sod2-22 [36]). Also, in the family of prokaryotic NhaP-type transporters, antiporters with substrate specificity for K + exhibit low or no capacity to transport Li + (reviewed in Ref. [46]). Lithium binding was suggested to cause different conformational changes from the binding of larger cations [47–49]. Thus, the mutations/conformations that are more favorable for the transport of potassium cations are less convenient for lithium transport. Resch et al. hypothesize that the preferential transport of the larger K + cation over the smaller Li + can be explained by the “size-exclusion principle” in combination with the idea of “ligand shading”, taking into account that protons and lithium compete for different subsets of ligands than larger alkali-metal cations (Na + and K + ) at a common ion binding site during the catalytic cycle [46]. The most significant effect of the studied residues was observed with alanine 179 in TMS 5. Sitedirected mutagenesis of Ala179 revealed that only particular side chains at this position were permissible if transporter functionality was to be maintained (replacements for serine or glycine; Fig. 3). A long positively charged side chain at position 179 inactivated Zr Sod2-22 completely, a negatively charged aspartate produced only a low transport capacity and the replacement of Ala179 with a threonine with its β-branched side chain resulted in an antiporter with residual activity for Li + alone. The Zr Sod2-22 3D model shows that alanine 179 faces threonine 141 (Fig. 7) and our empirical data confirmed the close proximity of these two residues. The A179T substitution results in a positioning of two

Fig. 7. Detailed view of impact of T141A or A179T mutations on structure of Zr Sod2-22 cation filter. The 3D model of Zr Sod2-22 is seen from the top, centered among helices 2, 4, 5 and 11. Residues Thr141, Ala179, Phe180 and Val375 forming the cation filter are shown in a stick-and-surface representation; the putative cation binding residue Asp176 is represented in sticks and indicated with green arrow. Mutated residues are highlighted in yellow boxes. The effect of mutations on cation transport and substrate specificity is indicated below each protein, based on the results shown in Figs. 3 and 5. Drawn with PyMOL 1.6.

Cation Selectivity of the Na+/H + Antiporter

β-branched side chains of threonyl residues close to each other, which decreases the size of the hydrophobic hole (Fig. 7). Moreover, molecular modeling revealed that an additional hydrogen bond between the side chains of these two threonyl residues can be established, together with a hydrophobic interaction between the β-branched methyl groups of the threonines. Both interactions may strongly reduce the flexibility of the core region and lock the protein in a conformation preventing the exchange of large cations: small cations such as lithium being still able to pass through. The E. coli NhaA antiporter, in which the corresponding residue Ala167 was replaced with proline, had a strongly reduced transport capacity compared to the native antiporter [50,51]. Remarkably, in our experiments, removal of the β-branched methyl group at position 179 (A179S version; Fig. 4) restored Li + and Na + activity to almost the wild-type level. Steric constraints caused by the threonyl β-branched methyl group at position 179 could also be compensated for by introducing a smaller non-branched amino acid (serine and alanine) at position 141 instead of the threonine (Figs. 5 and 6). In the triple-mutated version T141S + A179T + V375I, the short β-branched side chain of valine 375 was replaced with that of isoleucine, longer by a single methyl group, which may also alter the structure of the hydrophobic filter. With the resulting conformation, the antiporter also gained the ability to transport the largest K + cation in addition to Na + and Li + (Fig. 6). Taking into consideration the overall empirical results in the light of the 3D model, it seems that the cation specificity of yeast plasma-membrane Na +/H + antiporters may be given by a combination of the size of the filter, its hydrophobicity and the position of the filter relative to the aspartic residue 176. We found that there is an interaction among TMS 4, 5 and 11 via residues Thr141, Ala179 and Val375. The studied point mutations do not significantly affect the global structure of the antiporter molecule, but they have more local significance for the protein. It is evident that the displacement of a methyl group within the protein structure influences its ability to transport particular alkali-metal cations. Similar examples have been already found in other types of transporters. The length of the side chains in the core of the molecule was shown to be a critical determinant of substrate specificity for a high-affinity glucose transporter [52]. A single mutation of essential tryptophan to glycine was shown to change the mechanism of the transporter from a drug/H + antiporter to a polyamine importer [53]. Recently, the single substitution T456S enabled a specific Can1 arginine permease to transport lysine [54]. In conclusion, this study combining protein modeling and experimentation demonstrates that the subtle replacement of residues forming the hydrophobic filter in close proximity to the putative cation binding site can fine tune the cation selectivity of

1691 yeast plasma-membrane Nha/Sod antiporters. It also validates the use of the structure of bacterial homologues to construct 3D models of eukaryotic Na +/H + antiporters, and it can help in understanding the structural code of alkali-metal cation recognition and the translocation mechanism in other members of the CPA family.

Materials and Methods Strains, media and plasmids The S. cerevisiae W303-1A derived alkali-metal cation sensitive strain BW31 (ena1Δ∷HIS3∷ena4Δ nha1Δ∷LEU2) [27], lacking genes encoding main Na + and K + efflux systems in the plasma membrane (Na +-ATPases Ena and Na + ,K + /H + antiporter Nha1), was used to characterize the antiporter activity in all assays. Yeast cells were grown aerobically in standard YNB (yeastnitrogen-based) media supplemented with 2% glucose and appropriate supplements at 30 °C. The ZrSOD2-22 gene and all its mutated versions were expressed under the control of the S. cerevisiae NHA1 promoter from a multicopy plasmid derived from YEp352 [31,55]. Zr Sod2-22 versions tagged with GFP at their N termini were expressed under the control of the Cu 2 +-inducible S. cerevisiae CUP1 promoter from the multicopy plasmid pYEX-GFP-Zr SOD2-22. Plasmids were constructed as described previously [28,56].

Site-directed mutagenesis Point mutations were introduced into the ZrSOD2-22 gene using a QuikChange XL Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA). For each mutation, two overlapping complementary oligonucleotides containing the corresponding nucleotide changes were designed. Primers were purchased from Sigma/Genosys (Steinheim, Germany). The accuracy of the mutation was confirmed by sequencing. Lasergene99 (DNASTAR Inc., Madison, WI, USA) was used for standard DNA and protein sequence analyses.

Salt tolerance determination The tolerance of yeast cells to alkali-metal cation salts was estimated by spotting 3 μl of serial 10-fold dilutions of saturated yeast cultures on solid YNB media supplemented with increasing amounts of salts (300–2000 mM NaCl, 800–2000 mM KCl and 10–140 mM LiCl). The pH of media was adjusted to 3.5 with tartaric acid after autoclaving. Solid media with pH 7.0 were supplemented with 20 mM Hepes, and the required pH was adjusted with NaOH. Cell growth was recorded at 24-h intervals over a 2- to 7-day period. Growth assays were repeated at least three times with similar results. Data from a representative experiment are presented.

1692 Cation loss measurement Cation efflux was estimated as described previously [27]. Cells were grown in YNB media to the early exponential phase of growth (OD600 ≅ 0.2). The intracellular Na + content in S. cerevisiae cells is negligible under normal growth conditions in minimal YNB medium and the efflux activity of yeast Na +/H + antiporters is lower at neutral pH than at acidic pH [36]. Therefore, to measure the efflux of Na +, we transferred cells to YNB medium of pH 7.0 (adjusted with NH4OH) supplemented with 100 mM NaCl and incubated them at 30 °C for 60 min. For the K + efflux measurements, since yeast cells accumulate a high intracellular K + concentration, cells grown in YNB media were directly transferred to a K +-free incubation buffer. For cation loss measurements, cells were harvested, washed and resuspended in a buffer of pH 4.5 consisting of 10 mM Tris, 0.1 mM MgCl2 and 2% glucose [the pH was adjusted to 4.4 with citric acid and Ca(OH)2 was added to increase the pH up to 4.5] and were supplemented with 10 mM KCl (RbCl) when Na + (K +) loss was measured to prevent Na + (K +) reuptake, respectively. Cell samples were withdrawn at intervals within the indicated period, and the intracellular concentration of Na + or K + was estimated by atomic absorption spectrophotometry [21,57]. Each experiment was repeated at least three times. Data presented either are from a representative experiment or are the mean of the replicative values plus or minus standard deviation.

Cation Selectivity of the Na+/H + Antiporter

default settings and then optimized manually. The resulting multiple alignment (Fig. S1) was used to build a 3D model of the membrane domain, based on the X-ray structure of EcNhaA (PDB code 1ZCD, chain A) using Swiss PDB Viewer 4.1 [59]. The model was first optimized with the SWISS-MODEL Web site [60] and then with the CHIRON server to remove clashes and minimize the structure † [61]. Protein Model Database accession code The 3D model of Z. rouxii Sod2-22 transmembrane part has been deposited in the PMDB database as PM0079562. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jmb.2015.02.012.

Acknowledgements This work was supported within the framework of COST CM 0902 and by MSMT COST LD 13037, by GA ČR P503/10/0307 and with the institutional support RVO: 67985823. The work of Pierre Falson was supported by the Ligue Contre le Cancer, ANR-Sve5 CLAMP and VLTAVA 2012. We wish to thank J. Zahrádka for a stimulating discussion on Zr Sod2-22 modeling and P. Herynková for excellent technical assistance. Available online 18 February 2015 Keywords: yeast; plasma membrane; sodium proton exchanger; substrate specificity; potassium transport

Fluorescence microscopy Cells exponentially growing in liquid YNB media containing 20 mM CuSO4 were viewed with an Olympus AX70 microscope with an Olympus DP70 digital camera (Olympus Optical Co., Ltd., Tokyo, Japan). Nomarski optics was used for whole-cell pictures. A U-MWB fluorescent cube with an excitation filter of 450–480 nm and a barrier filter of 515 nm was used for GFP visualization. Images were processed with the program Corel Paint Shop Pro X6 (Ottawa, Ontario, Canada). Model construction and validation The primary sequence corresponding to the membrane domain of the Na + /H + antiporter Sod2-22 of Z. rouxii (region 1–435 over 806 amino acids; ExPASy reference code Q9UUT4) was aligned with those of the same proteins from S. cerevisiae Nha1 (region 1–437 over 985 amino acids; ExPASy reference code Q99271), S. pombe Sod2 (region 1–431 over 468 amino acids; ExPASy reference code P36606) and E. coli NhaA (region 1– 388 over 388 amino acids; ExPASy reference code C9QSD0). The primary sequence alignment was first generated with CLUSTAL W [58] using the

†http://troll.med.unc.edu/chiron/index.php. Abbreviations used: 3D, three-dimensional; TMS, transmembrane segments; CPA, cation/proton antiporter.

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