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. 2014 Aug 5;9(8):e104147.
doi: 10.1371/journal.pone.0104147. eCollection 2014.

Functional analysis of the Na+,K+/H+ antiporter PeNHX3 from the tree halophyte Populus euphratica in yeast by model-guided mutagenesis

Liguang Wang  1 , Xueying Feng  1 , Hong Zhao  1 , Lidong Wang  1 , Lizhe An  1 , Quan-Sheng Qiu  1
Affiliations

Functional analysis of the Na+,K+/H+ antiporter PeNHX3 from the tree halophyte Populus euphratica in yeast by model-guided mutagenesis

Liguang Wang et al. PLoS One. .

Erratum in

Abstract

Na+,K+/H+ antiporters are H+-coupled cotransporters that are crucial for cellular homeostasis. Populus euphratica, a well-known tree halophyte, contains six Na+/H+ antiporter genes (PeNHX1-6) that have been shown to function in salt tolerance. However, the catalytic mechanisms governing their ion transport remain largely unknown. Using the crystal structure of the Na+/H+ antiporter from the Escherichia coli (EcNhaA) as a template, we built the three-dimensional structure of PeNHX3 from P. euphratica. The PeNHX3 model displays the typical TM4-TM11 assembly that is critical for ion binding and translocation. The PeNHX3 structure follows the 'positive-inside' rule and exhibits a typical physicochemical property of the transporter proteins. Four conserved residues, including Tyr149, Asn187, Asp188, and Arg356, are indentified in the TM4-TM11 assembly region of PeNHX3. Mutagenesis analysis showed that these reserved residues were essential for the function of PeNHX3: Asn187 and Asp188 (forming a ND motif) controlled ion binding and translocation, and Tyr149 and Arg356 compensated helix dipoles in the TM4-TM11 assembly. PeNHX3 mediated Na+, K+ and Li+ transport in a yeast growth assay. Domain-switch analysis shows that TM11 is crucial to Li+ transport. The novel features of PeNHX3 in ion binding and translocation are discussed.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The TM segments and topology of PeNHX3.
A. The TM helices were analyzed with FFAS03, FUGUE, HMMTOP, and TMHMM. The TM helix numbers are marked at the corresponding regions. The TM boundaries for structure modeling are highlighted in gray. B. The TM topology of PeNHX3. Amino acid residues are colored based on the hydrophobicity scale of Kessel and Ben-Tal (Schushan et al., 2010).
Figure 2
Figure 2. Hydrophilic residues are localized in the core of PeNHX3.
The PeNHX3 model is colored based on the hydrophobicity scale of Kessel and Ben-Tal (Schushan et al., 2010). A. A side view parallel to the membrane with the intracellular side facing upward. B. A top view from the extracellular side.
Figure 3
Figure 3. The PeNHX3 model follows the ‘positive-inside’ rule.
The cytoplasmic side is at the top. The approximate boundaries of the hydrocarbon region of the membrane are shown in orange. The Cα atoms of lysines and arginines in the PeNHX3 model are shown as blue spheres.
Figure 4
Figure 4. PeNHX3 facilitate Na+, K+ and Li+ transport and confer tolerance to hygromycin B in yeast.
The cDNAs of PeNHX3, AtNHX2, AtNHX2, and ScNHX1 were subcloned into the yeast expression vector pDR196 and transformed into the AXT3 mutant (ena1-4 nha1 nhx1). The yeast strains were grown overnight in AP or YPD medium. Cells were normalized in water to A600 of 0.12. Aliquos (4 µL) from the normalized yeast cultures or 10-fold serial dilutions were spotted onto AP plates supplemented with KCl (B) or onto YPD plates with NaCl (A), LiCl (C), or Hygromycin B (D). The strains were grown at 30°C for 3 days.
Figure 5
Figure 5. Schematic diagram of the yeast expression constructs for the domain-switch analysis.
(A) The CDS of AtNHX1and PeNHX3, and the C-terminal domain of PeNHX3. (B) The constructs of AtNHX1-PeNHX3-C537 and AtNHX1-PeNHX3-C399. Construct diagrams are not drawn to scale.
Figure 6
Figure 6. TM11 is crucial to Li+ transport in PeNHX3.
The yeast strains were grown overnight in AP medium. Cells were normalized in water to A600 of 0.12 Aliquos (4 µL) and 10-fold serial dilutions were spotted on AP plates supplemented with KCl (B) or YPD plates with NaCl (A), LiCl (C), Hyg B (D) as indicated on each panel. The strains were grown at 30°C for 3 days.
Figure 7
Figure 7. Comparison of the four conserved residues in the TM4-TM11 assembly regions of PeNHX3 and EcNhaA.
The cytoplasmic side is at the top and TM12 was omitted for clarity in all panels. The Cα atoms of the four conserved titratable residues in the TM4-TM11 assembly region are shown as spheres. A. The predicated structure of PeNHX3 (light green) is aligned to the crystal structure of EcNhaA (gray) (Hunte et al., 2005). The center of the TM4-TM11 assembly and flanking region is marked by a square. B and C. The marked regions of EcNhaA and PeNHX3.
Figure 8
Figure 8. Functional analysis of the conserved residues in the TM4-TM11 assembly region of PeNHX3 (dosage response).
The cDNAs of the wild type PeNHX3 and the PeNHX3 mutants were subcloned into the yeast expression vector pDR196 and transformed into the AXT3 mutant (ena1-4 nha1 nhx1). Yeast cells were grown in APG media containing different concentrations of NaCl. Yeast growth was determined at 600 nm (OD600) after 48 h at 30°C. Data are the average of three experiments. Asterisks indicate significant difference (P≤0.05; t test).
Figure 9
Figure 9. Functional analysis of the conserved residues in the TM4-TM11 assembly region of PeNHX3 (pH profile).
NaCl concentration was kept at 300Figure 6. Asterisks indicate significant difference (P≤0.05; t test).
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Grants and funding

This work was supported by the National Basic Research Program of China (973 project, 2013CB429904 to QSQ), the National Natural Science Foundation of China (NSFC) (31371438, 31070222 to QSQ), the Research Fund for the Doctoral Program of Higher Education of China (RFDP) (20130211110001 to QSQ), and the Fundamental Research Funds for the Central Universities (lzujbky-2013-k10). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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