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

Abstract

Na+,K+/H+ antiporters are H +-coupled cotransporters that are crucial for cellular homeostasis. Populus euphratica, a wellknown 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. Domainswitch analysis shows that TM11 is crucial to Li+ transport. The novel features of PeNHX3 in ion binding and translocation are discussed. © 2014 Wang et al.