Functional characterization of the NhaA Na+/H+ antiporter from the green picoalga Ostreococcus tauri.

Transmembrane ion transport is a critical process in the cellular response to salt stress. Among the known functional membrane transporters that are involved in the salt stress response, Na+/H+ antiporters have been extensively studied. These ubiquitous membrane proteins are crucial for salt tolerance and are associated with the regulation of internal pH, cell volume, morphogenesis, and vesicular trafficking. Molecular and functional analyses of Na+/H+ antiporters have been characterized among taxa but little is known about algal Na+/H+ antiporters. Here, we analyzed putative Na+/H+ antiporters from the complete genome sequence of the marine picoalga Ostreococcus tauri. At least 10 putative Na+/H+ antiporters belonging to the SOS1, NHX, and KEA/Kef families were found. Surprisingly, a bacterial type NhaA sequence (OtNhaA) was also found. Topological modeling of OtNhaA predicted 12 possible transmembrane segments with a long N-terminus. The full-length (FL_OtNhaA) and N-terminal truncated (ΔN112_OtNhaA) versions of OtNhaA were constructed, expressed in the salt-sensitive mutant Escherichia coli TO114, and functionally characterized. Complementation analysis revealed that FL_OtNhaA- and ΔN112_OtNhaA-expressing cells exhibited increased tolerance to high NaCl concentrations up to 700 mM. Antiporter activity assays showed that both FL_OtNhaA and ΔN112_OtNhaA proteins predominantly exhibited Na+/H+ and Ca2+/H+ antiporter activities at alkaline pH conditions. Intriguingly, the ΔN112_OtNhaA exhibited higher Na+/H+ and Ca2+/H+ antiporter activities compared to FL_OtNhaA. Kinetic analysis revealed that FL_OtNhaA has a high affinity for Na+ and Ca2+ ions with a Km of 1.1 ±â€¯0.23 mM for Na+ (at pH 8.5) and a Km of 0.3 ±â€¯0.07 mM for Ca2+ (at pH 8.5). Since NhaA has shown striking diversity among taxa, our results provide insight into the functional properties of the algal NhaA Na+/H+ antiporter. These results will contribute to the understanding of Na+/H+ antiporters that have various implications in all kingdoms of life.