CLONING AND EXPRESSION OF Na+/H+ ANTIPORTER GENE FROM MICROALGAE

Abstract

Na+/H+ antiporter is ubiquitous transmembrane protein. It is secondary active transporter, found in all living organisms. Fundamental role of this membrane protein is to regulate Na+. In addition, it is involved in pH homeostasis, cell cycle and proliferation, morphogenesis, and vesicle trafficking. To date, Na+/H+ antiporters have been extensively studied in bacteria, higher plants, and mammals. In contrast, little is known on microalgal Na+/H+antiporters. In this study, the putative NhaA Na+/H+ antiporter from marine microalga Ostreococcus tauri (OtNhaA) was cloned and expressed in the salt-sensitive mutant Escherichia coli TO114 (∆nhaA∆nhaB∆chaA). Topological model of OtNhaA predicted 10 transmembrane segments with a long N terminus. Both full length OtNhaA (FL_OtNhaA) and N –terminal truncation OtNhaA (∆N112_OtNhaA) proteins were constructed. Western blotting revealed that these proteins could be expressed and assembed in Escherichia coli TO114. Complementation test revealed that FL_OtNhaA and ∆N112_OtNhaA expressing cells increased tolerance to high concentration up to 700 mM NaCl (at pH 7). Antiporter assay using acridine quenching method revealed both FL_OtNhaA and ∆N112_OtNhaA predominantly exhibited Na+/H+ and Ca2+/H+ antiporter activities with maximal dequenching at pH 8.5. Interestingly, the ∆N112_OtNhaA could exhibit Na+/H+ and Ca2+/H+ antiporter activities higher than those of FL_OtNhaA. Kinetic analysis revealed that OtNhaA encodes a high affinity Na+/H+ or Ca2+/H+ antiporter with a Km of 1.1 ± 0.23 mM for Na+ (at pH 8.5) and a Km of 0.3 ± 0.07 mM for Ca2+ (at pH 8.5). To our knowledge, this is the first report of functional characterization of Na+/H+ antiporter from microalgae.