Journal
BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
Volume 1818, Issue 9, Pages 2362-2371Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbamem.2012.04.011
Keywords
Potassium transport; Arabidopsis; K+/H+ antiport; Yeast; Reconstitution
Categories
Funding
- Spanish Plan Nacional de Biotecnologia [BIO2008-01691]
- CSIC
- Ramon Areces Foundation
- NSF [IBN-209792]
- National Science Foundation [IBN0209788]
- Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy [DE-FG0207ER15883]
- Royal Thai Government
- University of Minnesota
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KEA genes encode putative K+ efflux antiporters that are predominantly found in algae and plants but are rare in metazoa; however, nothing is known about their functions in eukaryotic cells. Plant KEA proteins show homology to bacterial K+ efflux (Kef) transporters, though two members in the Arabidopsis thaliana family, AtKEA1 and AtKEA2, have acquired an extra hydrophilic domain of over 500 residues at the amino terminus. We show that AtKEA2 is highly expressed in leaves, stems and flowers, but not in roots, and that an N-terminal peptide of the protein is targeted to chloroplasts in Arabidopsis cotyledons. The full-length AtKEA2 protein was inactive when expressed in yeast: however, a truncated AtKEA2 protein (At5KEA2) lacking the N-terminal domain complemented disruption of the Na+(K+)/H+ antiporter Nhx1p to confer hygromycin resistance and tolerance to Na+ or K+ stress. To test transport activity, purified truncated AtKEA2 was reconstituted in proteoliposomes containing the fluorescent probe pyranine. Monovalent cations reduced an imposed pH gradient (acid inside) indicating AtsKEA2 mediated cation/H+ exchange with preference for K+ = Cs+ > Li+ > Na+. When a conserved Asp(721) in transmembrane helix 6 that aligns to the cation binding Asp(164) of Escherichia coli NhaA was replaced with Ala. AtsKEA2 was completely inactivated. Mutation of a Glu(835) between transmembrane helix 8 and 9 in AtsKEA2 also resulted in loss of activity suggesting this region has a regulatory role. Thus, AtKEA2 represents the founding member of a novel group of eukaryote K+/H+ antiporters that modulate monovalent cation and pH homeostasis in plant chloroplasts or plastids. (C) 2012 Elsevier B.V. All rights reserved.
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