Arabidopsis KEA2, a homolog of bacterial KefC, encodes a K+/H+ antiporter with a chloroplast transit peptide

Abstract

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 (AtsKEA2) 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.

Rapid hyperosmotic-induced Ca 2+ responses in Arabidopsis thaliana exhibit sensory potentiation and involvement of plastidial KEA transporters

“…Our analyses of the plastidial KEA transporters offer initial insight into this question. KEA1 and KEA2 are localized to the inner envelope of the plastid, whereas KEA3 is localized to the thylakoid membrane (39,41,42). Although kea1-2kea2-2 double mutants display phenotypes associated with severely impaired chloroplast functions (41), the kea3-1 mutant line does not affect chloroplast morphology and is known to display electron transport phenotypes under dynamic light situations (42).…”

“…We measured hyperosmotic-induced Ca 2+ responses in plastidial-localized kea mutant lines. KEA1 (AT1G01790) and KEA2 (AT4G00630) are targeted to the inner plastid envelope membrane and are required for plastid ion homeostasis and osmo-regulation (39)(40)(41). Loss of function results in morphologically swollen plastids and decreased photosynthetic activity (41).…”

Envelope K+/H+ Antiporters AtKEA1 and AtKEA2 Function in Plastid Development

“…Recently, AtKEA1 and AtKEA2 were shown to act as K + /H + antiporters (Aranda-Sicilia et al, 2012) and to be located in the inner envelope membranes (Ferro et al, 2010;Roston et al, 2012;Kunz et al, 2014). Lightinduced electrogenic K + uptake is expected to cause osmotic swelling of the organelle.…”

“…Coiled-coil proteins play important roles in chloroplast division, thylakoid membrane development, and chloroplast relocation in response to light, by interaction with cytoskeleton-like elements (Wada, 2013;Karim and Aronsson, 2014;Osteryoung and Pyke, 2014). Furthermore, although the AtKEA2 protein without the N-terminal domain constitutes an active antiporter (Aranda-Sicilia et al, 2012), it fails to complement the growth defects of the double mutant plant, suggesting that the specific localization of the protein is crucial for its function. The association of oligomers of AtKEA2 could depend on membrane tension or curvature, which is highest at the poles but more relaxed at the midcell, as in bacteria (Laloux and Jacobs-Wagner, 2014;Strahl et al, 2015).…”

“…The recent discovery of Arabidopsis (Arabidopsis thaliana) K + /H + antiporters, AtKEA1 and AtKEA2, at the inner envelope and AtKEA3 at the thylakoid membrane represented an important breakthrough in uncovering the molecular basis for chloroplast K + /H + homeostasis (Aranda-Sicilia et al, 2012;Armbruster et al, 2014;Kunz et al, 2014). AtKEA3 was shown to be involved in modulation of the thylakoid lumen pH, which is important for photosynthetic performance and nonphotochemical quenching under fluctuating light conditions (Armbruster et al, 2014).…”