K⁺ Efflux Antiporters 4, 5, and 6 Mediate pH and K⁺ Homeostasis in Endomembrane Compartments

Abstract

KEA4, KEA5, and KEA6 are members of the Arabidopsis (<i>Arabidopsis thaliana</i>) K⁺ efflux antiporter (KEA) family that share high sequence similarity but whose function remains unknown. Here, we show their gene expression pattern, subcellular localization, and physiological function in Arabidopsis. <i>KEA4</i>, <i>KEA5</i>, and <i>KEA6</i> had similar tissue expression patterns, and the three KEA proteins localized to the Golgi, the trans-Golgi network, and the prevacuolar compartment/multivesicular bodies, suggesting overlapping roles of these proteins in the endomembrane system. Phenotypic analyses of single, double, and triple mutants confirmed functional redundancy. The triple mutant <i>kea4 kea5 kea6</i> had small rosettes, short seedlings, and was sensitive to low K⁺ availability and to the sodicity imposed by high salinity. Also, the <i>kea4 kea5 kea6</i> mutant plants had a reduced luminal pH in the Golgi, trans-Golgi network, prevacuolar compartment, and vacuole, in accordance with the K/H exchange activity of KEA proteins. Genetic analysis indicated that KEA4, KEA5, and KEA6 as well as endosomal Na⁺/H⁺exchanger5 (NHX5) and NHX6 acted coordinately to facilitate endosomal pH homeostasis and salt tolerance. Neither cancelling nor overexpressing the vacuolar antiporters <i>NHX1</i> and <i>NHX2</i> in the <i>kea4 kea5 kea6</i> mutant background altered the salt-sensitive phenotype. The NHX1 and NHX2 proteins in the <i>kea4 kea5 kea6</i> mutant background could not suppress the acidity of the endomembrane system but brought the vacuolar pH close to wild-type values. Together, these data signify that KEA4, KEA5, and KEA6 are endosomal K⁺ transporters functioning in maintaining pH and ion homeostasis in the endomembrane network.