The S. cerevisiae cation translocation protein Trk1 is functional without its "long hydrophilic loop" but LHL regulates cation translocation activity and selectivity

Abstract

In Saccharomyces cerevisiae, K⁺-uptake under K⁺-limiting conditions is largely mediated by the cation translocation systems Trk1 and Trk2 belonging to the family of SKT proteins. They are related to two-transmembrane-domain (inward rectifying K-) channels but unlike the symmetrical tetrameric structure of K-channels, a single Trk contains four pore-forming domains (A-D) encoded on one polypeptide chain. Between domains A and B Trks contain large cytosolic regions dubbed "long hydrophilic loop" (LHL). LHLs are not homologous/similar to any other identified protein (domain) and also show little similarity between Trk1 and Trk2. Here we demonstrate that Trk1 is functional without LHL. However, in growth experiments NaCl sensitivity of Trk1[ΔLHL] expressing cells is increased under K⁺-limiting conditions compared to full-length Trk1. Non-invasive ion flux measurements showed that K⁺-influx through Trk1 and Trk1[ΔLHL] is decreased in the presence of surplus Na⁺ due to permeability of the proteins for both cations and competition between them. Trk1[ΔLHL] is less affected than full-length Trk1 because it is more selective for K⁺ over Na⁺. Furthermore, K⁺ re-uptake after starvation is delayed and decreased in Trk1[ΔLHL]. Thus, a role of LHL is regulating cation fluxes through Trk1 by (i) allowing also Na⁺ to pass if monovalent cations (mainly K⁺) are limiting and (ii) by accelerating/enhancing a switch from low to high affinity ion translocation. We propose that LHL could modulate Trk1 transport properties via direct influence on a transmembrane helix (M2_A) which can switch between bent and straight conformation, thereby directly modifying the radius of the pore and selectivity filter.

Keywords: Bimolecular Fluorescence Complementation – BiFC; K(+)-transport; Long hydrophilic loop, LHL; Non-invasive ion-flux measurements – FLISE; Saccharomyces cerevisiae; Trk1–potassium translocation system.