A Functional EXXEK Motif is Essential for Proton Coupling and Active Glucosinolate Transport by NPF2.11

The proton-dependent oligopeptide transporter (POT/PTR) family shares a highly conserved E(1)X(1)X(2)E(2)RFXYY (E(1)X(1)X(2)E(2)R) motif across all kingdoms of life. This motif is suggested to have a role in proton coupling and active transport in bacterial homologs. For the plant POT/PTR family, also known as the NRT1/PTR family (NPF), little is known about the role of the E(1)X(1)X(2)E(2)R motif. Moreover, nothing is known about the role of the X(1) and X(2) residues within the E(1)X(1)X(2)E(2)R motif. We used NPF2.11—a proton-coupled glucosinolate (GLS) symporter from Arabidopsis thaliana—to investigate the role of the E(1)X(1)X(2)E(2)K motif variant in a plant NPF transporter. Using liquid chromatography–mass spectrometry (LC-MS)-based uptake assays and two-electrode voltage clamp (TEVC) electrophysiology, we demonstrate an essential role for the E(1)X(1)X(2)E(2)K motif for accumulation of substrate by NPF2.11. Our data suggest that the highly conserved E(1), E(2) and K residues are involved in translocation of protons, as has been proposed for the E(1)X(1)X(2)E(2)R motif in bacteria. Furthermore, we show that the two residues X(1) and X(2) in the E(1)X(1)X(2)E(2)[K/R] motif are conserved as uncharged amino acids in POT/PTRs from bacteria to mammals and that introducing a positive or negative charge in either position hampers the ability to overaccumulate substrate relative to the assay medium. We hypothesize that introducing a charge at X(1) and X(2) interferes with the function of the conserved glutamate and lysine residues of the E(1)X(1)X(2)E(2)K motif and affects the mechanism behind proton coupling.