A functional EXXEK motif is essential for proton coupling and active glucosinolate transport by NPF_2.11

TY - JOUR

T1 - A functional EXXEK motif is essential for proton coupling and active glucosinolate transport by NPF2.11

AU - Jørgensen, Morten Egevang

AU - Olsen, Carl Erik

AU - Geiger, Dietmar

AU - Mirza, Osman Asghar

AU - Halkier, Barbara Ann

AU - Nour-Eldin, Hussam Hassan

N1 - © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

PY - 2015/4/9

Y1 - 2015/4/9

N2 - The proton-dependent oligopeptide transporter (POT/PTR) family shares a highly conserved E1X1X2E2RFXYY (E1X1X2E2R) 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 E1X1X2E2R motif. Moreover, nothing is known about the role of the X1 and X2 residues within the E1X1X2E2R motif. We used NPF2.11-a proton-coupled glucosinolate (GLS) symporter from Arabidopsis thaliana-to investigate the role of the E1X1X2E2K 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 E1X1X2E2K motif for accumulation of substrate by NPF2.11. Our data suggest that the highly conserved E1, E2 and K residues are involved in translocation of protons, as has been proposed for the E1X1X2E2R motif in bacteria. Furthermore, we show that the two residues X1 and X2 in the E1X1X2E2[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 X1 and X2 interferes with the function of the conserved glutamate and lysine residues of the E1X1X2E2K motif and affects the mechanism behind proton coupling.

AB - The proton-dependent oligopeptide transporter (POT/PTR) family shares a highly conserved E1X1X2E2RFXYY (E1X1X2E2R) 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 E1X1X2E2R motif. Moreover, nothing is known about the role of the X1 and X2 residues within the E1X1X2E2R motif. We used NPF2.11-a proton-coupled glucosinolate (GLS) symporter from Arabidopsis thaliana-to investigate the role of the E1X1X2E2K 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 E1X1X2E2K motif for accumulation of substrate by NPF2.11. Our data suggest that the highly conserved E1, E2 and K residues are involved in translocation of protons, as has been proposed for the E1X1X2E2R motif in bacteria. Furthermore, we show that the two residues X1 and X2 in the E1X1X2E2[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 X1 and X2 interferes with the function of the conserved glutamate and lysine residues of the E1X1X2E2K motif and affects the mechanism behind proton coupling.

U2 - 10.1093/pcp/pcv145

DO - 10.1093/pcp/pcv145

M3 - Journal article

C2 - 26443378

SN - 0032-0781

VL - 56

SP - 2340

EP - 2350

JO - Plant and Cell Physiology

JF - Plant and Cell Physiology

IS - 12

ER -