title: A Functional EXXEK Motif is Essential for Proton Coupling and Active Glucosinolate Transport by NPF2.11.
abstract: 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.
authors.affiliation: DynaMo Center, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, 1871 Frederiksberg C, Denmark Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, 1871 Frederiksberg C, Denmark.
authors.forename: Morten Egevang
authors.initials: ME
authors.lastname: Jørgensen
authors.affiliation: Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, 1871 Frederiksberg C, Denmark.
authors.forename: Carl Erik
authors.initials: CE
authors.lastname: Olsen
authors.affiliation: Julius-von-Sachs-Institute, Molecular Plant Physiology and Biophysics, University of Würzburg, D-97082 Würzburg, Germany.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Geiger
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
authors.forename: Osman
authors.initials: O
authors.lastname: Mirza
authors.affiliation: DynaMo Center, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, 1871 Frederiksberg C, Denmark Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, 1871 Frederiksberg C, Denmark.
authors.forename: Barbara Ann
authors.initials: BA
authors.lastname: Halkier
authors.affiliation: DynaMo Center, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, 1871 Frederiksberg C, Denmark Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, 1871 Frederiksberg C, Denmark huha@plen.ku.dk.
authors.forename: Hussam Hassan
authors.initials: HH
authors.lastname: Nour-Eldin
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism*; D001692:Biological Transport; D003470:Culture Media; D000939:Epitopes / Q000378:metabolism; D005961:Glucosinolates / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011522:Protons; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D003470:Culture Media; D000939:Epitopes; C575186:GTR2 protein, Arabidopsis; D005961:Glucosinolates; D009004:Monosaccharide Transport Proteins; D050505:Mutant Proteins; D011522:Protons; D013912:Threonine
keywords: Defense compound transporters; EXXERFYY motif; NPF2.11; POTs; Proton-coupling; TEVC electrophysiology