title: A Barley Efflux Transporter Operates in a Na+-Dependent Manner, as Revealed by a Multidisciplinary Platform.
abstract: Plant growth and survival depend upon the activity of membrane transporters that control the movement and distribution of solutes into, around, and out of plants. Although many plant transporters are known, their intrinsic properties make them difficult to study. In barley (Hordeum vulgare), the root anion-permeable transporter Bot1 plays a key role in tolerance to high soil boron, facilitating the efflux of borate from cells. However, its three-dimensional structure is unavailable and the molecular basis of its permeation function is unknown. Using an integrative platform of computational, biophysical, and biochemical tools as well as molecular biology, electrophysiology, and bioinformatics, we provide insight into the origin of transport function of Bot1. An atomistic model, supported by atomic force microscopy measurements, reveals that the protein folds into 13 transmembrane-spanning and five cytoplasmic α-helices. We predict a trimeric assembly of Bot1 and the presence of a Na(+) ion binding site, located in the proximity of a pore that conducts anions. Patch-clamp electrophysiology of Bot1 detects Na(+)-dependent polyvalent anion transport in a Nernstian manner with channel-like characteristics. Using alanine scanning, molecular dynamics simulations, and transport measurements, we show that conductance by Bot1 is abolished by removal of the Na(+) ion binding site. Our data enhance the understanding of the permeation functions of Bot1.
authors.affiliation: Australian Centre for Plant Functional Genomics, School of Agriculture, Food, and Wine, University of Adelaide, Glen Osmond, South Australia 5064, Australia.
authors.forename: Yagnesh
authors.initials: Y
authors.lastname: Nagarajan
authors.affiliation: Australian Centre for Plant Functional Genomics, School of Agriculture, Food, and Wine, University of Adelaide, Glen Osmond, South Australia 5064, Australia.
authors.forename: Jay
authors.initials: J
authors.lastname: Rongala
authors.affiliation: Australian Centre for Plant Functional Genomics, School of Agriculture, Food, and Wine, University of Adelaide, Glen Osmond, South Australia 5064, Australia.
authors.forename: Sukanya
authors.initials: S
authors.lastname: Luang
authors.affiliation: Department of Materials Science and Engineering, North Carolina State University, Raleigh, North Carolina 27695-7907.
authors.forename: Abhishek
authors.identifier: http://orcid.org/0000-0002-9450-7229
authors.initials: A
authors.lastname: Singh
authors.affiliation: Australian Centre for Plant Functional Genomics, School of Agriculture, Food, and Wine, University of Adelaide, Glen Osmond, South Australia 5064, Australia.
authors.forename: Nadim
authors.initials: N
authors.lastname: Shadiac
authors.affiliation: Australian Centre for Plant Functional Genomics, School of Agriculture, Food, and Wine, University of Adelaide, Glen Osmond, South Australia 5064, Australia.
authors.forename: Julie
authors.identifier: http://orcid.org/0000-0001-8716-5024
authors.initials: J
authors.lastname: Hayes
authors.affiliation: Australian Centre for Plant Functional Genomics, School of Agriculture, Food, and Wine, University of Adelaide, Glen Osmond, South Australia 5064, Australia.
authors.forename: Tim
authors.initials: T
authors.lastname: Sutton
authors.affiliation: Australian Research Council Centre of Excellence in Plant Energy Biology, School of Agriculture, Food, and Wine, University of Adelaide, Glen Osmond, South Australia 5064, Australia.
authors.forename: Matthew
authors.identifier: http://orcid.org/0000-0003-0666-3078
authors.initials: M
authors.lastname: Gilliham
authors.affiliation: Australian Research Council Centre of Excellence in Plant Energy Biology, School of Agriculture, Food, and Wine, University of Adelaide, Glen Osmond, South Australia 5064, Australia.
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Tyerman
authors.affiliation: Mawson Institute, University of South Australia, Mawson Lakes, South Australia 5095, Australia.
authors.forename: Gordon
authors.initials: G
authors.lastname: McPhee
authors.affiliation: Mawson Institute, University of South Australia, Mawson Lakes, South Australia 5095, Australia.
authors.forename: Nicolas H
authors.initials: NH
authors.lastname: Voelcker
authors.affiliation: Small- and Wide-Angle X-Ray Scattering Beamline, Australian Synchrotron, Clayton, Victoria 3168, Australia.
authors.forename: Haydyn D T
authors.identifier: http://orcid.org/0000-0002-5664-744X
authors.initials: HD
authors.lastname: Mertens
authors.affiliation: Small- and Wide-Angle X-Ray Scattering Beamline, Australian Synchrotron, Clayton, Victoria 3168, Australia.
authors.forename: Nigel M
authors.identifier: http://orcid.org/0000-0002-2791-4684
authors.initials: NM
authors.lastname: Kirby
authors.affiliation: Department of Materials Science and Engineering, North Carolina State University, Raleigh, North Carolina 27695-7907.
authors.forename: Jung-Goo
authors.initials: JG
authors.lastname: Lee
authors.affiliation: Department of Materials Science and Engineering, North Carolina State University, Raleigh, North Carolina 27695-7907.
authors.forename: Yaroslava G
authors.identifier: http://orcid.org/0000-0002-8557-9992
authors.initials: YG
authors.lastname: Yingling
authors.affiliation: Australian Centre for Plant Functional Genomics, School of Agriculture, Food, and Wine, University of Adelaide, Glen Osmond, South Australia 5064, Australia maria.hrmova@adelaide.edu.au.
authors.forename: Maria
authors.identifier: http://orcid.org/0000-0002-3545-0605
authors.initials: M
authors.lastname: Hrmova
mesh_terms: D000838:Anions / Q000378:metabolism; D001665:Binding Sites; D001881:Borates / Q000378:metabolism; D002474:Cell-Free System; D003198:Computer Simulation; D001467:Hordeum / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D010539:Permeability; D010843:Pichia / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D055503:Protein Multimerization; D012964:Sodium / Q000378:metabolism*; D014908:Triticum / Q000378:metabolism
chemical_list: D000838:Anions; D001881:Borates; D008051:Lipid Bilayers; D008081:Liposomes; D026901:Membrane Transport Proteins; D010940:Plant Proteins; D012964:Sodium