title: Purification and biochemical characterization of NpABCG5/NpPDR5, a plant pleiotropic drug resistance transporter expressed inNicotiana tabacumBY-2 suspension cells.
abstract: Pleiotropic drug resistance (PDR) transporters belong to the ABCG subfamily of ATP-binding cassette (ABC) transporters and are involved in the transport of various molecules across plasma membranes. During evolution,PDRgenes appeared independently in fungi and in plants from a duplication of a half-size ABC gene. The enzymatic properties of purified PDR transporters from yeast have been characterized. This is not the case for any plant PDR transporter, or, incidentally, for any purified plant ABC transporter. Yet, plant PDR transporters play important roles in plant physiology such as hormone signaling or resistance to pathogens or herbivores. Here, we describe the expression, purification, enzymatic characterization and 2D analysis by electron microscopy of NpABCG5/NpPDR5 fromNicotiana plumbaginifolia, which has been shown to be involved in the plant defense against herbivores. We constitutively expressed NpABCG5/NpPDR5, provided with a His-tag in a homologous system: suspension cells fromNicotiana tabacum(Bright Yellow 2 line). NpABCG5/NpPDR5 was targeted to the plasma membrane and was solubilized by dodecyl maltoside and purified by Ni-affinity chromatography. The ATP-hydrolyzing specific activity (27 nmol min-1mg-1) was stimulated seven-fold in the presence of 0.1% asolectin. Electron microscopy analysis indicated that NpABCG5/NpPDR5 is monomeric and with dimensions shorter than those of known ABC transporters. Enzymatic data (optimal pH and sensitivity to inhibitors) confirmed that plant and fungal PDR transporters have different properties. These data also show thatN. tabacumsuspension cells are a convenient host for the purification and biochemical characterization of ABC transporters.
authors.affiliation: Institut des Sciences de la Vie, Université catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Toussaint
authors.affiliation: Institut des Sciences de la Vie, Université catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium.
authors.forename: Baptiste
authors.initials: B
authors.lastname: Pierman
authors.affiliation: Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, CNRS UMR168, 75005 Paris, France.|Sorbonne Universités, UPMC Univ Paris 06, 75005 Paris, France.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Bertin
authors.affiliation: Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, CNRS UMR168, 75005 Paris, France.|Sorbonne Universités, UPMC Univ Paris 06, 75005 Paris, France.
authors.forename: Daniel
authors.initials: D
authors.lastname: Lévy
authors.affiliation: Institut des Sciences de la Vie, Université catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium marc.boutry@uclouvain.be.
authors.forename: Marc
authors.initials: M
authors.lastname: Boutry
mesh_terms: D000071018:ATP Binding Cassette Transporter, Subfamily G, Member 5 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D061249:Batch Cell Culture Techniques; D019149:Bioreactors; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003902:Detergents / Q000737:chemistry; D005960:Glucosides / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007091:Image Processing, Computer-Assisted; D049990:Membrane Transport Modulators / Q000494:pharmacology; D008854:Microscopy, Electron; D008970:Molecular Weight; D010713:Phosphatidylcholines / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D014026:Nicotiana / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*
chemical_list: D000071018:ATP Binding Cassette Transporter, Subfamily G, Member 5; D003902:Detergents; D005960:Glucosides; D049990:Membrane Transport Modulators; D010713:Phosphatidylcholines; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C040358:dodecyl maltoside; C024990:asolectin; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases
keywords: ABC transport proteins; Nicotiana tabacum; electron microscopy; kinetics; quaternary structure; suspension cells