Purification and biochemical characterization of NpPDR5, a plant ATP-Binding Cassette transporter expressed in Nicotiana tabacum BY-2 cells

Toussaint, Frédéric [UCL]

ATP-Binding Cassette (ABC) transporters are ubiquitous membrane proteins found in all living organisms, from bacteria to higher eukaryotes. They are involved in a wide variety of biological processes such as nutrients uptake, macromolecules export or ions transport. Some ABC transporters, particularly those involved in human diseases, have been well studied since decades. However, less is known about plant ABC transporters. Our laboratory is focused on one of the three subfamilies of full-size ABC transporters found in plants: ABCG or pleiotropic drug resistance (PDR) transporters. PDRs have been shown to be involved in many physiological processes at the whole plant level. Nevertheless, their biochemical properties have been barely characterized. In this work, we aimed to characterize two PDR transporters, MpPDR1 from Mentha x piperita and NpPDR5 from Nicotiana plumbaginifolia at the biochemical level. We sought to produce and purify both transporters, and then identify their enzymatic and structural properties. Nicotiana tabacum BY-2 suspension cells combined with a strong transcription promoter were used as an expression system. Both PDR genes provided with a double purification tag sequence (10-His/StrepII) were successfully expressed in N. tabacum BY-2 cells and solubilized from a microsomal fraction with dodecyl maltoside. Unlike MpPDR1, NpPDR5 was purified to homogeneity at a concentration (15 mg/ml) suitable for structural analysis. Exclusion chromatography and single particle analysis by electron microscopy showed that purified NpPDR5 is monodisperse and monomeric. At pH 7.5, NpPDR5 displayed an ATPase activity of 27.0 nmol.min-1.mg-1, which was increased up to 0.25 µmol.mg-1.min-1 in the presence of 0.1 % (w/v) asolectin and 0.1 % (w/v) dodecyl maltoside. This activity was inhibited by vanadate, miconazole, and verapamil. In conclusion, N. tabacum BY-2 suspension cells were shown to be a convenient expression system for PDR transporters. Moreover, the first structural and enzymatic properties of a purified plant ABC transporter were identified.