Functional Reconstitution of a Bicomponent ABC Transporter

Abstract

The ATP-binding-cassette (ABC) transporters are transmembrane protein nanomachineries present in all living systems. ABC transporters utilize the energy of ATP hydrolysis to transport a variety of solutes across the membrane. Pseudomonas aeruginosa, a Gram-negative pathogenic bacterium, employs a bi-component ABC transporter as an active efflux of polysaccharides during the biogenesis of endotoxic lipopolysaccharides. We reconstituted the ABC transporter in various systems, including microsomes, planar lipid bilayers, and transfected mammalian N2a cell lines to obtain a mechanistic understanding of the functional properties of this nanomachinery. We employed single-channel electrical recordings to show that the transmembrane domain (TMD) of the ABC transporter features pore-forming activity. Further, our biochemical characterization of purified components sheds light on the structural assembly and stoichiometry of this bi-component ABC transporter. Our long-term goal is to detect, explore and characterize the translocation of polysaccharides at single-protein complex resolution. The use of a broad range of reconstitution systems enables a comprehensive examination of the ATP-dependent transport kinetics and thermodynamics of the large-size substrates from one side of the membrane to the other. These studies might also contribute to drug design against Pseudomonas aeruginosa.Acknowledgements. This research was supported by grants from the National Science Foundation (DMR-0706517 and HRD-0703452) and the National Institutes of Health (R01 GM088403) as well as by Syracuse Biomaterials Institute (SBI) for Liviu Movileanu and the National Institutes of Health (HL042220) for Richard Veenstra.