Membrane reconstitution of ABC transporters and assays of translocator function.
In this protocol, we describe a procedure for incorporating ATP-binding cassette (ABC) transporters into large unilamellar vesicles (LUVs) and assays to determine ligand binding and solute translocation by these membrane-reconstituted systems. The reconstitution technique as described has been optimized for ABC transporters but can be readily adapted for other types of transport systems. Purified transporters are inserted into detergent-destabilized preformed liposomes and detergent is subsequently removed by adsorption onto polystyrene beads. Next, Mg-ATP or an ATP-regenerating system is incorporated into the vesicle lumen by one or more cycles of freezing-thawing, followed by extrusion through polycarbonate filters to obtain unilamellar vesicles. Binding and translocation of substrates are measured using isotope-labeled ligands and rapid filtration to separate the proteoliposomes from the surrounding medium. Quantitative information is obtained about dissociation constants (K(d)) for ligand binding, number of binding-sites, transport affinities (K(m)), rates of transport, and the activities of transporter molecules with opposite orientations in the membrane. The full protocol can be completed within 4-5 d.
Geertsma
Eric R
ER
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute & Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Nik Mahmood
N A B
NA
Schuurman-Wolters
Gea K
GK
Poolman
Bert
B
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D001692:Biological Transport; D003902:Detergents; D008563:Membrane Lipids; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008566:Membranes / Q000502:physiology*; D017830:Octoxynol; D053835:Unilamellar Liposomes / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D003902:Detergents; D008563:Membrane Lipids; D026901:Membrane Transport Proteins; D053835:Unilamellar Liposomes; D017830:Octoxynol