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Membrane reconstitution of ABC transporters and assays of translocator function

Nature Protocols volume 3, pages 256–266 (2008)Cite this article

Abstract

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 (Kd) for ligand binding, number of binding-sites, transport affinities (Km), 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.

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Figure 1: Outline of the described procedures for membrane reconstitution and functional analysis of ATP-binding cassette (ABC) transporters.
Figure 2: Titration of preformed liposomes with Triton X-100.
Figure 3: Architecture of ATP-binding cassette transport systems.
Figure 4: Glycine-betaine binding and transport by OpuA.

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Acknowledgements

The authors greatly acknowledge the past and present members of the Poolman laboratory: without their contributions the protocols presented here would not have been established. The EU-FP6 program (E-MeP; 504601), Netherlands Proteomics Centre (NPC) and the National Science Foundation (NWO-Top Subsidy, grant 700.56.302) are acknowledged for funding.

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Authors and Affiliations

  1. Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute & Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, Groningen, 9747 AG, The Netherlands

    Eric R Geertsma, N A B Nik Mahmood, Gea K Schuurman-Wolters & Bert Poolman

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  1. Eric R Geertsma
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  3. Gea K Schuurman-Wolters
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Correspondence to Bert Poolman.

Supplementary information

Supplementary Figure 1

Glycine betaine transport by OpuA. [14C]-glycine betaine uptake by OpuA proteoliposomes containing an ARS in 100 mM KPi, pH7.0 without (○; isotonic) or with 250 mM KCl (□; hyperosmotic). The data is corrected for background binding of [14C]-glycine betaine to OpuA liposomes without the ARS. (PDF 805 kb)

Supplementary Figure 2

Demonstration of glycine betaine uptake by RSO-reconstituted OpuA and efflux by ISO-reconstituted OpuA. Following [14C]glycine betaine preloading of OpuA proteoliposomes (containing 10 mM Mg-ATP in the vesicle lumen) under activating conditions (80 mM KPi, pH 7.0 with 450 mM sucrose) and assaying for actual uptake (•), the proteoliposomes were stored on ice for subsequent measurements of glycine betaine efflux via inside-out reconstituted OpuA. Following storage on ice (no leakage of [14C]glycine betaine was observed up to 4 h of storage), samples were diluted 5-fold with 10 mM KPi, pH 7.0, and equilibrated for 3 min at 30 °C. Subsequently, 0.3 M KCl was added to activate OpuA; the addition was done by a 2-fold dilution with pre-warmed medium of the appropriate composition (for further details, see a); this step, the ionic activation, is required for osmoregulatory ABC transporters a, b but will not be necessary for other ABC transport systems. After another 2 min of incubation, 10 mM Mg-ATP was added to energize inside-out oriented OpuA (▪). Samples were taken at the indicated time points and processed in the same way as for uptake measurements. (PDF 264 kb)

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Geertsma, E., Nik Mahmood, N., Schuurman-Wolters, G. et al. Membrane reconstitution of ABC transporters and assays of translocator function. Nat Protoc 3, 256–266 (2008). https://doi.org/10.1038/nprot.2007.519

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