Monitoring the function of membrane transport proteins in detergent-solubilized form
- 27 February 2007
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 104 (9), 3603-3608
- https://doi.org/10.1073/pnas.0609573104
Abstract
Transport proteins constitute approximately 10% of most proteomes and play vital roles in the translocation of solutes across membranes of all organisms. Their (dys)function is implicated in many disorders, making them frequent targets for pharmacotherapy. The identification of substrates for members of this large protein family, still replete with many orphans of unknown function, has proven difficult, in part because high-throughput screening is greatly complicated by endogenous transporters present in many expression systems. In addition, direct structural studies require that transporters be extracted from the membrane with detergent, thereby precluding transport measurements because of the lack of a vectorial environment and necessitating reconstitution into proteoliposomes for activity measurements. Here, we describe a direct scintillation proximity-based radioligand-binding assay for determining transport protein function in crude cell extracts and in purified form. This rapid and universally applicable assay with advantages over cell-based platforms will greatly facilitate the identification of substrates for many orphan transporters and allows monitoring the function of transport proteins in a nonmembranous environment.Keywords
This publication has 28 references indexed in Scilit:
- State-dependent Conformations of the Translocation Pathway in the Tyrosine Transporter Tyt1, a Novel Neurotransmitter:Sodium Symporter from Fusobacterium nucleatumJournal of Biological Chemistry, 2006
- Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transportersNature, 2005
- Perturbation Analysis of the Voltage-sensitive Conformational Changes of the Na+/Glucose CotransporterThe Journal of general physiology, 2004
- Quorum sensing-controlled gene expression in lactic acid bacteriaJournal of Biotechnology, 1998
- A Conserved Aspartate Residue, Asp187, Is Important for Na+-Dependent Proline Binding and Transport by the Na+/Proline Transporter of Escherichia coliBiochemistry, 1998
- Over-production of Proteins inEscherichia coli: Mutant Hosts that Allow Synthesis of some Membrane Proteins and Globular Proteins at High LevelsJournal of Molecular Biology, 1996
- Orphanin FQ: A Neuropeptide That Activates an Opioidlike G Protein-Coupled ReceptorScience, 1995
- Fluorescence of native single‐Trp mutants in the lactose permease from Escherichia coli: Structural properties and evidence for a substrate‐induced conformational changeProtein Science, 1995
- Ligand‐Induced conformational changes in the lactose permease of escherichia coli: Evidence for two binding sitesProtein Science, 1994
- A method for rapid, continuous monitoring of solute uptake and bindingBiochemistry, 1982