Monitoring the Function of Membrane Transport Proteins in Detergent-solubilized Form

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2007 Mar 16

PMID 17360689

Citations 122

Authors

Matthias Quick

Jonathan A Javitch

Affiliations

Soon will be listed here.

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.

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References

Reinscheid R, Nothacker H, Bourson A, Ardati A, Henningsen R, Bunzow J . Orphanin FQ: a neuropeptide that activates an opioidlike G protein-coupled receptor. Science. 1995; 270(5237):792-4. DOI: 10.1126/science.270.5237.792. View

Wu J, Frillingos S, Voss J, Kaback H . Ligand-induced conformational changes in the lactose permease of Escherichia coli: evidence for two binding sites. Protein Sci. 1994; 3(12):2294-301. PMC: 2142758. DOI: 10.1002/pro.5560031214. View

Miroux B, Walker J . Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol. 1996; 260(3):289-98. DOI: 10.1006/jmbi.1996.0399. View

de Ruyter P, Kuipers O, de Vos W . Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl Environ Microbiol. 1996; 62(10):3662-7. PMC: 168174. DOI: 10.1128/aem.62.10.3662-3667.1996. View

Sonders M, Zhu S, Zahniser N, Kavanaugh M, Amara S . Multiple ionic conductances of the human dopamine transporter: the actions of dopamine and psychostimulants. J Neurosci. 1997; 17(3):960-74. PMC: 6573182. View

Quick M, Jung H . A conserved aspartate residue, Asp187, is important for Na+-dependent proline binding and transport by the Na+/proline transporter of Escherichia coli. Biochemistry. 1998; 37(39):13800-6. DOI: 10.1021/bi980562j. View

Janowski B, Grogan M, Jones S, Wisely G, Kliewer S, Corey E . Structural requirements of ligands for the oxysterol liver X receptors LXRalpha and LXRbeta. Proc Natl Acad Sci U S A. 1999; 96(1):266-71. PMC: 15128. DOI: 10.1073/pnas.96.1.266. View

Loo D, Hirayama B, Cha A, Bezanilla F, Wright E . Perturbation analysis of the voltage-sensitive conformational changes of the Na+/glucose cotransporter. J Gen Physiol. 2004; 125(1):13-36. PMC: 2217483. DOI: 10.1085/jgp.200409150. View

Yamashita A, Singh S, Kawate T, Jin Y, Gouaux E . Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters. Nature. 2005; 437(7056):215-23. DOI: 10.1038/nature03978. View

10.

Quick M, Yano H, Goldberg N, Duan L, Beuming T, Shi L . State-dependent conformations of the translocation pathway in the tyrosine transporter Tyt1, a novel neurotransmitter:sodium symporter from Fusobacterium nucleatum. J Biol Chem. 2006; 281(36):26444-54. DOI: 10.1074/jbc.M602438200. View

11.

Beuming T, Shi L, Javitch J, Weinstein H . A comprehensive structure-based alignment of prokaryotic and eukaryotic neurotransmitter/Na+ symporters (NSS) aids in the use of the LeuT structure to probe NSS structure and function. Mol Pharmacol. 2006; 70(5):1630-42. DOI: 10.1124/mol.106.026120. View

12.

Saier Jr M . A functional-phylogenetic system for the classification of transport proteins. J Cell Biochem. 2000; Suppl 32-33:84-94. DOI: 10.1002/(sici)1097-4644(1999)75:32+<84::aid-jcb11>3.0.co;2-m. View

13.

Quick M, Loo D, Wright E . Neutralization of a conserved amino acid residue in the human Na+/glucose transporter (hSGLT1) generates a glucose-gated H+ channel. J Biol Chem. 2000; 276(3):1728-34. DOI: 10.1074/jbc.M005521200. View

14.

Li X, Villa A, Gownley C, Kim M, Song J, Auer M . Monomeric state and ligand binding of recombinant GABA transporter from Escherichia coli. FEBS Lett. 2001; 494(3):165-9. DOI: 10.1016/s0014-5793(01)02334-1. View

15.

Quick M, Wright E . Employing Escherichia coli to functionally express, purify, and characterize a human transporter. Proc Natl Acad Sci U S A. 2002; 99(13):8597-601. PMC: 124325. DOI: 10.1073/pnas.132266599. View

16.

Androutsellis-Theotokis A, Goldberg N, Ueda K, Beppu T, Beckman M, Das S . Characterization of a functional bacterial homologue of sodium-dependent neurotransmitter transporters. J Biol Chem. 2003; 278(15):12703-9. DOI: 10.1074/jbc.M206563200. View

17.

Goldberg N, Beuming T, Soyer O, Goldstein R, Weinstein H, Javitch J . Probing conformational changes in neurotransmitter transporters: a structural context. Eur J Pharmacol. 2003; 479(1-3):3-12. DOI: 10.1016/j.ejphar.2003.08.052. View

18.

Schaffner W, Weissmann C . A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973; 56(2):502-14. DOI: 10.1016/0003-2697(73)90217-0. View

19.

Gruner S, Kirk G, Patel L, Kaback H . A method for rapid, continuous monitoring of solute uptake and binding. Biochemistry. 1982; 21(13):3239-43. DOI: 10.1021/bi00256a033. View

20.

UDENFRIEND S, Gerber L, Nelson N . Scintillation proximity assay: a sensitive and continuous isotopic method for monitoring ligand/receptor and antigen/antibody interactions. Anal Biochem. 1987; 161(2):494-500. DOI: 10.1016/0003-2697(87)90479-9. View