A Mass Spectrometry Based Transport Assay for Studying EmrE Transport of Unlabeled Substrates

Overview

Journal Anal Biochem

Publisher Elsevier

Specialty Biochemistry

Date 2018 Mar 22

PMID 29559333

Citations 3

Authors

Anne E Robinson

Jeffrey P Henderson

Katherine A Henzler-Wildman

Affiliations

Soon will be listed here.

Abstract

Membrane transporters are an important class of proteins which remain challenging to study. Transport assays are crucial to developing our understanding of such proteins as they allow direct measurement of their transport activity. However, currently available methods for monitoring liposomal loading of organic substrates primarily rely on detection of radioactively or fluorescently labeled substrates. The requirement of a labeled substrate significantly restricts the systems and substrates that can be studied. Here we present a mass spectrometry based detection method for liposomal uptake assays that eliminates the need for labeled substrates. We demonstrate the efficacy of the assay with EmrE, a small multidrug resistance transporter found in E. coli that has become a model transport system for the study of secondary active transport. Furthermore, we develop a method for differentiation between bound and transported substrate, enhancing the information gained from the liposomal uptake assay. The transport assay presented here is readily applicable to other transport systems and substrates.

Citing Articles

Cooperative Gating of a K Channel by Unmodified Biological Anionic Lipids Viewed by Solid-State NMR Spectroscopy.

Yekefallah M, van Aalst E, A M van Beekveld R, Eason I, Breukink E, Weingarth M J Am Chem Soc. 2024; 146(7):4421-4432.

PMID: 38334076 PMC: 10885140. DOI: 10.1021/jacs.3c09266.

Highly coupled transport can be achieved in free-exchange transport models.

Hussey G, Thomas N, Henzler-Wildman K J Gen Physiol. 2019; 152(1).

PMID: 31816638 PMC: 7034097. DOI: 10.1085/jgp.201912437.

Inducing conformational preference of the membrane protein transporter EmrE through conservative mutations.

Leninger M, Her A, Traaseth N Elife. 2019; 8.

PMID: 31637997 PMC: 6805155. DOI: 10.7554/eLife.48909.

References

Rotem D, Schuldiner S . EmrE, a multidrug transporter from Escherichia coli, transports monovalent and divalent substrates with the same stoichiometry. J Biol Chem. 2004; 279(47):48787-93. DOI: 10.1074/jbc.M408187200. View

Morrison E, Robinson A, Liu Y, Henzler-Wildman K . Asymmetric protonation of EmrE. J Gen Physiol. 2015; 146(6):445-61. PMC: 4664823. DOI: 10.1085/jgp.201511404. View

Padan E, Schuldiner S . Intracellular pH and membrane potential as regulators in the prokaryotic cell. J Membr Biol. 1987; 95(3):189-98. DOI: 10.1007/BF01869481. View

Rideout D, Bustamante A, Siuzdak G . Cationic drug analysis using matrix-assisted laser desorption/ionization mass spectrometry: application to influx kinetics, multidrug resistance, and intracellular chemical change. Proc Natl Acad Sci U S A. 1993; 90(21):10226-9. PMC: 47747. DOI: 10.1073/pnas.90.21.10226. View

Yerushalmi H, Mordoch S, Schuldiner S . A single carboxyl mutant of the multidrug transporter EmrE is fully functional. J Biol Chem. 2001; 276(16):12744-8. DOI: 10.1074/jbc.M010979200. View

Fitzgerald G, Mulligan C, Mindell J . A general method for determining secondary active transporter substrate stoichiometry. Elife. 2017; 6. PMC: 5305207. DOI: 10.7554/eLife.21016. View

Morrison E, DeKoster G, Dutta S, Vafabakhsh R, Clarkson M, Bahl A . Antiparallel EmrE exports drugs by exchanging between asymmetric structures. Nature. 2011; 481(7379):45-50. PMC: 3253143. DOI: 10.1038/nature10703. View

Dutta S, Morrison E, Henzler-Wildman K . Blocking dynamics of the SMR transporter EmrE impairs efflux activity. Biophys J. 2014; 107(3):613-620. PMC: 4129506. DOI: 10.1016/j.bpj.2014.06.030. View

Cheng Z, Winant R, Gambhir S . A new strategy to screen molecular imaging probe uptake in cell culture without radiolabeling using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. J Nucl Med. 2005; 46(5):878-86. View

10.

Adam Y, Tayer N, Rotem D, Schreiber G, Schuldiner S . The fast release of sticky protons: kinetics of substrate binding and proton release in a multidrug transporter. Proc Natl Acad Sci U S A. 2007; 104(46):17989-94. PMC: 2084284. DOI: 10.1073/pnas.0704425104. View

11.

Jardetzky O . Simple allosteric model for membrane pumps. Nature. 1966; 211(5052):969-70. DOI: 10.1038/211969a0. View

12.

Gayen A, Leninger M, Traaseth N . Protonation of a glutamate residue modulates the dynamics of the drug transporter EmrE. Nat Chem Biol. 2016; 12(3):141-5. PMC: 4755857. DOI: 10.1038/nchembio.1999. View

13.

Morrison E, Henzler-Wildman K . Transported substrate determines exchange rate in the multidrug resistance transporter EmrE. J Biol Chem. 2014; 289(10):6825-6836. PMC: 3945343. DOI: 10.1074/jbc.M113.535328. View

14.

Yerushalmi H, Schuldiner S . An essential glutamyl residue in EmrE, a multidrug antiporter from Escherichia coli. J Biol Chem. 2000; 275(8):5264-9. DOI: 10.1074/jbc.275.8.5264. View

15.

Morrison E, Henzler-Wildman K . Reconstitution of integral membrane proteins into isotropic bicelles with improved sample stability and expanded lipid composition profile. Biochim Biophys Acta. 2012; 1818(3):814-20. PMC: 3292179. DOI: 10.1016/j.bbamem.2011.12.020. View

16.

Krumpochova P, Sapthu S, Brouwers J, De Haas M, De Vos R, Borst P . Transportomics: screening for substrates of ABC transporters in body fluids using vesicular transport assays. FASEB J. 2011; 26(2):738-47. DOI: 10.1096/fj.11-195743. View

17.

Robinson A, Thomas N, Morrison E, Balthazor B, Henzler-Wildman K . New free-exchange model of EmrE transport. Proc Natl Acad Sci U S A. 2017; 114(47):E10083-E10091. PMC: 5703289. DOI: 10.1073/pnas.1708671114. View

18.

Yerushalmi H, Lebendiker M, Schuldiner S . EmrE, an Escherichia coli 12-kDa multidrug transporter, exchanges toxic cations and H+ and is soluble in organic solvents. J Biol Chem. 1995; 270(12):6856-63. DOI: 10.1074/jbc.270.12.6856. View

19.

Ryan R, Mindell J . The uncoupled chloride conductance of a bacterial glutamate transporter homolog. Nat Struct Mol Biol. 2007; 14(5):365-71. DOI: 10.1038/nsmb1230. View