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A general method for determining secondary active transporter substrate stoichiometry

Fitzgerald, Gabriel A, Mulligan, Christopher, Mindell, Joseph A (2017) A general method for determining secondary active transporter substrate stoichiometry. eLife, 6 . Article Number 21016. ISSN 2050-084X. (doi:10.7554/eLife.21016) (KAR id:61675)

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http://dx.doi.org/10.7554/eLife.21016

Abstract

The number of ions required to drive substrate transport through a secondary active transporter determines the protein's ability to create a substrate gradient, a feature essential to its physiological function, and places fundamental constraints on the transporter's mechanism. Stoichiometry is known for a wide array of mammalian transporters, but, due to a lack of readily available tools, not for most of the prokaryotic transporters for which high-resolution structures are available. Here, we describe a general method for using radiolabeled substrate flux assays to determine coupling stoichiometries of electrogenic secondary active transporters reconstituted in proteoliposomes by measuring transporter equilibrium potentials. We demonstrate the utility of this method by determining the coupling stoichiometry of VcINDY, a bacterial Na(+)-coupled succinate transporter, and further validate it by confirming the coupling stoichiometry of vSGLT, a bacterial sugar transporter. This robust thermodynamic method should be especially useful in probing the mechanisms of transporters with available structures.

Item Type: Article
DOI/Identification number: 10.7554/eLife.21016
Subjects: Q Science > QD Chemistry > QD431 Organic Chemistry- Biochemistry- Proteins, peptides, amino acids
Q Science > QP Physiology (Living systems) > QP517 Biochemistry
Divisions: Divisions > Division of Natural Sciences > School of Biosciences
Depositing User: Christopher Mulligan
Date Deposited: 10 May 2017 14:33 UTC
Last Modified: 24 Sep 2020 14:28 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/61675 (The current URI for this page, for reference purposes)
Mulligan, Christopher: https://orcid.org/0000-0001-5157-4651
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