1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
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A general method for determining secondary active transporter substrate stoichiometry

  1. Gabriel A Fitzgerald
  2. Christopher Mulligan
  3. Joseph A Mindell  Is a corresponding author
  1. National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States
Research Article
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Cite this article as: eLife 2017;6:e21016 doi: 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.

Article and author information

Author details

  1. Gabriel A Fitzgerald

    Membrane Transport Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Christopher Mulligan

    Membrane Transport Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Joseph A Mindell

    Membrane Transport Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States
    For correspondence
    mindellj@ninds.nih.gov
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6952-8247

Funding

NINDS Intramural Program

  • Joseph A Mindell

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Olga Boudker, Weill Cornell Medical College, United States

Publication history

  1. Received: August 28, 2016
  2. Accepted: January 17, 2017
  3. Accepted Manuscript published: January 25, 2017 (version 1)
  4. Accepted Manuscript updated: February 2, 2017 (version 2)
  5. Version of Record published: February 13, 2017 (version 3)

Copyright

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

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