1. Structural Biology and Molecular Biophysics
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A facile approach for the in vitro assembly of multimeric membrane transport proteins

  1. Erika A Riederer
  2. Paul J Focke
  3. Elka R Georgieva
  4. Nurunisa Akyuz
  5. Kimberly Matulef
  6. Peter P Borbat
  7. Jack H Freed
  8. Scott C Blanchard
  9. Olga Boudker
  10. Francis I Valiyaveetil  Is a corresponding author
  1. Oregon Health and Science University, United States
  2. Cornell University, United States
  3. Weill Cornell Medicine, United States
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Cite this article as: eLife 2018;7:e36478 doi: 10.7554/eLife.36478

Abstract

Membrane proteins such as ion channels and transporters are frequently homomeric. The homomeric nature raises important questions regarding coupling between subunits and complicates the application of techniques such as FRET or DEER spectroscopy. These challenges can be overcome if the subunits of a homomeric protein can be independently modified for functional or spectroscopic studies. Here, we describe a general approach for in vitro assembly that can be used for the generation of heteromeric variants of homomeric membrane proteins. We establish the approach using GltPh, a glutamate transporter homolog that is trimeric in the native state. We use heteromeric GltPh transporters to directly demonstrate the lack of coupling in substrate binding and demonstrate how heteromeric transporters considerably simplify the application of DEER spectroscopy. Further, we demonstrate the general applicability of this approach by carrying out the in vitro assembly of VcINDY, a Na+-coupled succinate transporter and CLC-ec1, a Cl-/H+ antiporter.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Erika A Riederer

    Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1011-6536
  2. Paul J Focke

    Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, United States
    Competing interests
    No competing interests declared.
  3. Elka R Georgieva

    Department of Chemistry and Chemical Biology, Cornell University, Ithaca, United States
    Competing interests
    No competing interests declared.
  4. Nurunisa Akyuz

    Weill Cornell Medicine, New York, United States
    Competing interests
    No competing interests declared.
  5. Kimberly Matulef

    Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5011-9064
  6. Peter P Borbat

    Department of Chemistry and Chemical Biology, Cornell University, Ithaca, United States
    Competing interests
    No competing interests declared.
  7. Jack H Freed

    Department of Chemistry and Chemical Biology, Cornell University, Ithaca, United States
    Competing interests
    No competing interests declared.
  8. Scott C Blanchard

    Weill Cornell Medicine, New York, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2717-9365
  9. Olga Boudker

    Weill Cornell Medicine, New York, United States
    Competing interests
    Olga Boudker, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6965-0851
  10. Francis I Valiyaveetil

    Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, United States
    For correspondence
    valiyave@ohsu.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3387-8018

Funding

National Institute of General Medical Sciences (R01 GM087546)

  • Francis I Valiyaveetil

Howard Hughes Medical Institute

  • Olga Boudker

National Institute of Neurological Disorders and Stroke (R37 NS085318)

  • Scott C Blanchard
  • Olga Boudker
  • Francis I Valiyaveetil

National Institute of General Medical Sciences (P41GM103521)

  • Jack H Freed

American Heart Association (12POST1910068)

  • Paul J Focke

National Institute of General Medical Sciences (R01 GM123779)

  • Elka R Georgieva
  • Jack H Freed

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

Reviewing Editor

  1. Kenton Jon Swartz, National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States

Publication history

  1. Received: March 7, 2018
  2. Accepted: June 8, 2018
  3. Accepted Manuscript published: June 11, 2018 (version 1)
  4. Version of Record published: June 29, 2018 (version 2)

Copyright

© 2018, Riederer et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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