A facile approach for the in vitro assembly of multimeric membrane transport proteins
- Oregon Health and Science University, United States
- Cornell University, United States
- Weill Cornell Medicine, United States
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
Francis I Valiyaveetil
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.
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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A facile approach for the in vitro assembly of multimeric membrane transport proteins
eLife 7:e36478.
https://doi.org/10.7554/eLife.36478
Further reading
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