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Kinetic mechanism of Na(+)-coupled aspartate transport catalyzed by Glt(Tk)
It is well-established that the secondary active transporters Glt(Tk) and Glt(Ph) catalyze coupled uptake of aspartate and three sodium ions, but insight in the kinetic mechanism of transport is fragmentary. Here, we systematically measured aspartate uptake rates in proteoliposomes containing purifi...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Nature Publishing Group UK
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8211817/ https://www.ncbi.nlm.nih.gov/pubmed/34140623 http://dx.doi.org/10.1038/s42003-021-02267-y |
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author | Trinco, Gianluca Arkhipova, Valentina Garaeva, Alisa A. Hutter, Cedric A. J. Seeger, Markus A. Guskov, Albert Slotboom, Dirk J. |
author_facet | Trinco, Gianluca Arkhipova, Valentina Garaeva, Alisa A. Hutter, Cedric A. J. Seeger, Markus A. Guskov, Albert Slotboom, Dirk J. |
author_sort | Trinco, Gianluca |
collection | PubMed |
description | It is well-established that the secondary active transporters Glt(Tk) and Glt(Ph) catalyze coupled uptake of aspartate and three sodium ions, but insight in the kinetic mechanism of transport is fragmentary. Here, we systematically measured aspartate uptake rates in proteoliposomes containing purified Glt(Tk), and derived the rate equation for a mechanism in which two sodium ions bind before and another after aspartate. Re-analysis of existing data on Glt(Ph) using this equation allowed for determination of the turnover number (0.14 s(−1)), without the need for error-prone protein quantification. To overcome the complication that purified transporters may adopt right-side-out or inside-out membrane orientations upon reconstitution, thereby confounding the kinetic analysis, we employed a rapid method using synthetic nanobodies to inactivate one population. Oppositely oriented Glt(Tk) proteins showed the same transport kinetics, consistent with the use of an identical gating element on both sides of the membrane. Our work underlines the value of bona fide transport experiments to reveal mechanistic features of Na(+)-aspartate symport that cannot be observed in detergent solution. Combined with previous pre-equilibrium binding studies, a full kinetic mechanism of structurally characterized aspartate transporters of the SLC1A family is now emerging. |
format | Online Article Text |
id | pubmed-8211817 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-82118172021-07-01 Kinetic mechanism of Na(+)-coupled aspartate transport catalyzed by Glt(Tk) Trinco, Gianluca Arkhipova, Valentina Garaeva, Alisa A. Hutter, Cedric A. J. Seeger, Markus A. Guskov, Albert Slotboom, Dirk J. Commun Biol Article It is well-established that the secondary active transporters Glt(Tk) and Glt(Ph) catalyze coupled uptake of aspartate and three sodium ions, but insight in the kinetic mechanism of transport is fragmentary. Here, we systematically measured aspartate uptake rates in proteoliposomes containing purified Glt(Tk), and derived the rate equation for a mechanism in which two sodium ions bind before and another after aspartate. Re-analysis of existing data on Glt(Ph) using this equation allowed for determination of the turnover number (0.14 s(−1)), without the need for error-prone protein quantification. To overcome the complication that purified transporters may adopt right-side-out or inside-out membrane orientations upon reconstitution, thereby confounding the kinetic analysis, we employed a rapid method using synthetic nanobodies to inactivate one population. Oppositely oriented Glt(Tk) proteins showed the same transport kinetics, consistent with the use of an identical gating element on both sides of the membrane. Our work underlines the value of bona fide transport experiments to reveal mechanistic features of Na(+)-aspartate symport that cannot be observed in detergent solution. Combined with previous pre-equilibrium binding studies, a full kinetic mechanism of structurally characterized aspartate transporters of the SLC1A family is now emerging. Nature Publishing Group UK 2021-06-17 /pmc/articles/PMC8211817/ /pubmed/34140623 http://dx.doi.org/10.1038/s42003-021-02267-y Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Trinco, Gianluca Arkhipova, Valentina Garaeva, Alisa A. Hutter, Cedric A. J. Seeger, Markus A. Guskov, Albert Slotboom, Dirk J. Kinetic mechanism of Na(+)-coupled aspartate transport catalyzed by Glt(Tk) |
title | Kinetic mechanism of Na(+)-coupled aspartate transport catalyzed by Glt(Tk) |
title_full | Kinetic mechanism of Na(+)-coupled aspartate transport catalyzed by Glt(Tk) |
title_fullStr | Kinetic mechanism of Na(+)-coupled aspartate transport catalyzed by Glt(Tk) |
title_full_unstemmed | Kinetic mechanism of Na(+)-coupled aspartate transport catalyzed by Glt(Tk) |
title_short | Kinetic mechanism of Na(+)-coupled aspartate transport catalyzed by Glt(Tk) |
title_sort | kinetic mechanism of na(+)-coupled aspartate transport catalyzed by glt(tk) |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8211817/ https://www.ncbi.nlm.nih.gov/pubmed/34140623 http://dx.doi.org/10.1038/s42003-021-02267-y |
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