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Studying the interaction between PEX5 and its full-length cargo proteins in living cells by a novel Försteŕs resonance energy transfer-based competition assay
The import of the majority of soluble peroxisomal proteins is initiated by the interaction between type-1 peroxisomal targeting signals (PTS1) and their receptor PEX5. PTS1 motifs reside at the extreme C-terminus of proteins and consist of a characteristic tripeptide and a modulatory upstream region...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9669585/ https://www.ncbi.nlm.nih.gov/pubmed/36407094 http://dx.doi.org/10.3389/fcell.2022.1026388 |
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author | Hochreiter, Bernhard Malagon-Vina, Hugo Schmid, Johannes A. Berger, Johannes Kunze, Markus |
author_facet | Hochreiter, Bernhard Malagon-Vina, Hugo Schmid, Johannes A. Berger, Johannes Kunze, Markus |
author_sort | Hochreiter, Bernhard |
collection | PubMed |
description | The import of the majority of soluble peroxisomal proteins is initiated by the interaction between type-1 peroxisomal targeting signals (PTS1) and their receptor PEX5. PTS1 motifs reside at the extreme C-terminus of proteins and consist of a characteristic tripeptide and a modulatory upstream region. Various PTS1-PEX5 interactions have been studied by biophysical methods using isolated proteins or in heterologous systems such as two-hybrid assays, but a recently established approach based on Försters resonance energy transfer (FRET) allows a quantifying investigation in living cells. FRET is the radiation-free energy transfer between two fluorophores in close proximity and can be used to estimate the fraction of acceptor molecules bound to a donor molecule. For PTS1-PEX5 this method relies on the measurement of FRET-efficiency between the PTS1-binding TPR-domain of PEX5 tagged with mCherry and EGFP fused to a PTS1 peptide. However, this method is less suitable for binding partners with low affinity and protein complexes involving large proteins such as the interaction between full-length PTS1-carrying cargo proteins and PEX5. To overcome this limitation, we introduce a life-cell competition assay based on the same FRET approach but including a fusion protein of Cerulean with the protein of interest as a competitor. After implementing the mathematical description of competitive binding experiments into a fitting algorithm, we demonstrate the functionality of this approach using known interaction partners, its ability to circumvent previous limitations of FRET-measurements and its ability to study the interaction between PEX5 and its full-length cargo proteins. We find that some proteins (SCP2 and AGXT) bind PEX5 with higher affinity than their PTS1-peptides alone, but other proteins (ACOX3, DAO, PerCR-SRL) bind with lower but reasonable affinity, whereas GSTK1 binds with very low affinity. This binding strength was not increased upon elongating the PEX5 TPR-domain at its N-terminus, PEX5(N-TPR), although it interacts specifically with the N-terminal domain of PEX14. Finally, we demonstrate that the latter reduces the interaction strength between PEX5(N-TPR) and PTS1 by a dose-dependent but apparently non-competitive mechanism. Altogether, this demonstrates the power of this novel FRET-based competition approach for studying cargo recognition by PEX5 and protein complexes including large proteins in general. |
format | Online Article Text |
id | pubmed-9669585 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-96695852022-11-18 Studying the interaction between PEX5 and its full-length cargo proteins in living cells by a novel Försteŕs resonance energy transfer-based competition assay Hochreiter, Bernhard Malagon-Vina, Hugo Schmid, Johannes A. Berger, Johannes Kunze, Markus Front Cell Dev Biol Cell and Developmental Biology The import of the majority of soluble peroxisomal proteins is initiated by the interaction between type-1 peroxisomal targeting signals (PTS1) and their receptor PEX5. PTS1 motifs reside at the extreme C-terminus of proteins and consist of a characteristic tripeptide and a modulatory upstream region. Various PTS1-PEX5 interactions have been studied by biophysical methods using isolated proteins or in heterologous systems such as two-hybrid assays, but a recently established approach based on Försters resonance energy transfer (FRET) allows a quantifying investigation in living cells. FRET is the radiation-free energy transfer between two fluorophores in close proximity and can be used to estimate the fraction of acceptor molecules bound to a donor molecule. For PTS1-PEX5 this method relies on the measurement of FRET-efficiency between the PTS1-binding TPR-domain of PEX5 tagged with mCherry and EGFP fused to a PTS1 peptide. However, this method is less suitable for binding partners with low affinity and protein complexes involving large proteins such as the interaction between full-length PTS1-carrying cargo proteins and PEX5. To overcome this limitation, we introduce a life-cell competition assay based on the same FRET approach but including a fusion protein of Cerulean with the protein of interest as a competitor. After implementing the mathematical description of competitive binding experiments into a fitting algorithm, we demonstrate the functionality of this approach using known interaction partners, its ability to circumvent previous limitations of FRET-measurements and its ability to study the interaction between PEX5 and its full-length cargo proteins. We find that some proteins (SCP2 and AGXT) bind PEX5 with higher affinity than their PTS1-peptides alone, but other proteins (ACOX3, DAO, PerCR-SRL) bind with lower but reasonable affinity, whereas GSTK1 binds with very low affinity. This binding strength was not increased upon elongating the PEX5 TPR-domain at its N-terminus, PEX5(N-TPR), although it interacts specifically with the N-terminal domain of PEX14. Finally, we demonstrate that the latter reduces the interaction strength between PEX5(N-TPR) and PTS1 by a dose-dependent but apparently non-competitive mechanism. Altogether, this demonstrates the power of this novel FRET-based competition approach for studying cargo recognition by PEX5 and protein complexes including large proteins in general. Frontiers Media S.A. 2022-11-03 /pmc/articles/PMC9669585/ /pubmed/36407094 http://dx.doi.org/10.3389/fcell.2022.1026388 Text en Copyright © 2022 Hochreiter, Malagon-Vina, Schmid, Berger and Kunze. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Cell and Developmental Biology Hochreiter, Bernhard Malagon-Vina, Hugo Schmid, Johannes A. Berger, Johannes Kunze, Markus Studying the interaction between PEX5 and its full-length cargo proteins in living cells by a novel Försteŕs resonance energy transfer-based competition assay |
title | Studying the interaction between PEX5 and its full-length cargo proteins in living cells by a novel Försteŕs resonance energy transfer-based competition assay |
title_full | Studying the interaction between PEX5 and its full-length cargo proteins in living cells by a novel Försteŕs resonance energy transfer-based competition assay |
title_fullStr | Studying the interaction between PEX5 and its full-length cargo proteins in living cells by a novel Försteŕs resonance energy transfer-based competition assay |
title_full_unstemmed | Studying the interaction between PEX5 and its full-length cargo proteins in living cells by a novel Försteŕs resonance energy transfer-based competition assay |
title_short | Studying the interaction between PEX5 and its full-length cargo proteins in living cells by a novel Försteŕs resonance energy transfer-based competition assay |
title_sort | studying the interaction between pex5 and its full-length cargo proteins in living cells by a novel försteŕs resonance energy transfer-based competition assay |
topic | Cell and Developmental Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9669585/ https://www.ncbi.nlm.nih.gov/pubmed/36407094 http://dx.doi.org/10.3389/fcell.2022.1026388 |
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