In vivo monitoring of the recruitment and activation of AP-1 by Arf1

Abstract AP-1 is a clathrin adaptor recruited to the trans-Golgi Network where it can interact with specific signals found in the cytosolic tail of cargo proteins to incorporate them into clathrin-coated vesicles for trafficking. The small G protein Arf1 regulates the spatiotemporal recruitment of A...

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Autores principales:	Etienne Sauvageau, Peter J. McCormick, Stephane Lefrancois
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/5c25b839927c4dc981e9a61bf3574f94
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spelling	oai:doaj.org-article:5c25b839927c4dc981e9a61bf3574f942021-12-02T15:05:50ZIn vivo monitoring of the recruitment and activation of AP-1 by Arf110.1038/s41598-017-07493-12045-2322https://doaj.org/article/5c25b839927c4dc981e9a61bf3574f942017-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-07493-1https://doaj.org/toc/2045-2322Abstract AP-1 is a clathrin adaptor recruited to the trans-Golgi Network where it can interact with specific signals found in the cytosolic tail of cargo proteins to incorporate them into clathrin-coated vesicles for trafficking. The small G protein Arf1 regulates the spatiotemporal recruitment of AP-1 and also drives a conformational change favoring an interaction with cargo proteins. A recent crystal structure and in vitro experiments highlighted potential residues mediating the AP-1/Arf1 interaction and the unlocking of the complex. We have used bioluminescence resonance energy transfer (BRET) to study the Arf1/AP-1 interaction and AP-1 conformational changes in vivo. We identified novel residues required for this interaction in addition to those predicted in the crystal structure. We also studied the conformational changes in AP-1 driven by Arf1 in live cells and found that opening of the complex is prerequisite for oligomerization. Using Arf1 knockout cells generated by CRISPR/Cas9, we demonstrated that residue 172 in Arf1 is necessary for AP-1 activation and is required for the efficient sorting of the lysosomal protein prosaposin. We have used BRET to study the in vivo activation of AP-1. The advantages of BRET include expressing full-length proteins in their native environment that have been fully post-translationally modified.Etienne SauvageauPeter J. McCormickStephane LefrancoisNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-14 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Etienne Sauvageau Peter J. McCormick Stephane Lefrancois In vivo monitoring of the recruitment and activation of AP-1 by Arf1
description	Abstract AP-1 is a clathrin adaptor recruited to the trans-Golgi Network where it can interact with specific signals found in the cytosolic tail of cargo proteins to incorporate them into clathrin-coated vesicles for trafficking. The small G protein Arf1 regulates the spatiotemporal recruitment of AP-1 and also drives a conformational change favoring an interaction with cargo proteins. A recent crystal structure and in vitro experiments highlighted potential residues mediating the AP-1/Arf1 interaction and the unlocking of the complex. We have used bioluminescence resonance energy transfer (BRET) to study the Arf1/AP-1 interaction and AP-1 conformational changes in vivo. We identified novel residues required for this interaction in addition to those predicted in the crystal structure. We also studied the conformational changes in AP-1 driven by Arf1 in live cells and found that opening of the complex is prerequisite for oligomerization. Using Arf1 knockout cells generated by CRISPR/Cas9, we demonstrated that residue 172 in Arf1 is necessary for AP-1 activation and is required for the efficient sorting of the lysosomal protein prosaposin. We have used BRET to study the in vivo activation of AP-1. The advantages of BRET include expressing full-length proteins in their native environment that have been fully post-translationally modified.
format	article
author	Etienne Sauvageau Peter J. McCormick Stephane Lefrancois
author_facet	Etienne Sauvageau Peter J. McCormick Stephane Lefrancois
author_sort	Etienne Sauvageau
title	In vivo monitoring of the recruitment and activation of AP-1 by Arf1
title_short	In vivo monitoring of the recruitment and activation of AP-1 by Arf1
title_full	In vivo monitoring of the recruitment and activation of AP-1 by Arf1
title_fullStr	In vivo monitoring of the recruitment and activation of AP-1 by Arf1
title_full_unstemmed	In vivo monitoring of the recruitment and activation of AP-1 by Arf1
title_sort	in vivo monitoring of the recruitment and activation of ap-1 by arf1
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/5c25b839927c4dc981e9a61bf3574f94
work_keys_str_mv	AT etiennesauvageau invivomonitoringoftherecruitmentandactivationofap1byarf1 AT peterjmccormick invivomonitoringoftherecruitmentandactivationofap1byarf1 AT stephanelefrancois invivomonitoringoftherecruitmentandactivationofap1byarf1
_version_	1718388706454274048

In vivo monitoring of the recruitment and activation of AP-1 by Arf1

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