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

Sauvageau, Etienne, McCormick, Peter and Lefrancois, Stephane (2017) In vivo monitoring of the recruitment and activation of AP-1 by Arf1 Scientific Reports, 7.

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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.

Item Type: Article
Divisions : Faculty of Health and Medical Sciences > School of Veterinary Medicine
Authors :
NameEmailORCID
Sauvageau, EtienneUNSPECIFIEDUNSPECIFIED
McCormick, Peterp.mccormick@surrey.ac.ukUNSPECIFIED
Lefrancois, StephaneUNSPECIFIEDUNSPECIFIED
Date : 2 August 2017
Identification Number : 10.1038/s41598-017-07493-1
Copyright Disclaimer : © The Author(s) 2017. 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/.
Depositing User : Clive Harris
Date Deposited : 14 Aug 2017 09:03
Last Modified : 14 Aug 2017 09:10
URI: http://epubs.surrey.ac.uk/id/eprint/841904

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