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Journal Articles Scientific Reports Year : 2017

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 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.
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Dates and versions

pasteur-01574612 , version 1 (15-08-2017)

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Attribution - CC BY 4.0

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Etienne Sauvageau, Peter J Mccormick, Stephane Lefrancois. In vivo monitoring of the recruitment and activation of AP-1 by Arf1.. Scientific Reports, 2017, 7 (1), pp.7148. ⟨10.1038/s41598-017-07493-1⟩. ⟨pasteur-01574612⟩

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