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

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Sauvageau, Etienne, McCormick, Peter J. et Lefrancois, Stephane (2017). In vivo monitoring of the recruitment and activation of AP-1 by Arf1 Scientific Reports , vol. 7 , nº 1. 7148-1-7148-14. DOI: 10.1038/s41598-017-07493-1.

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Résumé

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.

Type de document: Article
Mots-clés libres: -
Centre: Centre INRS-Institut Armand Frappier
Date de dépôt: 27 sept. 2017 16:04
Dernière modification: 27 sept. 2017 16:04
URI: https://espace.inrs.ca/id/eprint/6268

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