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A biophysical approach to monitor Arf1 mediated AP-1 activation in vivo

Sauvageau, Etienne; Lefrancois, Stephane; McCormick, Peter J. (2016). A biophysical approach to monitor Arf1 mediated AP-1 activation in vivo In: Annual Meeting of the American-Society-for-Cell-Biology (ASCB), December 03-07, 2016, San Francisco.

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Résumé

Dysfunction of vesicular traffic within the trans‐Golgi network (TGN)‐endosome‐lysosome system leads to a variety of human diseases. Moreover, several bacterial toxins and viruses hijack this transport system in order to infect cells. Therefore fundamental understanding of the mechanisms regulating this trafficking pathway could lead to novel therapeutic approaches to several diseases. AP‐1 is a clathrin adaptor complex that sorts cargo between the TGN and endosomes. Membrane‐bound AP‐1 interacts with the cytosolic tail of transmembrane proteins while also recruiting clathrin and inducing the formation of clathrin‐coated vesicles (CCVs). AP‐1 recruitment to membranes is mediated by the small GTPase Arf1. A recently published crystal structure of the AP‐1 core in complex with GTP‐bound Arf1, suggests a mechanism by which these proteins interact and the conformational changes associated with AP‐1 activation. So far, most of the experiments leading to the model of AP‐1 activation have been performed with purified proteins in vitro. We propose to use the bioluminescence resonance energy transfer (BRET) technology which will allow us to study the Arf1/AP‐1 interaction, the monomeric/oligomeric states of AP‐1, and the conformational changes inside AP‐1 in living cells. BRET titration curves between Arf1‐RlucII and the AP‐1 subunit β1 and γ fused to GFP10 confirmed the formation of an AP‐1/Arf1 complex in living cells. Mutations in Arf1, β1 and γ subunit previously shown to disrupt their interaction in vitro, lead to a loss of BRET signal in vivo. BRET titration curves between β1‐Rluc and β1‐GFP10 confirms the existence of AP‐1 oligomeric complexes in vivo and the recruitment and activation of AP‐1 by Arf1 increase its oligomerization. Changes in BRET between ϒ1‐Rluc and μ1‐GFP10 reveal conformational modifications of the AP‐1 complex related to its activation state and the importance of Arf1 in this process. Finally, rescue experiments in CRISPR generated Arf1, γ or β1 KO cells were performed to study the functional consequences of preventing AP‐1 binding to Arf1 and its activation. These results suggest that BRET is well suited to study the interaction between these proteins and how they are activated in living cells.

Type de document: Document issu d'une conférence ou d'un atelier
Informations complémentaires: Affiche scientifique Molecular Biology of the Cell 27: P1286
Mots-clés libres: -
Centre: Centre INRS-Institut Armand Frappier
Date de dépôt: 05 avr. 2019 15:51
Dernière modification: 05 avr. 2019 15:51
URI: http://espace.inrs.ca/id/eprint/5580

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