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In vivo monitoring of the recruitment and activation of AP-1 by Arf1.
Sauvageau, Etienne; McCormick, Peter J; Lefrancois, Stephane.
  • Sauvageau E; Centre INRS-Institut Armand-Frappier, INRS, Laval, Canada, H7V 1B7.
  • McCormick PJ; Faculty of Health and Medical Sciences, School of Veterinary Medicine, University of Surrey, Guildford, GU27XH, UK.
  • Lefrancois S; Centre INRS-Institut Armand-Frappier, INRS, Laval, Canada, H7V 1B7.
Sci Rep ; 7(1): 7148, 2017 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-28769048
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.





Texto completo: Disponível Coleções: Bases de dados internacionais Base de dados: MEDLINE Assunto principal: Fator de Transcrição AP-1 / Fator 1 de Ribosilação do ADP Limite: Humanos Idioma: Inglês Revista: Sci Rep Ano de publicação: 2017 Tipo de documento: Artigo