Flexible open conformation of the AP-3 complex explains its role in cargo recruitment at the Golgi

Autor(en): Schoppe, Jannis
Schubert, Evelyn
Apelbaum, Amir
Yavavli, Erdal
Birkholz, Oliver
Stephanowitz, Heike
Han, Yaping
Perz, Angela
Hofnagel, Oliver
Liu, Fan
Piehler, Jacob 
Raunser, Stefan
Ungermann, Christian 
Stichwörter: ADAPTER COMPLEX; APPENDAGE; BINDING-SITE; Biochemistry & Molecular Biology; COATED VESICLES; CRYO-EM; CRYSTAL-STRUCTURE; MEMBRANE-PROTEINS; RECOGNITION; STRUCTURAL BASIS; YEAST
Erscheinungsdatum: 2021
Herausgeber: ELSEVIER
Journal: JOURNAL OF BIOLOGICAL CHEMISTRY
Volumen: 297
Ausgabe: 5
Zusammenfassung: 
Vesicle formation at endomembranes requires the selective concentration of cargo by coat proteins. Conserved adapter protein complexes at the Golgi (AP-3), the endosome (AP-1), or the plasma membrane (AP-2) with their conserved core domain and flexible ear domains mediate this function. These complexes also rely on the small GTPase Arf1 and/or specific phosphoinositides for membrane binding. The structural details that influence these processes, however, are still poorly understood. Here we present cryo-EM structures of the full-length stable 300 kDa yeast AP-3 complex. The structures reveal that AP-3 adopts an open conformation in solution, comparable to the membrane-bound conformations of AP-1 or AP-2. This open conformation appears to be far more flexible than AP-1 or AP-2, resulting in compact, intermediate, and stretched subconformations. Mass spectrometrical analysis of the cross-linked AP-3 complex further indicates that the ear domains are flexibly attached to the surface of the complex. Using biochemical reconstitution assays, we also show that efficient AP-3 recruitment to the membrane depends primarily on cargo binding. Once bound to cargo, AP-3 clustered and immobilized cargo molecules, as revealed by single-molecule imaging on polymer-supported membranes. We conclude that its flexible open state may enable AP-3 to bind and collect cargo at the Golgi and could thus allow coordinated vesicle formation at the trans-Golgi upon Arf1 activation.
DOI: 10.1016/j.jbc.2021.101334

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