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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geprüft am 16.05.2024