ジャーナル: J Biol Chem / 年: 2021 タイトル: Flexible open conformation of the AP-3 complex explains its role in cargo recruitment at the Golgi. 著者: Jannis Schoppe / Evelyn Schubert / Amir Apelbaum / Erdal Yavavli / Oliver Birkholz / Heike Stephanowitz / Yaping Han / Angela Perz / Oliver Hofnagel / Fan Liu / Jacob Piehler / Stefan Raunser ...著者: Jannis Schoppe / Evelyn Schubert / Amir Apelbaum / Erdal Yavavli / Oliver Birkholz / Heike Stephanowitz / Yaping Han / Angela Perz / Oliver Hofnagel / Fan Liu / Jacob Piehler / Stefan Raunser / Christian Ungermann / 要旨: 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 ...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.
pH: 7.4 / 詳細: 20 mM Hepes pH 7.4 150 mM NaCl 1.5 mM MgCl2
凍結
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 277.15 K / 装置: FEI VITROBOT MARK III 詳細: The sample was blotted using a 2.5 s blotting time and 0 blotting force with 100% humidity at 277.15 K.