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

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dc.contributor.authorSchoppe, Jannis
dc.contributor.authorSchubert, Evelyn
dc.contributor.authorApelbaum, Amir
dc.contributor.authorYavavli, Erdal
dc.contributor.authorBirkholz, Oliver
dc.contributor.authorStephanowitz, Heike
dc.contributor.authorHan, Yaping
dc.contributor.authorPerz, Angela
dc.contributor.authorHofnagel, Oliver
dc.contributor.authorLiu, Fan
dc.contributor.authorPiehler, Jacob
dc.contributor.authorRaunser, Stefan
dc.contributor.authorUngermann, Christian
dc.date.accessioned2021-12-23T16:16:08Z-
dc.date.available2021-12-23T16:16:08Z-
dc.date.issued2021
dc.identifier.urihttps://osnascholar.ub.uni-osnabrueck.de/handle/unios/11740-
dc.description.abstractVesicle 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.
dc.description.sponsorshipDeutsche ForschungsgemeinschaftGerman Research Foundation (DFG) [UN111/5-2, RA1781/2-4]; iBiOs funds of the DFG; Max Planck SocietyMax Planck SocietyFoundation CELLEX; [Sonderforschungsbereich 944]; C. U. was supported by a grant of the Deutsche Forschungsgemeinschaft (UN111/5-2) and the Sonderforschungsbereich 944 (Project P11). J. P. was supported by the Sonderforschungsbereich 944 (Projekt P08, Z01) and the iBiOs funds of the DFG. S. R. received funds from the Max Planck Society and a grant of the Deutsche Forschungsgemeinschaft (RA1781/2-4).
dc.language.isoen
dc.publisherELSEVIER
dc.relation.ispartofJOURNAL OF BIOLOGICAL CHEMISTRY
dc.subjectADAPTER COMPLEX
dc.subjectAPPENDAGE
dc.subjectBINDING-SITE
dc.subjectBiochemistry & Molecular Biology
dc.subjectCOATED VESICLES
dc.subjectCRYO-EM
dc.subjectCRYSTAL-STRUCTURE
dc.subjectMEMBRANE-PROTEINS
dc.subjectRECOGNITION
dc.subjectSTRUCTURAL BASIS
dc.subjectYEAST
dc.titleFlexible open conformation of the AP-3 complex explains its role in cargo recruitment at the Golgi
dc.typejournal article
dc.identifier.doi10.1016/j.jbc.2021.101334
dc.identifier.isiISI:000723119000012
dc.description.volume297
dc.description.issue5
dc.identifier.eissn1083351X
dc.publisher.placeRADARWEG 29, 1043 NX AMSTERDAM, NETHERLANDS
dcterms.isPartOf.abbreviationJ. Biol. Chem.
dcterms.oaStatusGreen Published, gold
crisitem.author.deptFB 05 - Biologie/Chemie-
crisitem.author.deptidfb05-
crisitem.author.orcid0000-0002-2143-2270-
crisitem.author.parentorgUniversität Osnabrück-
crisitem.author.netidPiJa938-
crisitem.author.netidUnCh999-
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