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Conformation of the AP-3 complex explains its role in cargo recruitment at the Golgi

ABSTRACT: Crosslinking mass spectrometry study of an important adapter protein complex at the Golgi AP-3. We performed crosslinking mass spectrometry study using crosslinker DSSO and analyzed the digested peptides by LC-MS. The crosslinking study provided important information the subunit connectivity of AP-3, which facilitated the structural analysis of AP-3 by cyro-EM.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Heike Stephanowitz

LAB HEAD: Christian Ungermann

PROVIDER: PXD028005 | Pride | 2022-02-17

REPOSITORIES: Pride

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			Action	DRS
	HS1028_AP-3_Komplex.fasta	Fasta
	HS1028_results_sample_AP3_Komplex.xlsx	Xlsx
	L1_20190913_HS_1028_FL_AP3_1.raw	Raw
	L1_20190913_HS_1028_FL_AP3_2.raw	Raw
	L1_20190913_HS_1028_FL_AP3_3.raw	Raw

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Publications

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

Schoppe Jannis J Schubert Evelyn E Apelbaum Amir A Yavavli Erdal E Birkholz Oliver O Stephanowitz Heike H Han Yaping Y Perz Angela A Hofnagel Oliver O Liu Fan F Piehler Jacob J Raunser Stefan S Ungermann Christian C

The Journal of biological chemistry 20211022 5

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 pres ...[more]

PMID: 34688652

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

Project description: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.

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