Dataset Information

AP-4 cargo - Identification of cargo for adaptor protein complexes AP-3 and AP-4 by sucrose gradient profiling

ABSTRACT: Identificatoin of cargo protein of AP-4 using metabolic labeling in label-swap experiments

INSTRUMENT(S): Q Exactive

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Seedling

SUBMITTER: Waltraud Schulze

LAB HEAD: Waltraud Schulze

PROVIDER: PXD003894 | Pride | 2016-07-07

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	18-25_Col-015Nap414N_reziprok.raw	Raw
	18-25_Col014NAP415N_normal_2.raw	Raw
	18-25_Col015NAP414N_reciprocal.raw	Raw
	18-25_ap415NCol-014N_normal.raw	Raw
	25-30_Col-015Nap414N_reziprok.raw	Raw

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Publications

Identification of Cargo for Adaptor Protein (AP) Complexes 3 and 4 by Sucrose Gradient Profiling.

Pertl-Obermeyer Heidi H Wu Xu Na XN Schrodt Jens J Müdsam Christina C Obermeyer Gerhard G Schulze Waltraud X WX

Molecular & cellular proteomics : MCP 20160701 9

Intracellular vesicle trafficking is a fundamental process in eukaryotic cells. It enables cellular polarity and exchange of proteins between subcellular compartments such as the plasma membrane or the vacuole. Adaptor protein complexes participate in the vesicle formation by specific selection of the transported cargo. We investigated the role of the adaptor protein complex 3 (AP-3) and adaptor protein complex 4 (AP-4) in this selection process by screening for AP-3 and AP-4 dependent cargo pro ...[more]

PMID: 27371946

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Project description:The independently folded appendages of the large alpha and beta2 subunits of the endocytic adaptor protein (AP)-2 complex coordinate proper assembly and operation of endocytic components during clathrin-mediated endocytosis. The beta2 subunit appendage contains a common binding site for beta-arrestin or the autosomal recessive hypercholesterolemia (ARH) protein. To determine the importance of this interaction surface in living cells, we used small interfering RNA-based gene silencing. The effect of extinguishing beta2 subunit expression on the internalization of transferrin is considerably weaker than an AP-2 alpha subunit knockdown. We show the mild sorting defect is due to fortuitous substitution of the beta2 chain with the closely related endogenous beta1 subunit of the AP-1 adaptor complex. Simultaneous silencing of both beta1 and beta2 subunit transcripts recapitulates the strong alpha subunit RNA interference (RNAi) phenotype and results in loss of ARH from endocytic clathrin coats. An RNAi-insensitive beta2-yellow fluorescent protein (YFP) expressed in the beta1 + beta2-silenced background restores cellular AP-2 levels, robust transferrin internalization, and ARH colocalization with cell surface clathrin. The importance of the beta appendage platform subdomain over clathrin for precise deposition of ARH at clathrin assembly zones is revealed by a beta2-YFP with a disrupted ARH binding interface, which does not restore ARH colocalization with clathrin. We also show a beta-arrestin 1 mutant, which engages coated structures in the absence of any G protein-coupled receptor stimulation, colocalizes with beta2-YFP and clathrin even in the absence of an operational clathrin binding sequence. These findings argue against ARH and beta-arrestin binding to a site upon the beta2 appendage platform that is later obstructed by polymerized clathrin. We conclude that ARH and beta-arrestin depend on a privileged beta2 appendage site for proper cargo recruitment to clathrin bud sites.