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

ABSTRACT: 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 proteins. Specific cargo proteins are expected to be mis-targeted in knock-out mutants of adaptor protein complex components. Thus, we screened for altered distribution profiles across a density gradient of membrane proteins in wild type versus ap-3? and ap-4? knock-out mutants. In ap-3? mutants, especially proteins with transport functions, such as aquaporins and plasma membrane ATPase, as well as vesicle trafficking proteins showed differential protein distribution profiles across the density gradient. In the ap-4? mutant aquaporins but also proteins from lipid metabolism were differentially distributed. These proteins also showed differential phosphorylation patterns in ap-3? and ap-4? compared with wild type. Other proteins, such as receptor kinases were depleted from the AP-3 mutant membrane system, possibly because of degradation after mis-targeting. In AP-4 mutants, membrane fractions were depleted for cytochrome P450 proteins, cell wall proteins and receptor kinases. Analysis of water transport capacity in wild type and mutant mesophyll cells confirmed aquaporins as cargo proteins of AP-3 and AP-4. The combination of organelle density gradients with proteome analysis turned out as a suitable experimental strategy for large-scale analyses of protein trafficking.

SUBMITTER: Pertl-Obermeyer H

PROVIDER: S-EPMC5013305 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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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.

Dataset Information

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

Publications

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

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