title: 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-3β and ap-4β knock-out mutants. In ap-3β 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-4β mutant aquaporins but also proteins from lipid metabolism were differentially distributed. These proteins also showed differential phosphorylation patterns in ap-3β and ap-4β 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.
authors.affiliation: From the ‡Department of Plant Systems Biology, University of Hohenheim, 70593 Stuttgart, Germany;
authors.forename: Heidi
authors.initials: H
authors.lastname: Pertl-Obermeyer
authors.affiliation: From the ‡Department of Plant Systems Biology, University of Hohenheim, 70593 Stuttgart, Germany;
authors.forename: Xu Na
authors.initials: XN
authors.lastname: Wu
authors.affiliation: From the ‡Department of Plant Systems Biology, University of Hohenheim, 70593 Stuttgart, Germany;
authors.forename: Jens
authors.initials: J
authors.lastname: Schrodt
authors.affiliation: ¶Molecular Plant Physiology, University of Erlangen, Staudtstraβe 5, 91058 Erlangen, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Müdsam
authors.affiliation: §Molecular Plant Biophysics and Biochemistry, Department of Molecular Biology, University of Salzburg, Billrothstraβe 11, 5020 Salzburg, Austria;
authors.forename: Gerhard
authors.initials: G
authors.lastname: Obermeyer
authors.affiliation: From the ‡Department of Plant Systems Biology, University of Hohenheim, 70593 Stuttgart, Germany; wschulze@uni-hohenheim.de.
authors.forename: Waltraud X
authors.initials: WX
authors.lastname: Schulze
mesh_terms: D033963:Adaptor Protein Complex 3 / Q000235:genetics* / Q000378:metabolism; D033964:Adaptor Protein Complex 4 / Q000235:genetics* / Q000378:metabolism; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism; D055786:Gene Knockout Techniques; D009154:Mutation; D010766:Phosphorylation; D021381:Protein Transport; D040901:Proteomics / Q000379:methods*
chemical_list: D033963:Adaptor Protein Complex 3; D033964:Adaptor Protein Complex 4; D029681:Arabidopsis Proteins