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.
Highlights
From the ‡Department of Plant Systems Biology, University of Hohenheim, 70593 Stuttgart, Germany; §Molecular Plant Biophysics and Biochemistry, Department of Molecular Biology, University of Salzburg, Billrothstrae 11, 5020 Salzburg, Austria; ¶Molecular Plant Physiology, University of Erlangen, Staudtstrae 5, 91058 Erlangen, Germany
Recent work demonstrated a role of adaptor protein complexes (AP)-2 in clathrin-mediated endocytosis (CME), which is the major process by which receptors and other integral membrane proteins and lipids are removed from the plasma membrane and delivered into the endosomal system (8 –11)
We used pairwise experimental setups of wild type compared with ap-3 and wild type compared with ap-4, each repeated twice in label swap experimental design (Fig. 1)
Summary
The composition of the plasma membrane and vacuole is a dynamic and signal-dependent process requiring correct targeting and recycling of specific membrane components. Clathrin-coated vesicles are required for receptor-mediated endocytosis at the plasma membrane. Specificity in this process is achieved through complex formation of the large subunits with the -adaptin. Recent work demonstrated a role of AP-2 in clathrin-mediated endocytosis (CME), which is the major process by which receptors and other integral membrane proteins and lipids are removed from the plasma membrane and delivered into the endosomal system (8 –11). Mutants in the - and ␦-subunit of the AP-3 complex seemed to accumulate plasma membrane proteins in vacuolar compartments, and vacuole biogenesis was disturbed [13]. A complete set of adaptor protein complex 4 (AP-4) subunits has, besides in mammals, only been found in Arabidopsis thaliana [5] and recently, a role of the AP-4 complex in vacuolar protein sorting has been suggested [14]
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