Abstract
Clathrin-mediated endocytosis depends upon the coordinated assembly of a large number of discrete clathrin coat components to couple cargo selection with rapid internalization from the cell surface. Accordingly, the heterotetrameric AP-2 adaptor complex binds not only to clathrin and select cargo molecules, but also to an extensive family of endocytic accessory factors and alternate sorting adaptors. Physical associations between accessory proteins and AP-2 occur primarily through DP(F/W) or FXDXF motifs, which engage an interaction surface positioned on the C-terminal platform subdomain of the independently folded alpha subunit appendage. Here, we find that the WXX(F/W)X(D/E) interaction motif found in several endocytic proteins, including synaptojanin 1, stonin 2, AAK1, GAK, and NECAP1, binds a second interaction site on the bilobal alpha appendage, located on the N-terminal beta sandwich subdomain. Both alpha appendage binding sites can be engaged synchronously, and our data reveal that varied assemblies of interaction motifs with different affinities for two sites upon the alpha appendage can provide a mechanism for temporal ordering of endocytic accessory proteins during clathrin-mediated endocytosis.
Highlights
Clathrin-coated vesicles are a major portal of entry into eukaryotic cells, carrying macromolecular nutrients, ligands, select transmembrane proteins, and even viruses into the cell interior from the plasma membrane [1, 2]
In addition to binding YXXØ-type internalization sequences, AP-2 binds, via the independently folded ␣ and 2 subunit appendages that project off the heterotetrameric core, to at least 12 endocytic accessory proteins and alternate adaptors [2, 5, 13]
The sandwich site increases the number of ␣C appendage binding modes, and we propose a model for hierarchical protein recruitment based on the representation of different interaction motifs with different affinities positioned within intrinsically disordered domains of endocytic ␣C appendage-binding proteins
Summary
DNA and Plasmids—The GST2-fused ␣C appendage and its mutants W840A, R905A, and R916A; GST-SJ170M1, GST-SJ170C2, SJ170C2(FD 3 A), and SJ170C2(W 3 A); and GST-stonin 2-(1– 426) have been described previously [14, 16, 18]. Cytosol or PC12 cell lysates were adjusted to 25 mM Hepes-KOH (pH 7.2), 125 mM potassium acetate, 5 mM magnesium acetate, 2 mM EDTA, 2 mM EGTA, and 1 mM dithiothreitol (assay buffer) by addition of a 10ϫ stock and centrifuged at 245,000 ϫ gmax (TLA-100.4 rotor) for 20 min at 4 °C to remove insoluble particulate material. PC12 cell lysates, or purified thrombin-cleaved ␣C appendage (in the presence of 0.1 mg/ml carrier bovine serum albumin) was added, and the tubes were incubated at 4 °C for 60 min with continuous gentle mixing. Superdex S200-purified ␣C appendages were concentrated, and proteins and peptides were prepared for ITC by overnight dialysis against buffer containing 50 mM phosphate (pH 7.5), 100 mM sodium chloride, and 1 mM tris(2-carboxyethyl)phosphine. After washing with HKMgE buffer, the membranes were fixed in 2% glutaraldehyde in HKMgE buffer and prepared for freeze-etch analysis [21]
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