Abstract
The domains on clathrin responsible for interaction with the plasma membrane-associated assembly protein AP-2 have been studied using a novel cage binding assay. AP-2 bound to pure clathrin cages but not to coat structures already containing AP that had been prepared by coassembly. Binding to preassembled cages also occurred in the presence of elevated Tris-HCl concentrations (greater than or equal to 200 mM) which block AP-2 interactions with free clathrin. AP-2 interactions with assembled cages could also be distinguished from AP-2 binding to clathrin trimers by sodium tripolyphosphate (NaPPPi), which binds to the alpha subunit of AP-2 (Beck, K., and Keen, J. H. (1991) J. Biol. Chem. 266, 4442-4447). At concentrations of 1-5 mM, NaPPPi blocked clathrin-triskelion binding; in contrast, interactions with cages persisted in the presence of 25 mM NaPPPi. To begin to identify the region(s) of the clathrin molecule important in recognition by AP-2, clathrin cages were proteolyzed to remove heavy chain terminal domains and portions of the distal leg as well as all of the light chains. AP-2 bound to these "clipped cages"; however, unlike the interaction with native cages, binding of AP-2 to clipped cages was sensitive to the lower concentrations of both Tris-HCl and NaPPPi which disrupt interactions of AP-2 with clathrin trimers. Reconstitution of the clipped cages with clathrin light chains did not restore resistance of AP-2 binding to Tris-HCl. We conclude that one binding site for AP-2 resides on the hub and/or proximal part of the clathrin triskelion whereas a second site is likely to involve the terminal domain and/or distal leg; the second site is manifested only in the assembled lattice structure. We suggest that these two distinct binding interactions may be mediated by the two unique large subunits within the AP-2 complex, acting sequentially during assembly.
Highlights
The domains on clathrin responsible for interaction which the processing of lysosomal enzymes is perhaps the with the plasma membrane-associated assembly pro- best characterized
AP-2 bound to pure clathrin cages but not to with which coated membranes are involved, we have been coat structures already containing A P that had been engaged in an analysis of the protein-proteininteractions prepared by coassembly
AP-2 interactions with assembled cages could be distinguished from AP2 binding to clathrin trimers by sodium tripolyphosphate (NaPPPi), which binds to thaesubunit of AP-2
Summary
Would indicate that itis the hub and/or proximal segments of the clathrin triskelion that are involved in AP-2 recognition. Recent studies report fractionationof the large subunits of AP-2 and provide evidence thatthe separated’ CY and (3 (Ahle and Ungewickell, 1989) subunits of the complex exhibit clathrin binding activity althoughneither individually is active in promoting clathrin assembly These and relatedobservations (see “Discussion”) suggest that thebinding sites on the clathrin molecule which we report here may be distinct recognition sites for the a and Consistent with earlier observations (Zaremba and Keen, 1983)two distinct sizes of cages with apparent szo,w values of approximately 230 and 410 S were obtained These structures were entirely stable to buffer A containing 50 mM Tris-HC1, pH 6.5, for the time required (~90-130min) for binding and glycerol gradient ultracentrifugation (Fig. lA, dashed line).When AP preparations were added to preassem-
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