THE ALPHA-CHAIN OF THE AP-2 ADAPTER IS A CLATHRIN BINDING SUBUNIT

We have utilized a rabbit reticulocyte lysate coupled transcription-translation system to express the large subunits of the clathrin associated protein-2 (AP-2) complex so that their individual functions may be studied separately. Appropriate folding of each subunit into N-terminal core and C-terminal appendage domains was confirmed by limited proteolysis. Translated beta 2 subunit bound to both assembled clathrin cages and immobilized clathrin trimers, confirming and extending earlier studies with preparations obtained by chemical denaturation-renaturation. Translated alpha(a) exhibited rapid, reversible and specific binding to clathrin cages. As with native AP-2, proteolysis of alpha(a) bound to clathrin cages released the appendages, while cores were retained. Further digestion revealed a approximate to 29 kDa alpha(a) clathrin-binding fragment that remained tightly cage-associated. Translated alpha(a) also bound to immobilized clathrin trimers, although with greater sensitivity to increasing pH than the translated beta 2 subunit. Clathrin binding by both the alpha and beta subunits is consistent with a bivalent cross-linking model for lattice assembly (Keen, J. H. (1987) Cell Biol, 105, 1989). It also raises the possibility that the alpha clathrin interaction may have other consequences, such as modulation of lattice stability or shape, or other alpha functions.