The α chain of the AP-2 adaptor is a clathrin binding subunit

Abstract

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 β2 subunit bound to both assembled clathrin cages and immobilized clathrin trimers, confirming and extending earlier studies with preparations obtained by chemical denaturation-renaturation. Translated α(a) exhibited rapid, reversible and specific binding to clathrin cages. As with native AP-2, proteolysis of α(a) bound to clathrin cages released the appendages, while cores were retained. Further digestion revealed a κ29-kDa α(a) clathrin- binding fragment that remained tightly cage-associated. Translated α(a) also bound to immobilized clathrin trimers, although with greater sensitivity to increasing pH than the translated β2 subunit. Clathrin binding by both the α and β 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 α-clathrin interaction may have other consequences, such as modulation of lattice stability or shape, or other α functions.