title: The alpha 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 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 approximately 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.
authors.affiliation: Department of Pharmacology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
authors.forename: O B
authors.initials: OB
authors.lastname: Goodman
authors.forename: J H
authors.initials: JH
authors.lastname: Keen
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001665:Binding Sites; D002966:Clathrin / Q000378:metabolism*; D066298:In Vitro Techniques; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins