The Chain of the AP-2 Adaptor Is a Clathrin Binding Subunit

doi:10.1074/jbc.270.40.23768

The Chain of the AP-2 Adaptor Is a Clathrin Binding Subunit (*)

From the Department of Pharmacology and the Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

^¶ To whom correspondence should be addressed. Tel.: 215-955-4624; Fax: 215-923-1098; keen{at}lac.jci.tju.edu.

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 exhibited rapid, reversible and specific binding to clathrin cages. As with native AP-2, proteolysis of α bound to clathrin cages released the appendages, while cores were retained. Further digestion revealed a ≈29-kDa α clathrin-binding fragment that remained tightly cage-associated. Translated α 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 [Abstract] raises the possibility that the α-clathrin interaction may have other consequences, such as modulation of lattice stability or shape, or other α functions.

Footnotes

↵^§ Supported by NIH National Research Service Award #CA09662 and the Foerderer Foundation.
↵* This research was funded by National Institutes of Health GM-28526 (to J. H. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

APs

associated proteins

MES

4-morpholineethanesulfonic acid

PAGE

polyacrylamide gel electrophoresis.
↵² I. Gaidarov, Q. Chen and J. H. Keen, manuscript in preparation.
- Received April 28, 1995.
- Revision received July 13, 1995.