Assembly of Clathrin Coats Disrupts the Association between Eps15 and AP-2 Adaptors*
- From the Department of Cell Biology, Harvard Medical School and The Center for Blood Research, Boston, Massachusetts 02115
Abstract
Eps15 is a phosphorylation substrate of the epidermal growth factor receptor kinase. In vivo, it is largely found in complex with AP-2, the plasma membrane clathrin adaptor protein complex. Although AP-2 is uniformly distributed across the surface of clathrin-coated pits and vesicles, Eps15 is preferentially found in the rims of endocytic clathrin-coated pits (1). This observation suggests that Eps15 may disengage from AP-2 during coat formation. Here we use two new anti-Eps15 antibodies to show that, contrary to our own earlier suggestion, coated vesicles isolated from brain do not contain detectable amounts of Eps15. Furthermore, when AP-2 complexes that are saturated with Eps15 are used for in vitro assembly of clathrin-AP-2 coats, normal structures are formed that contain the expected amounts of clathrin and AP-2, but the amount of Eps15 present is dramatically lower than that of AP-2. We propose that during coated pit formation, addition of clathrin to the growing edge at the rim of the pit releases Eps15 from AP-2.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grant GM36548, by Special Funds from The Center for Blood Research and the Department of Cell Biology (to H. M. S.), and by a NATO fellowship (to P. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence addressed: Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-278-3140; Fax: 617-278-3131; E-mail: kirchhausen{at}crystal.harvard.edu.
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↵1 A. Contreras and T. Kirchhausen, manuscript in preparation.
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↵2 The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; MES, 4-morpholineethanesulfonic acid.
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- Received October 31, 1997.
- Revision received December 1, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
