Assembly of Clathrin Coats Disrupts the Association between Eps15 and AP-2 Adaptors

Philippe Cupers,Ashutosh P Jadhav,Tomas Kirchhausen

doi:10.1074/jbc.273.4.1847

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.

Highlights

During the past decade, several groups have extensively studied the protein components and the mechanism responsible for the formation of clathrin-coated pits
To further examine how Eps15 behaves during the process of coat formation, we made 1:1 complexes between recombinant Eps15 and purified AP-2 and determined their ability to incorporate into clathrin coats assembled in vitro
We have shown that the interaction between AP-2 complexes and clathrin is severely disrupted by the coat assembly process

Summary

Introduction

Several groups have extensively studied the protein components and the mechanism responsible for the formation of clathrin-coated pits. The membrane-bound Eps15/AP-2 complexes are mainly found within clathrin-coated pits [1, 10], usually at the rims and growing edges of coated pits [1]. To further examine how Eps15 behaves during the process of coat formation, we made 1:1 complexes between recombinant Eps15 and purified AP-2 and determined their ability to incorporate into clathrin coats assembled in vitro.

Results

Conclusion