Assembly of clathrin coats disrupts the association between Eps15 and AP-2 adaptors.
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.
Cupers
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Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.
Jadhav
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AP
Kirchhausen
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D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001923:Brain Chemistry; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism / Q000648:ultrastructure; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism*; D008854:Microscopy, Electron; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D002135:Calcium-Binding Proteins; D002966:Clathrin; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins