Inhibition of clathrin-coated pit assembly by an Eps15 mutant

Recent data have shown that Eps15, a newly identified component of clathrin -coated pits constitutively associated with the AP-2 complex, is required f or receptor-mediated endocytosis. However, its precise function remains unk nown, Interestingly, Eps15 contains three EH (Eps15-Homology) domains also found in proteins required for the internalization step of endocytosis in y east. Results presented here show that EH domains are required for correct coated pit targeting of Eps15. Furthermore, when cells expressed an Eps15 m utant lacking EH domains, the plasma membrane punctate distribution of both AP-2 and clathrin was lost, implying the absence of coated pits. This was further confirmed by the fact that dynamin, a GTPase found in coated pits, was homogeneously redistributed on the plasma membrane and that endocytosis of transferrin, a specific marker of clathrin-dependent endocytosis, was s trongly inhibited. Altogether, these results strongly suggest a role for Ep s15 in coated pit assembly and more precisely a role for Eps15 in the docki ng of AP-2 onto the plasma membrane. This hypothesis is supported by the fa ct that a GFP fusion protein encoding the ear domain of alpha-adaptin, the AP-2 binding site for Eps15, was efficiently targeted to plasma membrane co ated pits.