SH3-domain-containing proteins function at distinct steps in clathrin-coated vesicle formation

Several SH3-domain-containing proteins have been implicated in endocytosis by virtue of their interactions with dynamin; however, their functions rema in undefined. Here we report the efficient reconstitution of ATP-, GTP-, cy tosol- and dynamin-dependent formation of clathrin-coated vesicles in perme abilized 3T3-L1 cells. The SH3 domains of intersectin, endophilin I, syndap in I and amphiphysin II inhibit coated-vesicle formation in vitro through i nteractions with membrane-associated proteins. Most of the SH3 domains test ed selectively inhibit late events involving membrane fission, but the SH3A domain of intersectin uniquely inhibits intermediate events leading to the formation of constricted coated pits. These results suggest that interacti ons between SH3 domains and their partners function sequentially in endocyt ic coated-vesicle formation.