INDUCTION OF MUTANT DYNAMIN SPECIFICALLY BLOCKS ENDOCYTIC COATED VESICLE FORMATION

Dynamin is the mammalian homologue to the Drosophila shibire gene prod uct. Mutations in this 100-kD GTPase cause a pleiotropic defect in end ocytosis. To further investigate its role, we generated stable HeLa ce ll lines expressing either wild-type dynamin or a mutant defective in GTP binding and hydrolysis driven by a tightly controlled, tetracyclin e-inducible promoter. Overexpression of wild-type dynamin had no effec t. In contrast, coated pits failed to become constricted and coated ve sicles failed to bud in cells overexpressing mutant dynamin so that en docytosis via both transferrin (Tfn) and EGF receptors was potently in hibited. Coated pit assembly, invagination, and the recruitment of rec eptors into coated pits were unaffected. Other vesicular transport pat hways, including Tfn receptor recycling, Tfn receptor biosynthesis, an d cathepsin D transport to lysosomes via Golgi-derived coated vesicles , were unaffected. Bulk fluid-phase uptake also continued at the same initial rates as wild type. EM immunolocalization showed that membrane -bound dynamin was specifically associated with clathrin-coated pits o n the plasma membrane. Dynamin was also associated with isolated coate d vesicles, suggesting that it plays a role in vesicle budding. Like t he Drosophila shibire mutant, HeLa cells overexpressing mutant dynamin accumulated long tubules, many of which remained connected to the pla sma membrane. We conclude that dynamin is specifically required for en docytic coated vesicle formation, and that its GTP binding and hydroly sis activities are required to form constricted coated pits and, subse quently,for coated vesicle budding.