DOMAIN-STRUCTURE AND INTRAMOLECULAR REGULATION OF DYNAMIN GTPASE

Dynamin is a 100 kDa GTPase required for receptor-mediated endocytosis , functioning as the key regulator of the late stages of clathrin-coat ed vesicle budding, It is specifically targeted to clathrin-coated pit s where it self-assembles into 'collars' required for detachment of co ated vesicles from the plasma membrane. Self-assembly stimulates dynam in GTPase activity. Thus, dynamin-dynamin interactions are critical in regulating its cellular function. We show by crosslinking and analyti cal ultracentrifugation that dynamin is a tetramer, Using limited prot eolysis, we have defined structural domains of dynamin and evaluated t he domain interactions and requirements for self-assembly and GTP bind ing and hydrolysis. We show that dynamin's C-terminal proline-and argi nine-rich domain (PRD) and dynamin's pleckstrin homology (PH) domain a re, respectively, positive and negative regulators of self-assembly an d GTP hydrolysis, Importantly, we have discovered that the a-helical d omain interposed between the PH domain and the PRD interacts with the N-terminal GTPase domain to stimulate GTP hydrolysis. We term this reg ion the GTPase effector domain (GED) of dynamin.