Supplementary Method

[32P]GTPgS filter binding assay. Dynamin (approximately 2uM) was incubated with 100uM GTPgS spiked with [32P]GTPgS (approximately 10uCi/ml) in GTPase assay buffer (see main paper materials and methods) for 20min. at room temperature. Sample volumes were 50ul. After incubation the samples were applied to a sheet of nitrocellulose in a slot-blotter, under vacuum. The slots to which samples had been applied were then washed with 3 applications of 150ul of ice cold GTPase assay buffer. After washing, the nitrocellulose was removed from the blotter, left to dry in a 37oC air oven and then placed in a phosphoimager cassette. Analysis was carried out using a Molecular Dynamics phosphoimager and ImageQuant software.

References 1. Sever, S., Muhlberg, A. B. & Schmid, S. L. Impairment of dynamin's GAP domain stimulates receptor-mediated endocytosis. Nature 398, 481-486 (1999). 2. Sever, S., Damke, H. & Schmid, S. L. Dynamin:GTP controls the formation of constricted coated pits, the rate limiting step in clathrin-mediated endocytosis. J Cell Biol 150, 1137-1148 (2000). 3. Muhlberg, A. B., Warnock, D. E. & Schmid, S. L. Domain structure and intramolecular regulation of dynamin GTPase. EMBO J. 16, 6676-6683 (1998). 4. Okamoto, P. M., Tripet, B., Litowski, J., Hodges, R. S. & Vallee, R. B. Multiple distinct coiled-coils are involved in dynamin self-assembly. J Biol Chem 274, 10277-10286 (1999). 5. Herskovits, J. S., Burgess, C. C., Obar, R. A. & Vallee, R. B. Effects of mutant rat dynamin on endocytosis. J. Cell Biol. 122, 565-578 (1993). 6. Owen, D. J. et al. Crystal structure of the Amphiphysin-2 SH3 domain and its role in prevention of dynamin ring formation. EMBO J. 17, 5273-5285 (1998). 7. Binns, D. D. et al. The mechanism of GTP hydrolysis by dynamin II: a transient kinetic study. Biochemistry 39, 7188-7196 (2000). 8. Hinshaw, J. E. & Schmid, S. L. Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding. Nature 374, 190-192 (1995). 9. Warnock, D. E., HInshaw, J. E. & Schmid, S. L. Dynamin self-assembly stimulates its GTPase activity. J. Biol. Chem. 271, 22310-22314 (1996).