Dynamin 1 supplemental data

This material is supplemental to Marks et al., "GTPase activity of dynamin and resulting conformation change are essential for endocytosis." Nature 2001: 410, 231-235 (.pdf) Please click on one of the links belo w to view the data

(Fig 1a) Reaction progress traces from a GTPase assay o f WT and R725A dynamins with PIP2 lipid nanotubes (LT). [Dyn] is 0.5uM, lipid nanotubes are 0.1mg/ml, [GTP] is 100uM.
(Fig1b) Reaction progress traces of a GTPase assay of WT and R725A dynamins with microtubules (MT). [Dyn] is 0.5mM, microtubules are 0.1mg/ml, [GTP] is 100mM. These assays were performed in low ionic strength buffer (24mM NaCl, 14mM Hepes pH7.4, 1mM MgCl2).
R725 and K694 mutants of dynamin1 show WT GTPase activity

(Fig2s) Filter binding assay of [32P]GTPgS binding to dynamin mutants.
This assay shows qualitatively the binding of [32P]GTPgS to various dynamin mutants. The experiment was done several times and this is a representative experiment done in duplicate.

Binding of mutants to lipid tubules
(Fig3s) Binding of dynamin mutants to lipid tubules.
[Dyn] is 0.5mM, PIP2 lipid nanotubes are 0.1mg/ml. Dynamin bound to lipid tubules after 5 min was assessed by pelleting the tubules in a benchtop ultracentrifuge followed by PAGE and Coomassie staining.
Supplementary Method and References