49] Mapping dynamin interdomain interactions with yeast two-hybrid and glutathione S-transferase pulldown experiments

Abstract

Dynamin is a 100-kDa GTPase that contributes to the formation of clathrin-coated vesicles. Dynamin assembles into multimeric spirals at the necks of budding vesicles. The chapter investigates protein interactions that in all likelihood form the basis for dynamin self-assembly using yeast two-hybrid and glutathione S-transferase (GST) pulldown techniques. These techniques were chosen because they allowed to systematically test binding between individual dynamin protein domains and they allowed to determine the boundaries of those domains with deletion series. The binding interactions between three different domains of dynamin are discovered. The binding data support a new model that describes how dynamin and other members of the dynamin family might assemble into multimeric spirals. In addition, dimerization of the dynamin assembly domain is tested using an in vitro cross-linking protocol. This procedure is based on the ability of nickel to catalyze cross-linking by magnesium monoperoxyphthalic acid (MMPP). Nickel is kept in association with the dynamin protein fragments using the hexahistidines that were also used for protein purification.