Entwicklung und Anwendung von Technologie für die Gewinnung und strukturelle Untersuchung von membranverbindenden Multiproteinkomplexen

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Rab/Ypt proteins comprise the largest group of the Ras superfamily of small GTPases and are key regulators of multiple steps of membrane transport. Numerous regulatory factors and effector molecules cooperate with Rab proteins in controlling docking an fusion steps in vesicular transport. Several of them either interact with all Rab proteins or form families of interacting proteins. The aim of this proposal is to investigate the structural basis of Rab interactions with two types of the cycling of Rabs between the membrane and the cytosol an is functionally shared among most of them. The second is the Gyp domain containing protein that belongs to the family of Rab GTPase activating proteins (RabGAP). We propose to solve the structure of a Rab: GDI complex and of a catalytic domain of Gyp1 (a yeast rab GAP). A novel methodology is proposed for the generation of preparative amounts of prenylated proteins in vitro that should enable production of Rab:GDI complex in large amounts for crystallization trials. Using similar approach we plan to generate fluorescently labeled Rab:GDI complexes and analyze kinetics of Rab interaction with membranes in vitro and in vivo. This integrated approach should signifigantly further our understanding of Rab protein function.