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Haas, Alexander (2008): Regulation of endocytosis and secretion by Rab GTPase activating proteins. Dissertation, LMU München: Fakultät für Biologie



Vesicle traffic in eukaryotic cells is a tightly organized process involving a multitude of regulatory proteins. Key regulators of this traffic are small GTPases called Rabs. With about 60 members in the human genome, they constitute the largest subgroup in the superfamily of Ras like monomeric GTPases. They recruit effector proteins to specific membranes and thus define the identity of organelles. Rabs switch between an active, GTP bound state and an inactive GDP bound state. Key regulators of this conversion are RabGAPs, which accelerate the hydrolysis of bound GTP. All RabGAPs are characterized by the presence of a TBC domain. In the human genome 40 RabGAPs were identified, most of which had not been studied so far. To assign them to their specific Rab proteins, a novel reverse yeast two-hybrid screening method was developed. This identified a GAP for Rab5 termed RabGAP-5. RabGAP-5 stimulated the GTPase activity of Rab5. Its expression inactivated Rab5 and redistributed the Rab5 effector EEA1 from early endosomes to the cytoplasm. RabGAP-5 also blocked the Rab5 dependent uptake of EGF and transferrin from the plasma membrane. When RabGAP-5 was depleted, the size of endosomes was increased, indicating elevated Rab5-GTP levels. Endocytosed EGF was unable to exit the endosome, indicating that trafficking through endosomes was also blocked. To identify GAPs and Rabs implicated in the regulation of early secretory events simultaneously, a second novel screening method was established. It involved the analysis of phenotypes caused by the inactivation of endogenous target Rabs via the overexpression of RabGAPs. Changes in Golgi morphology, ERGIC organisation and the proceeding of secretion were only observed with one candidate RabGAP, the highly conserved protein TBC1D20. TBC1D20 showed activity towards Rab1 and Rab2 in vitro, and acted primarily on Rab1 in vivo. In contrast to all other RabGAPs it has a transmembrane domain, which localises it to the ER. TBC1D20 interacts with RTN-1 on ER membranes. This interaction modulates the activity of TBC1D20. These data indicate a novel function for Rab1 in regulating ER exit, and thus extend the classical view of RabGAPs as regulators of active Rab lifetime.