Funktionelle Analyse von Rho-spezifischen Guaninnukleotidaustauschfaktoren in Ustilago maydis

Small GTPases of the Rho-family act as molecular switches in a plethora of cellular processes, including actin reorganisation, cell polarity and vesicular trafficking. This work addresses the problem how a specific GTPase is recognized by its activating proteins and how signalling fidelity is maintained. In Ustilago maydis, the small GTPases Cdc42 and Rac1 share a high degree of sequence homology but exert different cellular functions. Using an in vitro system, it could be demonstrated that the GEF Don1 is a specific activator of Cdc42, while the GEF Cdc24 is an activator of Rac1. Previous works demonstrated that the amino acid at position 56 of Cdc42 and Rac1 is critical for recognition of these GTPases by their specific GEFs. Exchange of the amino acid at position 56 between Cdc42 and Rac1 (Rac1W56F andd Cdc42F56W) lead to a changed activation pattern by Don1 and Cdc24. Rac1W56F is effectively activated by Don1 while Cdc42F56W is a good substrate for Cdc24. Exchange of these amino acid leads also to a switch of the in vivo functions. Rac1W56F is able to replace Cdc42 during cytokinesis, while Cdc42F56W is able to replace Rac1 during polar growth of Ustilago maydis. Expression of chimeric GEFs demonstrated that the activation of Cdc42 or Rac1 by Cdc24 is sufficient to ensure correct signalling fidelity during polar growth. During cytokinesis, signalling fidelity in not only ensured by a specific activation of Cdc42 but also by interaction with specific effectors. A second aspect of this work elucidates the role of the lipid-binding FYVE domain for the function of the GEF Don1. During cytokinesis, Ustialgo maydis forms two distinct septa that are both necessary for proper cell separation. The GEF Don1 activates Cdc42 and thereby regulates formation of the secondary septum. Don1 belongs to the FGD1-family of Rho-GEFs that are characterized by the presence of a C-terminal FYVE domain. This work demonstrates that the FYVE domain of Don1 binds to PtdIns(3)P and is critical for targetting Don1 to endosomal vesicles. These vesicles accumulate at the site of septation, thereby ensuring a high local concentration of Don1. Here, Don1 triggers reorganisation of the actin cytoskeleton during secondary septum formation. Finally, the role of the exocyst complex during cell separation and polar growth of Ustilago maydis was investigated. The landmark protein Sec3 is an effector of both Cdc42 and Rac1. Deletion of Sec3 leads to defects in polar growth and cell separation. Since the exocyst complex is needed for localized fusion of secretory vesicles with the plasma membrane, the dynamic of these vesicles was analysed. It could be shown that in U. maydis, secretory vesicles are transported bidirectionally along microtubules and that both kinesin and dynein motor proteins are involved in these processes.