Die selektive Aktivierung der kleinen GTPasen Cdc42 undRac1 durch Guaninnukleotid-Austauschfaktorenbestimmt deren Signalspezifität in Ustilago maydis

Rho GTPases act as molecular switches within complex signalling networks. They regulate many cellular processes like gene expression, cell proliferation, vesicle trafficking and reorganization of the cytoskeleton. This work addresses the problem how signalling specificity of Rho GTPases is guaranteed. Far more activators and effectors are known than GTPases themselves. Additionally, some effectors can be activated by more than one GTPase. So it needs to be strictly regulated, that a certain stimulus only induces the cognate cellular answer. Despite a very high degree of sequence similarity of the GTPases Cdc42 and Rac1, deletion of the corresponding genes leads to clearly distinguishable phenotypes in U. maydis. Complementation assays with chimeric constructs, consisting of different portions of Cdc42- and Rac1- Sequence, revealed that the region between amino acid residue 41 and 56 is necessary and sufficient to confer signalling specificity to Cdc42 and Rac1. A GTPase with a single amino acid substitution (Rac1W56F) exerts Cdc42 and Rac1 function in vivo. In vitro GEF-Assays demonstrated, that the amino acid substitution at this position leads to a recognition of Rac1W56F by the Cdc42 specific GEF Don1. Therefore activation of Rac1W56F by Don1 is the critical factor that enables this GTPase to replace Cdc42. The Rho GEFs Its1 and Cdc24 were analyzed regarding their cellular functions and their specificity towards the GTPases. In vitro GEF-Assays indicated that Its1 is Cdc42 specific and Cdc24 is a Rac1 specific GEF. Interestingly, the single amino acid exchange at position 56 of the GTPases leads to an altered responsiveness towards Its1 and Cdc24. The analysis of a conditional its1-mutant showed, that this GEF is essential in U. maydis. Mutant cells displayed an altered cell wall morphology, defects in endocytosis and vesicle fusion. Quantitative real time PCR detected two different isoforms of its1-mRNA, which are generated by the usage of alternative polyadenylation signals. Only the long isoform of Its1 possesses the catalytical GEF domain. The generation of a conditional cdc24-mutant revealed that also Cdc24 is an essential GEF. Overexpression leads to the formation of long filaments, via activation of Rac1. Furthermore, an additional regulator of GTPases was investigated in this work. A deletion of the gene rdi1, coding for the Rho-GDI Rdi1, interfered with the filamentation induced by Ras1-, Rac1- and Cdc24-overexpression. Interestingly, it did not inhibit filament formation of the dikaryotic form, which is Rac1-dependent. This shows that two different mechanisms are regulating filamentous growth in U. maydis.