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Small GTPases of the Rho-family play crucial roles during several cellular processes, e.g. polar growth and secretion. The conformation of these GTPases can change between GDP- and GTP-bound forms resulting in inactive and active enzymes, respectively. The active GTPases are able to interact with a variety of effectors and exchange of GDP and GTP is catalyzed by guanine nucleotide exchange factors (GEF). One unusual GEF is represented by SmgGDS which is neither belonging to the Dbl- nor the Dock180-family of Rho-GEFs. This GEF contains a series of armadillo repeats and is highly conserved within mammals and fungi. This study of Gds1, the SmgGDS homolog in Ustilago maydis, is the first characterization of this GEF in fungi and shows the specific activation of the small GTPase Rac1. Through this interaction, Gds1 also plays a role for the filamentous growth of the phytopathogenic organism U. maydis. The biochemical analysis conducted during this study show that the activation of Rac1 by Gds1 is facilitated in a different manner compared to the GEFs of the Dbl-family. While the members of the Dbl-family employ a specific amino acid residue at position 56 to distinguish between Rac1 and Cdc42, specific recognition by Gds1 is dependent on the first 157 residues of Rac1. The GEF itself uses the two conserved residues asparagine N600 and arginine R603 for its catalytic function.
Gds1, however, also showed an influence on the pathogenicity as a deletion of gds1 resulted in a reduced virulence. Interactions of Gds1 with the membrane proteins Msb2 and Sho1, the MAPKKK Kpp4 and Ubc2, all components of the pathogenic pathways in U. maydis, further show the influence on the pathogenicity. Therefore, it can be assumed that Gds1 might be a potential scaffold protein.
Another part of this study focused on the exocyst complex. This amongst plants, animals and fungi highly conserved effector of small GTPases is formed by eight components. The two landmark proteins Sec3 and Exo70, components of the complex, mark the location for exocytosis. The localization and function of the two proteins is partially regulated by interactions with Rho-GTPases. After localization to the membrane, the exocyst complex enables a successful fusion of secretory vesicles. While Exo70 plays an important role as a landmark protein in Saccharomyces cerevisiae, no similar function was identified for the homolog of U. maydis, which was characterized within this study. Yet a partially functional redundancy to Sec3 cannot be excluded. Interestingly, it was shown that Sec3 of U. maydis interacts with the inactive GDP-bound form of Rac1 and not with the active GTP-bound conformation as it was shown for the Sec3 homologs of other organisms. Detailed analysis of the interplay of Rac1 and Sec3 during polar growth of U. maydis revealed that Sec3 localization is independent of Rac1.