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Genomics 35: 403-404 West M, Kung HF, Kamata T (1990) A novel membrane factor stimulates guanine nucleotide exchange reaction of ras proteins. FEBS Lett 259: 245-248 Schulz B, Banuett F, Dahl M, Schlesinger R, Schafer W, Martin T, Herskowitz I, Kahmann R (1990) The b alleles of U. maydis, whose combinations program pathogenic development, code for polypeptides containing a homeodomain-related motif. Cell 60: 295-306 GTPasen Ustilago maydis GTPases 2014 Wehr, Michaela Wehr Michaela https://doi.org/10.17192/z2014.0410 GEF Kleine GTPasen der Rho-Familie dienen als molekulare Schalter an einer Vielzahl zellulärer Prozesse, wie zum Beispiel der Sekretion und dem polaren Wachstum. Dabei wechseln sie von einer inaktiven GDP-gebundenen zu einer aktiven GTP-gebundenen Konformation, in der sie mit Effektoren interagieren. Der Austausch von GDP zu GTP wird durch Guanin-Nukleotid-Austauschfaktoren (GEFs) katalysiert. Neben den bekannten Rho-GEFs der Dbl- und Dock180-Familie gibt es einen weiteren, ungewöhnlichen GEF, SmgGDS, welcher aus einer Reihe von Armadillo-Wiederholungen besteht. In der vorliegenden Arbeit wurde zum ersten Mal die Funktion dieses in Säugern und Pilzen stark konservierten GEFs in einem pilzlichen Organismus untersucht. Dabei konnte gezeigt werden, dass Gds1 in dem phytopathogenen Pilz U. maydis als ein spezifischer Aktivator der kleinen GTPase Rac1 wirkt und an der Ausbildung des filamentösen Wachstum beteiligt ist. Biochemische Analysen zeigten, dass die Aktivierung von Rac1 durch Gds1 grundsätzlich auf eine andere Art und Weise erfolgt, als es von den GEFs der Dbl-Familie bekannt war. Diese unterscheiden zwischen den in ihrer Sequenz sehr ähnlichen GTPasen Rac1 und Cdc42 durch eine konservierte Aminosäure an Position 56. Gds1 benötigt dagegen für die Spezifitätsbestimmung die ersten 157 Aminosäuren von Rac1. Auf Seiten des GEFs konnte gezeigt werden, dass zwei konservierte Aminosäuren, Asparagin N600 und Arginin R603, eine wichtige Rolle für die katalytische Funktion von Gds1 spielen. Neben dem filamentösen Wachstum, konnte für Gds1 auch eine Funktion während der pathogenen Entwicklung ermittelt werden, da eine Deletion von gds1 zu einer reduzierten Virulenz führt. Zudem konnten Interaktionen von Gds1 mit mehreren Komponenten der pathogenen Entwicklung, wie beispielweise den Plasmamembranproteinen Msb2 und Sho1, der MAPKKK Kpp4 und dessen interagierendem Protein Ubc2, gezeigt werden. Es wird daher vermutet, dass Gds1 während der pathogenen Entwicklung als ein potentielles Gerüstprotein agiert. Im zweiten Teil dieser Arbeit wurde mit dem Exozyst-Komplex ein Effektor kleiner GTPasen untersucht. Dieser aus 8 Proteinen bestehende Komplex ist in Pflanzen, Pilzen und Tieren sehr konserviert. Mit Hilfe der beiden Landmark-Proteine Sec3 und Exo70, die beide Teil des Komplexes sind, wird an der Plasmamembran der Ort für eine Exozytose markiert. Dabei wird die Lokalisation und Funktion der Landmark-Proteine unter anderem durch Interaktionen mit Rho-GTPasen reguliert. Gleichzeitig vermittelt der Exozyst-Komplex die Fusion von sekretorischen Vesikeln mit der Zielmembran. In der vorliegenden Arbeit konnte gezeigt werden, dass es sich bei Exo70 aus U. maydis um kein essentielles Landmark-Protein für die Exozytose handelt. Es erscheint aber wahrscheinlich, dass Exo70 vermutlich eine teilweise redundante Funktion zu Sec3 besitzt. Weiterhin hat sich gezeigt, dass Sec3 aus U. maydis im Gegensatz zu den Varianten anderer Organismen nicht mit der aktiven Form der Rac1 GTPase sondern bevorzugt mit der GDP-gebundenen Form interagiert. Nähere Untersuchungen des Zusammenspiels von Sec3 und der interagierenden GTPase Rac1 während des polaren Wachstums haben ergeben, dass die Lokalisation von Sec3 unabhängig von Rac1 ist. doctoralThesis Exozyst-Komplex Life sciences Biowissenschaften, Biologie monograph 2015-09-23 ppn:364326433 Untersuchung der Regulierung kleiner GTPasen während des filamentösen Wachstums und der Sekretion in Ustilago maydis Ustilago maydis GEF Fachbereich Biologie https://archiv.ub.uni-marburg.de/diss/z2014/0410/cover.png Exocyst complex Biologie Philipps-Universität Marburg Analysis of the regulation of small GTPases during filamentous growth and secretion in Ustilago maydis application/pdf 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. 2014-09-29 German