Rho-GTPases are essential signalling proteins which regulate a multitude of central cellular processes that are vital for organisms to thrive and adapt to changing environments. Many regulatory networks involving Rho proteins have first been elucidated in the model yeast Saccharomyces cerevisiae, in which Rho5 emerges as a central hub connecting different signalling pathways, such as the responses to cell wall stress, high medium osmolarity, and oxidative stress. In this work, the rapid translocation of Rho5 to mitochondria as reaction to oxidants and glucose starvation was thoroughly investigated. The studies on structure-function relationships was focussed on the C-terminal region of the Rho5 which in other Rho-type GTPases determines their spatio-temporal distribution and contributes to their physiological function. The C-terminal end of these GTPases is considered to be a hypervariable region (HPR) that consists of a polybasic region (PBR) and its preceding amino acid residues, followed by the CAAX motif which becomes prenylated at its cysteine residue. These motifs are conserved in the yeast Rho5 where the PBR contains a serine residue as a putative phosphorylation target. Moreover, Rho5 of S. cerevisiae is characterized by an extension preceding the PBR that comprises 98 amino acid residues. While substitutions of the serine residue within the PBR for either phosphomimetic or non-phosphorylatable residues indicate that it is of minor physiological importance, deletion analyses of the yeast-specific extension showed that it is required for proper localization of Rho5 to the plasma membrane. As expected, substitution of the cysteine residue within the CAAX motif also prevented proper plasma membrane localization, accompanied by a loss of function both with respect to oxidative stress response and glucose starvation. Results from studies employing a trapping-device of GFP-Rho5 to the mitochondrial surface indicate that the GTPase needs to be activated at the plasma membrane by its dimeric GDP/GTP exchange factor (GEF) which is composed of Dck1 and Lmo1, in response to stress conditions. The trimeric DLR complex is then capable of rapidly translocate to mitochondria and fulfil its functions at the organelle. This view was supported by the finding that a constitutively active Rho5 variant restored function when trapped to mitochondria. Interestingly, Rho5 requires the dimeric GEF for the translocation process under oxidative stress while Dck1 and Lmo1 can reach the mitochondria independent from each other. Finally, the human Rho5 homolog Rac1 cannot complement the defects of a rho5 deletion and does not show a proper intracellular distribution, unless its C-terminal end is equipped with the yeast-specific extension. Taken together, the results of this thesis contributed to a better understanding of the structure-function relationships of Rho5 and its human homolog Rac1.
| Identifer | oai:union.ndltd.org:uni-osnabrueck.de/oai:repositorium.ub.uni-osnabrueck.de:urn:nbn:de:gbv:700-202106034890 |
| Date | 03 June 2021 |
| Creators | Sterk, Carolin Christin |
| Contributors | Prof. Dr. Jürgen Heinisch, Prof. Dr. Joost Holthuis |
| Source Sets | Universität Osnabrück |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis |
| Format | application/pdf, application/zip |
| Rights | Attribution-NonCommercial-NoDerivs 3.0 Germany, http://creativecommons.org/licenses/by-nc-nd/3.0/de/ |
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