Characterization and physiological role of the yeast Dck1/Lmo1/Rho5 complex

• Applicant: Professor Dr. Jürgen J. Heinisch
• Subject Area: Cell Biology
• Term: from 2016 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 287022530

Small GTPases and their regulatory proteins play essential roles in eukaryotic cells from yeast to humans. Budding yeast has six Rho-type GTPases, Rho1-Rho5 and Cdc42. While five of those have been studied in considerable detail, literature on Rho5 is scarce. We previously demonstrated that Rho5 affects cell wall integrity and recently identified Dck1/Lmo1 as its putative dimeric GEF (GDP/GTP exchange factor). Dck1 and Lmo1 are homologs of the human DOCK and ELMO proteins, which function in the organization of the actin cytoskeleton and are related to neurodenerative diseases. Thus, malfunctions of the mammalian Rho5 homolog Rac1 have been implicated in neurobiological disorders and cell death. We found that the Dck1/Lmo1/Rho5 (DLR) complex in the model yeast Saccharomyces cerevisiae relocates rapidly from the plasma membrane to mitochondria under oxidative stress and mediates both mitophagy and apoptotic cell death. The work proposed here will investigate the roles of the DLR complex in oxidative stress response, mitophagy and apoptosis, and in regulating cytokinesis, using S. cerevisiae and Kluyveromyes lactis as yeast model systems. We will address three interconnected questions:1. How is the activity of the DLR complex regulated at the molecular level and integrated into cellular signal transduction under oxidative stress? - Here, we will first determine if Dck1/Lmo1 are a specific GEF for Rho5. We will then eliminate ubiquitination site in Rho5 and determine whether it is necessary for its activity in mitophagy. Trapping experiments to different membranes in vivo will clarify the role of mitochondrial localization of the complex for mitophagy and apoptosis. Genetic screens will then identify new up- and downstream signaling components which act on the complex.2. How are cell wall integrity signaling and DLR function coordinated in mitophagy? - This part will focus on the role of Wsc1-sensor clustering in signaling to mitophagy and ask, whether Slt2 kinase activity is necessary to mediate the signal. Epistasis analyses will reveal, whether the two signaling pathways converge or act in parallel to each other.3. Is the activity of the DLR complex required for cytokinesis? - Evidence for a role of the DLR complex in organizing the actin cytoskeleton and the actomyosin ring during cytokinesis in yeast is scarce. Morphological defects observed in the respiratory-oriented milk yeast K. lactis in a mutant lacking KlDck1 make it a good model to study effects on cytokinesis in parallel to the experiments in S. cerevisiae. In summary, since the multiple roles of Rho-GTPases in cell death, mitophagy and cytokinesis are still poorly understood, these model studies in yeast are expected to yield new insights with possible implications for human biology and medicine.

• DFG Programme: Research Grants