My research project

My project will address the still elusive mechanism of how the Rag GTPases control TORC1 in yeast. Unlike in mammalian cells where the Rag GTPases serve to tether mTORC1 to lysosomes, yeast Rag GTPases control vacuolar TORC1 primarily via an elusive mechanism that neither involves membrane recruitment nor the regulation via the Rheb-orthologous Rhb1 (own unpublished data). An appealing explanation for this mystery relates to the recently discovered non-canonical Rag GTPase-mTORC1 (NC-mTORC1) pathway.

We speculate that also the RagC/D orthologue Gtr2 in yeast locally couples TORC1 to specific substrates. To address this, the doctoral candidate will probe both the interactome and proxisome by MS-coupled Turbo-BioID of wild-type and GDP- or GTP-locked alleles of Rag GTPases. Our preliminary data with Gtr2 identify all known TORC1 effectors, and hitherto unknown candidates. Doctoral candidate will validate them by co-IP and in vitro TORC1 kinase assays followed by MS-based identification of the TORC1 target residues. Using a combination of two-hybrid, co-IP, and Alpha-Fold-assisted modeling, DC2 will map the interaction surfaces on both the Rag GTPase and the novel effectors to denominate a common Rag GTPase interaction motif in the effector proteins. Emerging models will be tested by CRISPR/Cas9-mediated introduction of point mutations in the interaction domains that should affect binding and the biological function of the respective TORC1 effectors.