DC 2: Non-canonical Rag GTPase-TORC1 signalling in yeast

Project part of Work Package 1

Objectives: DC2 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an elusive mechanism that neither involves membrane recruitment nor the regulation via the Rheb-orthologous Rhb1 (own unpublished data).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.

Expected Results: The doctoralcandidate will discover new TORC1 targets in yeast and provide new insight into the conservation of Rag-dependent substrate-specificity determinants.

Prospects of career development: For the past two decades, alumni from the De Virgilio lab have achieved remarkable success, securing positions as group leaders and tenured researchers at prestigious universities, leading projects in both established pharmaceutical companies and innovative startups, and even contributing to the advancement of scientific literature as a journal portfolio manager. The interdisciplinary nature of our Research and Educational program fosters a diverse skillset in our doctoral candidates. This multifaceted training expands their career horizons significantly, opening doors to exciting opportunities in emerging fields such as genetic testing, biomarker analysis, cellular modeling, and drug discovery and development. These highly sought-after skills are invaluable assets in both academic and industrial settings.

Planned secondments: 3 months at Fondazione Telethon (TIGEM) for validation of new yeast regulators of NC-TORC1 in mammalian systems. 3 months at BioSolveIT for drug modelling of RAGs.