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an elusive mechanism that neither involves membrane recruitment nor the regulation via the Rheb-orthologous Rhb1 (own unpublished data).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

Eligibility Criteria: Equivalent of Master Degree before the start date and international mobility. The candidate cannot have spent more than 12 months in Switzerland in the last 3 years.

Knowledge and Competence required:

• Qualified, creative, and exceptionally ambitious individuals with a MSc/diploma in life sciences are encouraged to apply.
• Previous experience in the following areas would be an advantage, but not essential: bioinformatics, yeast genetics, standard molecular biological techniques (DNA cloning, construct design, etc.), and fluorescence microscopy.
• Proficiency in English.

Description of the working conditions: The Department of Biology (Biochemistry) at the University of Fribourg invites researchers of all levels to join a vibrant and dynamic academic community. Founded in 1889, the University boasts a rich scientific heritage and a unique bilingual environment where over 10,000 students and 200 professors collaborate across five faculties, including Science and Medicine. This fosters an interdisciplinary atmosphere conducive to groundbreaking research. We are equipped with state-of-the-art facilities, including genomic, metabolomic, and proteomic platforms, confocal and electron microscopy, flow cytometry, and bioinformatics platforms. These advanced tools empower researchers to address complex scientific questions and make significant contributions to the field. The Department of Biology provides a comprehensive support system for its researchers. A well-coordinated graduate program offers guidance and support throughout the academic journey. Furthermore, researchers have free access to essential research infrastructure, including office space, computers, IT support, wet labs, and microscopy. To facilitate cutting-edge research, the department provides access to central facilities for metabolomic, proteomic, imaging, and bioinformatic analyses, with opportunities for training in these specialized areas. The department also actively fosters a stimulating research environment through weekly seminars where students, postdocs, and faculty members engage with renowned speakers, enriching the overall research experience. Researchers interested in joining this vibrant community are encouraged to explore our facilities further:

• Bioinformatic and statistical analyses
• Metabolomics and Proteomics
• Bioimaging
• Cell analytics

Salary: Living allowance – 4372,4€/month (Gross salary including employer contributions), Mobility allowance – 600€/month, Family allowance (if applicable) – 660€/month

Entitlements offered by the recruiting Institution: PhD degree in Biochemistry.

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.

Fondazione Teletogn (TIGEM)

3 months

Pozzuoli, Italy

BioSolveIT

3 months

Sankt Augustin, Germany