Identifying novel genes involved in amino acid sensing

TOR (target of rapamycin) is a conserved serine/threonine kinase that forms two key complexes, TORC1 and TORC2 that regulate many different pathways in the cell including cell growth and autophagy. The TOR pathway promotes growth under favorable conditions and activates (derepresses) autophagy during times of less nutrient availability. TORC1 is rapamycin sensitive and is also known to be sensitive to nutrients including amino acids. However, how low amino acid levels are sensed by TORC1 remains poorly understood. Misregulation of these processes can lead to many different human diseases including cancer, metabolic disorders and neurodegeneration. Previous studies by our lab have uncovered the yeast gene WHI2 to be an important potential negative regulator of TOR, and may be part of an amino acid sensing mechanism. Deletion mutants of whi2 have an overgrowth phenotype when grown on low amino acid medium and this overgrowth is sensitive to the drug rapamycin, which indicates that whi2 deletion mutants have a defect in TORC1 regulation. To determine the specificity of WHI2 for low amino acids, phosphorylated S6, a downstream target of TORC1, was used to monitor TOR activity in yeast during various nutrient depravations. My research shows that whi2 deletion mutants are specifically defective in responding to amino acid deprivation, particularly to the amino acid leucine. Deletion mutants of whi2 are still able to regulate TOR in response to low glucose levels, indicating that they do not have a general nutrient-sensing or TOR defect. However, activation of autophagy is impaired in these whi2 deletion mutants in response to both low amino acids and low glucose. To identify additional genes that might be important in amino acid sensing in yeast, 116 genes from the knockout collection with a whi2-like phenotype (including overgrowth on low amino acid media) were screened. An additional 50 genes were identified to be responsible for the low amino acid over growth phenotype. From those, three mutants (Δvma13, Δsrb5 and w1) were confirmed to have WHI2 like TOR activity and are candidates for involvement in the WHI2 pathway. The remaining genes could be involved in parallel pathways contributing to amino acid sensing in yeast