Project Summary/Abstract The mechanistic target of rapamycin complex 1 (mTORC1) licenses cell growth, division, and anabolism in response to changing growth factor and nutrient availability. Underscoring the critical role of mTOR signaling in both cellular and organismal physiology, the mTOR pathway is deregulated in cancer, diabetes, and neurological disorders. Substantial progress has been made in the last two and a half decades on the protein players and molecular mechanisms through which changing growth factor and nutrient availabilities modulate mTORC1 activity. These developments have been driven almost entirely by discoveries made using protein biochemistry and proteomics. However, unanswered questions exist about the molecular regulation of mTORC1 that have remained recalcitrant to these approaches. For example, no known input exists into the positive mTORC1 regulator FLCN, the mechanism or substrate of predicted ubiquitin ligase GATOR2 has not been described, and no known GTPase activating protein (GAP) exists for the RagC GTPase. Additionally, glucose availability and electron transport chain (ETC) flux have energetic-independent sufficiency inputs into mTORC1, but experimental handles on these sensing mechanisms have not been identified. Furthermore, it is not certain studying mTORC1 regulatory inputs in lower organisms will inform regulatory mechanisms in mammals, as recently discovered upstream inputs are not conserved. To address the discovery `bottleneck' that exists for mammalian regulators of mTORC1, we propose the implementation of a functional genomic-based approach for identifying novel regulators of mTORC1 in somatic mammalian cells. We have developed and optimized a highly stringent flow- cytometry protocol for assaying mTORC1 activation states directly. We propose the implementation of a cell sorting-based screening platform to find new regulators of mTORC1, both those that function generally and those that are responsible for signaling glucose availability and continual ETC flux. We propose the following aims: Aim 1. Develop and implement an mTORC1 activity based genetic screening platform ! Aim 2. Identify genes and gene products necessary for signaling glucose availability and ETC flux to mTORC1 ! Aim 3. Genetically and biochemically characterize novel genes and gene products necessary for mTORC1 inactivation with homeostatic perturbations