One of the gaps in our understanding of cellular regulation is the detailed mechanisms through which eukaryotic cells detect nutrients in their environment and respond to them in an integrated manner. This is particularly important with respect to the Target of Rapamycin Complex 1 (TorC1), a global regulator that integrates multiple environmental signals and controls a wide range of basic cellular processes in response to them. The growing clinical potential of rapamycin-family drugs for treating tissue rejection, a variety of cancers, progeria and aging-related diseases requires that we accurately understand the mechanistic details of mTorC1 regulation and in turn its control of downstream processes. For example, an incomplete understanding of how mTorC1 regulates downstream events (increases Tissue Factor production which triggers the coagulation cascade) has raised high concern about late stent thrombosis in rapamycin-derivative eluting stents used to treat acute myocardial infarction. The proposed experiments in this application seek to identify such missed or incompletely understood Tor regulatory mechanisms. The plan first utilizes the powerful genetics and well understood cellular and molecular biology of S. cerevisiae to gain a more accurate and in depth understanding of TorC1 regulation by investigating TorC1-dependent (rapamycin-inducible) and -independent mechanisms regulating the GATA transcription activators Gln3 and Gat1. They are among the most widely used reporters of TorC1 activity in non-pathogenic and pathogenic yeast. The mechanistic principles discovered will then be applied to investigate analogous central questions of mTorC1 nutrient sensing and responses to it in mammalian cells in collaboration with a recognized mammalian cell biologist and an expert in the field of mTor regulation. Specifically, the 1st and 2nd Specific Aims test the hypothesis that Gln3 and Gat1 are individually controlled by TorC1-dependent and -independent regulatory pathways with different nitrogen inputs, protein phosphatase (Sit4 and PP2A) and kinase requirements. The phosphatase and kinase requirements for each pathway will be established. The research strategy achieves this goal using gln3 amino acid substitution mutants that genetically isolate the hypothesized pathways from one another, thereby permitting one pathway's components and regulatory mechanisms to be rigorously analyzed without input or interference from the second pathway. Pivotal, proof-of-principle mutants demonstrating the success of this approach have already been isolated and preliminarily characterized. The 3rd Aim challenges and analyzes fundamental assumptions upon which most TorC1/mTorC1 investigations depend and tests predictions that emanate from the exciting new model describing Vam6-Gtr1/2-Ego1/3 activation of TorC1. This information is then used to design and perform experiments that investigate these questions in mammalian cells. If the proposed experiments substantiate the conclusions derived from recently acquired data by my group, significant reconsideration of existing TorC1 &mTorC1 data will be required, and new pathways of nutrient-responsive regulation will likely be identified.

Public Health Relevance

The importance and increasing clinical use of rapamycin-family drugs to treat tissue rejection in transplant patients, multiple types of cancer and potentially progeria make it imperative that we accurately and completely understand how the target of these drugs, the mammalian Target Of Rapamycin Complex 1 (mTorC1), is regulated and in turn regulates downstream cellular processes. Despite impressive advances, one of the least well understood aspects of mTorC1 regulation are the mechanisms through which the presence of nitrogenous nutrients, such as amino acids, are detected and appropriate, integrated responses to them subsequently implemented. The proposed research will significantly contribute to elucidating these mechanisms.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Cellular Signaling and Regulatory Systems Study Section (CSRS)
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Reddy, Michael K
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University of Tennessee Health Science Center
Schools of Medicine
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Georis, Isabelle; Isabelle, Georis; Tate, Jennifer J et al. (2015) Premature termination of GAT1 transcription explains paradoxical negative correlation between nitrogen-responsive mRNA, but constitutive low-level protein production. RNA Biol 12:824-37
Tate, Jennifer J; Georis, Isabelle; Rai, Rajendra et al. (2015) GATA Factor Regulation in Excess Nitrogen Occurs Independently of Gtr-Ego Complex-Dependent TorC1 Activation. G3 (Bethesda) 5:1625-38
Tate, Jennifer J; Rai, Rajendra; Cooper, Terrance G (2015) Nitrogen starvation and TorC1 inhibition differentially affect nuclear localization of the Gln3 and Gat1 transcription factors through the rare glutamine tRNACUG in Saccharomyces cerevisiae. Genetics 199:455-74
Rai, Rajendra; Tate, Jennifer J; Shanmuganatham, Karthik et al. (2015) Nuclear Gln3 Import Is Regulated by Nitrogen Catabolite Repression Whereas Export Is Specifically Regulated by Glutamine. Genetics 201:989-1016
Rai, Rajendra; Tate, Jennifer J; Georis, Isabelle et al. (2014) Constitutive and nitrogen catabolite repression-sensitive production of Gat1 isoforms. J Biol Chem 289:2918-33
Fayyadkazan, Mohammad; Tate, Jennifer J; Vierendeels, Fabienne et al. (2014) Components of Golgi-to-vacuole trafficking are required for nitrogen- and TORC1-responsive regulation of the yeast GATA factors. Microbiologyopen 3:271-87
Rai, Rajendra; Tate, Jennifer J; Shanmuganatham, Karthik et al. (2014) A domain in the transcription activator Gln3 specifically required for rapamycin responsiveness. J Biol Chem 289:18999-9018
Feller, Andre; Georis, Isabelle; Tate, Jennifer J et al. (2013) Alterations in the Ure2 ýýCap domain elicit different GATA factor responses to rapamycin treatment and nitrogen limitation. J Biol Chem 288:1841-55
Tate, Jennifer J; Cooper, Terrance G (2013) Five conditions commonly used to down-regulate tor complex 1 generate different physiological situations exhibiting distinct requirements and outcomes. J Biol Chem 288:27243-62
Kumaraswami, Muthiah; Avanigadda, Lakshmi; Rai, Rajendra et al. (2011) Genetic analysis of phage Mu Mor protein amino acids involved in DNA minor groove binding and conformational changes. J Biol Chem 286:35852-62

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