Activation of rapamycin-sensitive TORC1 by endomembrane amino acid transporters

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DESCRIPTION (provided by applicant): The target of rapamycin complex 1 (TORC1) plays a prominent role in a ubiquitously conserved signal transduction cascade that responds to nutrients and growth factor cues to control cell growth and proliferation. Dysregulation of the TORC1 cascade results in multiple types of human malignancies. The central TORC1 members are the Tor protein kinases, which were discovered in the yeast Saccharomyces cerevisiae, a model system that has been crucial in elucidating the TORC1 signaling cascade. Based on its potent antiproliferative activity, rapamycin is in use in several clinical areas including immunosuppression, cancer chemotherapy, and interventional cardiology. The TORC1 pathway remains partially characterized despite much study. We uncovered an unexpected role for the endomembrane vesicular trafficking system in regulating TORC1 signaling in yeast; mutations in protein complexes with roles in vesicular trafficking and protein sorting in combination with mutation of the nonessential Tor1 kinase, render cells inviable or severely growth impaired. We demonstrated that the HOPS complex is required to provide amino acid homeostasis for efficient TORC1 signaling and for normal expression of TORC1-governed genes. These studies also revealed a novel facet in the rapamycin mechanism of action: rapamycin bypasses vesicular trafficking events to activate TORC1- controlled transactivators. Moreover, we showed that mutations in the Ego complex (EGOC) compromise TORC1 signaling and both EGOC and TORC1 activity are required for optimal cell growth. The EGOC possesses evolutionary conserved amino acid-sensitive GTPase subunits and orthologs of these (Gtr1,2 and RagA-D proteins) mediate TORC1 activation in yeast, insect, and mammalian cells. However, the identity of the amino acid sensors and the underlying mechanisms by which amino acids activate EGOC GTPase are unknown. Intriguingly, TORC1 and the EGOC colocalize to endomembranes including those of endosomes and vacuoles, along with vacuolar amino acid transporters. The aims of this proposal are: 1) to elucidate the roles and underlying mechanism by which the endomembrane system enables TORC1 signaling and 2) to determine whether the vacuolar amino acid transporters, which are conserved in mammals, are integrated into the molecular cascade that conveys amino acid signals to evoke TORC1 activation. Our working model is that the endomembrane network provides a platform to facilitate molecular interactions that activate and enable TORC1 signaling. Similar to yeast TORC1, mammalian TORC1 is also localized to endomembranes, in particular lysosomes, which are the counterparts of yeast vacuoles. Thus, we submit that our research will continue to uncover fundamental, conserved TORC1 signaling aspects that could be directly extrapolated to mammalian models and guide pharmacological intervention in select TORC1 pathway defects in human cancer and other diseases. PUBLIC HEALTH RELEVANCE: The rapamycin sensitive-TORC1 is the central component of an evolutionary-conserved signaling cascade that in response to nutrients and growth factors regulates cell growth and proliferation. Defects in mTORC1 signaling lead to cancer and other human diseases. Rapamycin and its analogs are being developed as therapeutic agents for the treatment of a wide range of malignances and could also find indication in cognitive- and aging-related diseases. The model yeast S. cerevisiae has been crucial in the discovery of the TORC1 pathway and elucidation of rapamycin mechanism of action. Characterization of this pathway in yeast is continuing to reveal basic aspects of TORC1 signaling that could foster analogous studies in mammals and lead to improved anticancer therapies.