SUMMARY/ABSTRACTThe Target of Rapamycin kinase Complex I (TORC1) is a master regulator of cell growth and metabolism ineukaryotes. Work carried out over the last 20 years has shed light on the mechanisms underlying hormoneand amino acid signaling to TORC1 but it is still unclear how other key signals such as glucose starvation aretransmitted to this highly conserved complex. In the last grant period we examined TORC1 signaling inbudding yeast and found that the PKC Gcn2 Sit4 and CK2 signaling pathways work together with the GAPSEAC (GATOR1/2 in humans) to inhibit TORC1 via the highly-conserved GTPases Gtr1/2 (Rag A/B and C/Din humans). This in turn releases TORC1 to move into a single inactive body at the edge of thevacuole/lysosomean event that depends on the TORC1 binding protein Pib2. Building on this frameworkwe now wish to: (1) Identify and characterize the proteins and pathways work in parallel with Gtr1/2 to regulateTORC1 and (2) determine how the conserved Gcn2 PKC Sit4 CK2 pathways regulate TORC1 via Gtr1/2.To address the first question we purified TORC1 from cells exposed to a variety of stress and starvationconditions and identified numerous new interactors. The most notable are the uncharacterizedvacuolar/lysosomal membrane proteins Ydl180w Ygr125w and Syg1 since they bind tightly to TORC1 and arerequired for its movement into or out of the inactive bodies. We now propose to study the function of theseTORC1 binding proteins in detail testing the hypotheses that: (i) Ydl180w is repressor of TORC1 andcompetes with Gtr1/2 to control TORC1 activity (ii) Ygr125w is a sulfur dependent activator of TORC1 and (iii)Syg1 is a phosphate dependent activator of TORC1. To address the second question we purified the majorGtr1/2 regulator SEAC and mapped its phosphorylation in glucose and nitrogen starvation conditions. This ledto the identification of over 150 phosphorylation sites many of which are hyper- or hypo-phosphorylated duringglucose and/or nitrogen starvation. Building on these data we now wish to test the hypothesis that theconserved Gcn2 PKC Sit4 CK2 and other kinases/phosphatases inhibit TORC1 by (de)phosphorylating andactivating SEAC. We also plan to explore a new connection we identified between the key serine synthesisenzymes Ser3/33 (PHGDH in humans) and the TORC1 regulator Pib2testing the hypothesis that Ser3/33activate TORC1 via Pib2 in the presence but not absence of serine. Our proposal is innovative in that westudy new and unexplored aspects of TORC1 signaling using state-of-the-art systems proteomic andbiochemical approaches. The proposed research is significant in that it promises to shed light on themechanisms underlying cell growth control and complex signal integration in an important model organismwith implications for (a) understanding TORC1 related diseases such as cancer epilepsy diabetes andobesity since many of the proteins and pathways under investigation are conserved and (b) developing drugsthat selectively block the growth of pathogenic fungi.