Dissection of the TORC1 Signaling Network in Yeast

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SUMMARY/ABSTRACT The Target of Rapamycin kinase Complex I (TORC1) is a master regulator of cell growth and metabolism in eukaryotes. Work carried out over the last 20 years has shed light on the mechanisms underlying hormone and amino acid signaling to TORC1, but it is still unclear how other key signals, such as glucose starvation, are transmitted to this highly conserved complex. In the last grant period, we examined TORC1 signaling in budding yeast, and found that the PKC, Gcn2, Sit4, and CK2 signaling pathways work together with the GAP SEAC (GATOR1/2 in humans) to inhibit TORC1 via the highly-conserved GTPases, Gtr1/2 (Rag A/B and C/D in humans). This in turn releases TORC1 to move into a single inactive body at the edge of the vacuole/lysosome—an event that depends on the TORC1 binding protein, Pib2. Building on this framework, we now wish to: (1) Identify and characterize the proteins and pathways work in parallel with Gtr1/2 to regulate TORC1, 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 starvation conditions, and identified numerous new interactors. The most notable are the uncharacterized vacuolar/lysosomal membrane proteins Ydl180w, Ygr125w and Syg1, since they bind tightly to TORC1 and are required for its movement into, or out of, the inactive bodies. We now propose to study the function of these TORC1 binding proteins in detail, testing the hypotheses that: (i) Ydl180w is repressor of TORC1 and competes 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 major Gtr1/2 regulator SEAC, and mapped its phosphorylation in glucose and nitrogen starvation conditions. This led to the identification of over 150 phosphorylation sites, many of which are hyper- or hypo-phosphorylated during glucose and/or nitrogen starvation. Building on these data, we now wish to test the hypothesis that the conserved Gcn2, PKC, Sit4, CK2, and other kinases/phosphatases inhibit TORC1 by (de)phosphorylating and activating SEAC. We also plan to explore a new connection we identified between the key serine synthesis enzymes Ser3/33 (PHGDH in humans) and the TORC1 regulator Pib2—testing the hypothesis that Ser3/33 activate TORC1 via Pib2 in the presence, but not absence, of serine. Our proposal is innovative in that we study new and unexplored aspects of TORC1 signaling using state-of-the-art systems, proteomic, and biochemical approaches. The proposed research is significant in that it promises to shed light on the mechanisms underlying cell growth control, and complex signal integration, in an important model organism— with implications for (a) understanding TORC1 related diseases such as cancer, epilepsy, diabetes and obesity, since many of the proteins and pathways under investigation are conserved and (b) developing drugs that selectively block the growth of pathogenic fungi.