Multilayered Regulation of TORC1 Signaling in Saccharomyces cerevisiae

Abstract

The Target of Rapamycin Complex 1 (TORC1) is a master regulator of cellular growth in eukaryotes. Much insight has been gained into how amino acid and nitrogen levels regulate TORC1 through the escape from rapamycin-induced growth arrest complex (EGOC), and its regulators including the Seh1-associated complex (SEAC). However, other nutrient levels and environmental stresses also act on TORC1, and far less is known about how these signals are transmitted to the complex. In two projects presented here we investigate the osmotic stress signaling network acting on TORC1 as well as regulators of TORC1 agglomeration that act in glucose and nitrogen starvation conditions. In the first investigation, we introduce a novel and reproducible high-throughput assay to screen for genes that affect TORC1 activity in stress conditions. We then use these methods to measure the expression of a TORC1 dependent ribosome biogenesis gene, NSR1, in ~4700 strains from the yeast knock-out library during osmotic stress. We show that 440 of these strains are not able to properly repress NSR1 transcription. The genes identified in the screen form a highly-connected network including 17 proteins that directly interact with TORC1. Secondary rapamycin-based assays performed on these strains allowed us to further characterize the network and show that more than 50 of the proteins act downstream of TORC1. The data derived from this work serve as a resource for our lab and others studying TORC1, and the assay itself is customizable and can be used to characterize any gene regulatory network. In the second study, we sought to further our understanding of the movement of TORC1 from its position distributed across the surface of the vacuolar membrane to a single agglomerate (TORC1-body) in starvation conditions. Previous work suggested that the AMPK in yeast, Snf1, indirectly promoted the phosphorylation of the TORC1 component Kog1. This phosphorylation event sped up aggregation of the complex by ~20 fold. In order to identify other signaling proteins that regulate TORC1-body formation we performed a screen examining the impact that nearly all non-essential kinases and phosphatases in yeast, as well as selected proteins from the previous high-throughput network, have on TORC1 agglomeration. We identified 13 new regulators of TORC1 body formation, including the PI(3)P binding protein Pib2. We also examined the impact of EGOC deletions and mutants had on body formation and discovered that active EGOC was an inhibitor of TORC1 aggregation. Together, we show that seven of the new regulators likely act at or above the EGOC dependent inhibition of TORC1 body formation; while others act at a later step to assist in body formation.