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Systematic identification of proteins regulated by the TOR Complex 2-dependent kinase Ypk1 in Saccharomyces cerevisiae


Eukaryotic plasma membranes are highly complex structures, both with respect to molecular composition and spatial organization. This composition and organization is essential for cellular function and must be maintained during dramatic shifts in extracellular environment that challenge membrane homeostasis. In the model eukaryote Saccharomyces cerevisiae, signaling by TOR Complex 2 (TORC2) and its effector kinases Ypk1 and Ypk2 is modulated in response to various membrane stresses and cells deficient in TORC2-Ypk signaling have membrane defects, suggesting that this signaling pathway is important for maintaining membrane homeostasis. To understand the molecular mechanisms by which TORC2-Ypk1 signaling regulates membrane homeostasis, I devised a three-tiered genome-wide screen to identify substrates of Ypk kinases. This screen identified 12 novel Ypk substrates in a variety of processes linking TORC2-Ypk signaling to membrane homeostasis including: lipid metabolism, glycerol metabolism, peroxisome function and autophagy. I then performed detailed studies of the regulation of identified substrates Lac1 and Lag1, components of the ceramide synthase complex, a lipid metabolism enzyme. In response to various membrane stresses, TORC2-Ypk1 activates ceramide synthase. This activation is essential in allowing cells to survive these stresses. Interestingly, it appears that activation of ceramide synthase promotes stress survival not by increasing ceramide levels, but rather by preventing the accumulation of ceramide precursors. Lastly, I characterized the TORC2-Ypk regulation of identified substrates Fps1, Gpt2 and Smp1, proteins involved in glycerol metabolism and the cellular response to hyperosmotic shock. I show that TORC2-Ypk is a novel hyperosmotic shock responsive pathway and that TORC2-Ypk coordinately regulates glycerol metabolism substrates to promote accumulation of glycerol, balance osmolarity and promote cellular survival. Thus, this work has identified at least some of the molecular mechanisms by which TORC2-Ypk signaling restores membrane homeostasis in the face of environmental stress.

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