Modulation of TOR Complex 2 signaling and maintenance of plasma membrane homeostasis in Saccharomyces cerevisiae

Target of Rapamycin (TOR) Complex 2 (TORC2) is a conserved multi-subunit protein kinase associated with the plasma membrane that is an essential regulator of growth. In Saccharomyces cerevisiae, TORC2 regulates the lipid composition and organization of the plasma membrane during normal cell growth and, in turn, responds to environmental insults (such as changes in osmotic conditions) that exert stress on the plasma membrane to maintain homeostasis. Ample genetic and biochemical evidence indicates that TORC2 exerts its effects solely via direct phosphorylation and stimulation of the activity of the downstream protein kinase Ypk1 (and its paralog Ypk2). Ypk1 action modulates plasma membrane lipid homeostasis in multiple ways, including up-regulation of sphingolipid synthesis and inhibition of aminoglycero-phospholipid flipping. Ypk1 also controls glycerol production and efflux, allowing cells to adapt to osmotic changes. Prior work demonstrated that TORC2 phosphorylates Ypk1 at two conserved sequence elements near its C-terminus, dubbed the "turn" and "hydrophobic" motifs. However, this study documents that TORC2 also phosphorylates Ypk1 at four additional C-terminal sites that are also critical for full TORC2-mediated stimulation of Ypk1 activity. Ala substitutions at the four new sites abrogated the ability of Ypk1 to rescue the phenotypes of Ypk1 deficiency, whereas Glu substitutions had no ill effect. Combining the Ala substitutions with an N-terminal mutation (D242A) that has been shown to bypass the need for TORC2 phosphorylation restored the ability to complement a Ypk1-deficient cell. These findings provide new insights about the molecular basis for TORC2-mediated activation of Ypk1. Moreover, TORC2 phosphorylation of Ypk1 changes differentially in response to different plasma membrane stresses; it is elevated in a sustained manner upon sphingolipid depletion, but rapidly and greatly diminished, although only transiently, upon hyperosmotic shock. In this work