# The TORC2-Dependent Signaling Network in the Yeast _Saccharomyces cerevisiae_
## 5 Substrates of Ypk1
### Control of Intracellular Glycerol Concentration  
In _S. cerevisiae_, glycerol-3-phosphate (Glo3P) derived by NADH-dependent reduction of the glycolytic intermediate dihydroxyacetone-phosphate (DHAP) sits at an important metabolic node. Under normal growth conditions, Glo3P can be esterified at its \(sn\)-1 and \(sn\)-2 hydroxyls with fatty acyl groups, catalyzed by Gpt2 and Sct1, to form phosphatidic acid (PtdOH), which, in turn, can be converted to various classes of glycerophospholipids either via Cds1-catalyzed CDP-DAG formation (leading to PtdIns, PtdSer, and PtdGlo) or via Pah1-catalyzed dephosphorylation to DAG (leading to PtdEth and PtdCho via the Kennedy pathway) [165; 166]. Alternatively, DAG can be esterified with a fatty acyl group at its \(sn\)-3 hydroxyl, catalyzed by Dga1 (and Lro1), to generate triacylglycerglycerol that is stored in lipid droplets [167; 168; 169]. However, when subjected to the stress of being placed in hypertonic conditions, Glo3P in the yeast cell has a completely different fate--it is dephosphorylated by two phosphatases (Gpp1/Rhr2 and Gpp2/Hor1) whose cognate genes are upregulated upon hyperosmotic stress [170]. Generation of high internal glycerol is the strategy yeast has evolved to provide a sufficient concentration of an inocuous intracellular osmolyte to combat the loss of water [171]. The effector of a signaling pathway, the HOG (high osmolarity glycerol) response, that controls the processes needed to cope with hyperosmotic stress, including induction of the expression of appropriate genes, is the MAPK Hog1 [172]  
Given the above considerations, it is not surprising that the reduction of DHAP, catalyzed by two paralogous dehydrogenases (Gpd1 and Gpd2) [173], is regulated by mechanisms that allow for a rapid switch between the two distinct metabolic uses for the Glo3P produced. Gpd2, which is located in the cytosol and inside the mitochondrion, is constitutively active, but is inactivated when glucose is limiting via phosphorylation at an N-terminal site by the energy stress-responsive protein kinase Snf1 (mammalian ortholog is AMPK) [137]. Thus, it is ensured that little Glo3P is made under conditions where fatty acids need to be \"burned\" for energy generation rather than diverted into either phospholipid or triacylglycerolglycerol production. In contrast, Gpd1, which is found in the cytosol and inside peroxisomes, is kept inactive via phosphorylation by Ypk1 at a site analogous to that in Gpd2; however, TORC2-Ypk1 signaling is dramatically and rapidly decreased when cells are subjected to hyperosmotic shock [137]. Thus, under conditions were Glo3P is needed for glycerol production, Ypk1-mediated inhibition of Gpd1 function is rapidly alleviated. Furthermore, this effect is potentiated by the fact that, upon hyperosmotic stress, _GPD1_ mRNA and protein expression are markedly upregulated in a Hog1-dependent manner [174], allowing the level of Glo3P for glycerol production to ramp up quickly. However, glycerol will only accumulate inside the cell if a PM-localized channel, the aquaglyceroporin Fps1, closes. A primary mechanism for channel closure involves Hog1-mediated phosphorylation and displacement of its positive regulators, Rgc1 and Rgc2 [175]. However, that effect takes some time and, moreover, Fps1 still closes in response to hyperosmotic shock even in _hog1\(\Delta\)_ cells [176; 177], indicating another mechanism to prevent Fps1-mediated glycerol efflux. Strikingly, in our proteome-wide screen [23], Fps1 was identified as a likely target of Ypk1. This conclusion was corroborated by showing that Fps1 is an authentic Ypk1 substrate in vitro and in vivo and that the open channel state of Fps1 requires its phosphorylation by Ypk1 at three sites [24]. We further showed that under hyperosmotic conditions, where TORC2-Ypk1 signaling is rapidly decreased, Fps1 phosphorylation is lost, causing channel closure, glycerol accumulation, and enhanced survival under hyperosmotic stress [24].