# The TORC2-Dependent Signaling Network in the Yeast _Saccharomyces cerevisiae_
## 5 Substrates of Ypk1
### Control of Sphingolipid Biosynthesis  
The first clue that Ypk1 action was centrally involved in regulating sphingolipid production came from the fact that _SLI2_, one of the genes isolated in a screen for dosage suppressors of \"SphingoLipid inhibition by ISP-1\", was isogenic to _YPK1_[154]. It had been established previously that the antibiotic ISP-1, more commonly known as myriocin [155; 156; 157], inhibits eukaryotic cell growth because it is a transition state mimic that potentially blocks l-serine:palmitoyl-CoA C-palmitoyltransferase (decarboxylolating) (SPT; Lcb1-Lcb2-Tsc3 heterotrimer in yeast), the first enzyme unique to the sphingolipid biosynthetic pathway in all eukaryotes [101; 158; 159; 160]. Overproduction of a Ypk1 mutant lacking catalytic activity did not suppress growth inhibition by myriocin. These findings suggested that elevating the amount of Ypk1 activity was able to somehow overcome the limitation for sphingolipids caused by reducing the rate of their synthesis with the inhibitor; however, no targets of Ypk1 were identified. The next evidence that a protein kinase downstream of TORC2 was important for controlling the rate of sphingolipid production came from the observations, first, that an _av03-30\({}^{ts}\)_ mutation, which cripples TORC2 function at the non-permissive temperature, also greatly diminished the amount of ceramides in the cell under the same conditions and, second, that expression of the hyperactive D239A allele of the Ypk1 paralog Ypk2 was able to suppress the growth defect of _av03-30\({}^{ts}\)_ cells at the restrictive temperature [68]. It had already been established that Ypk2 was a downstream target of TORC2 [67]. These findings suggested that sphingolipid depletion stimulated TORC2-dependent phosphorylation of Ypk2, which, in turn, somehow promoted more sphingolipid production; however, no direct substrate of Ypk2 was pinpointed. In 2002, we carried out a comprehensive screen for both dosage (gain-of-function) suppressors and transposon insertion (loss-of-function) suppressors of the temperature-sensitive lethality of a _ypk1\({}^{ts}\)__\(ypk2\Delta\)_ strain [46]. The former potentially represent substrates whose function requires stimulation by Ypk1-mediated phosphorylation and the latter potentially represent substrates whose function requires inhibition by Ypk1-mediated phosphorylation. One of the best suppressors was a transposon insertion in open-reading-frame YLR350w (now designated _ORM2_). However, none of the suppressors were able to rescue the inviability of a _ypk1\(\Delta\)__\(ypk2\Delta\)_ double mutant, emphasizing the importance of the residual Ypk1 activity in the _ypk1\({}^{ts}\)__\(ypk2\Delta\)_ strain. That observation, combined with the fact that no single suppressor or combination of two different suppressors was able to fully rescue either the _ypk1\({}^{ts}\)__\(ypk2\Delta\)_ cells or the _ypk1\(\Delta\)__\(ypk2\Delta\)_ mutant, implied that Ypk1 must have multiple targets. At that time, Orm2 and its paralog Orm1 were so-called \"pioneer proteins\" because nothing was known about them, or their orthologs in other organisms, with regard to their molecular function. However, that picture changed dramatically in 2010, when it was demonstrated that Orm1 and Orm2 are small, ER-localized tetraspannies that physically associate with and act as negative regulators of SPTand that, upon sphingolipid depletion, both Orm1 and Orm2 become heavily phosphorylated, which alleviates their inhibitory effect on SPT [161]. Moreover, Orm2 appears to be the major isoform [162; 163]. These findings immediately suggested that Ypk1 is the protein kinase responsible for phosphorylating Orm1 and Orm2 when sphingolipids become limiting. Indeed, we established that myriocin-induced hyperphosphorylation of Orm1 and Orm2 does not occur in \(ypk1\Delta\) cells, that immuno-purified Ypk1 phosphorylated Orm1 and Orm2 robustly in vitro and did so exclusively on the residues that correspond to its myriocin-induced sites [22]. These conclusions were corroborated by others [28].