# The TORC2-Dependent Signaling Network in the Yeast _Saccharomyces cerevisiae_
## 5 Substrates of Ypk1  
### Control of Aminoglycerophospholipid Asymmetry in the Plasma Membrane Bilayer  
The first bona fide targets of Ypk1 discovered were Fpk1 and Fpk2. They were likely candidates for several reasons. First, Ypk1- and Ypk2-deficient cells display defects in PM-associated functions [32; 46; 111]. Second, Fpk1 was shown to phosphorylate and thereby stimulate at least two PM-localized aminoglycerophospholipid flippases [144]. Flippases are P-type ATPases (class IV) that translocate lipid clients from the outer leaflet to the inner leaflet of the PM bilayer [145]. Third, in a screen for transposon insertion mutations that suppressed the temperature-sensitive growth defect of _ypk1 ts_\(ypk2\Delta\) cells, one insertion that was isolated disrupted the coding sequence for a flippase [46], suggesting that one role of Ypk1 is to negatively regulate flippase function, either directly or indirectly. Finally, as already mentioned above, Fpk1 has three consensus Ypk1 phosphorylation motifs in its N-terminal regulatory domain and its minor paralog Fpk2 has one (Figure 6). Indeed, it was demonstrated that Fpk1 (and Fpk2) are phosphorylated robustly by Ypk1 in vitro and at the predicted sites and, likewise, that Fpk1 is phosphorylated in a Ypk1-dependent manner in vivo at the expected sites [52]. Moreover, it was shown that an Fpk1(S37A T244A S481A) mutant that is immune to Ypk1 phosphorylation is hyperactive both in vitro and in vivo and, conversely, that the absence of Fpk1 and Fpk2 suppresses the temperature-sensitive growth of _ypk1 ts_\(ypk2\Delta\) cells [52]. These results and other more recent findings [26] support the conclusion that TORC2-Ypk1 signaling down regulates the rate of inward aminoglycerophospholipid translocation (mainly PtdEth) by inhibiting the ability of Fpk1 (and Fpk2) to phosphorylate and thereby stimulate the flippases. Moreover, based on the observation that Fpk1(S37A T244A S481A) activity was prevented when sphingolipid synthesis was blocked, revealed that there was a separate Ypk1-independent mechanism for controlling Fpk1 function, which was traced to the need for MITC [52], as was already discussedin a preceding section. Overall, this regulatory circuit, with its interplay between Ypk1 and Fpk1, provides a mechanism to couple the rate of aminophospholipid flipping within the PM to the rate of synthesis of a complex sphingolipid. The more MIPC available, the faster the internalization of outer leaflet PtdEth. Maintaining this balance in PM lipid composition and leaflet distribution appears to be crucial for many aspects of membrane function because defects in flippase activity have been reported to cause problems in the vesicle-mediated transport of proteins in both the endocytic and exocytic pathways [146], in the non-vesicular trafficking of sterols [147], and in maintaining the axis of polarized growth through effects on PM recruitment of Cdc42 [148; 149]. Because counterparts of TORC2, Ypk1, and Fpk1 are found throughout phylogeny (although TORC2 core proteins seem to be missing in photosynthetic organisms [150]), it is possible that analogous regulation occurs in mammals. Indeed, proper distribution of PtdEth (and PtdSer) between the outer and inner leaflets of the plasma membrane in animal cells is necessary for membrane protein activity, for vesicle biogenesis, and for cell signaling, morphology, and movement [151]. Elevated levels of aminoglycerophospholipids (especially PtdSer) in the extracellular leaflet can initiate many responses, including phagocytosis, platelet activation, and apoptotic death [151; 152; 153].