# The TORC2-Dependent Signaling Network in the Yeast _Saccharomyces cerevisiae_
## 5 Substrates of Ypk1
### Control of Integral Plasma Membrane Protein Endocytosis  
In addition to modulating the levels and leaflet distribution of glycerophospholipids and the rate of sphingolipid biosynthesis, Ypk1 also influences PM homeostasis by down regulating the rate of endocytosis of integral membrane proteins in at least two ways. Using the \(\alpha\)-factor pheromone receptor Ste2 as a model polytopic membrane protein, which is internalized in response to both its constitutive and agonist-induced ubiquitinylation [178], we found that Ypk1-mediated phosphorylation at two sites blocked the ability of the endocytic adaptor (\(\alpha\)-arrestin) Rod1/Art4 to mediate ligand-induced internalization of Ste2 [25]. The \(\alpha\)-arrestins promote ubiquitinylation of their respective cargo molecules and their engagement by the clathrin-dependent endocytic machinery because these adaptors recruit the membrane-associated HECT-domain ubiquitin ligase Rsp5. The _S. cerevisiae_ genome encodes 14 recognized \(\alpha\)-arrestins, most of which have been implicated in endocytosis and trafficking of a wide variety of nutrient permeases [179; 180; 181]. The majority of these adaptors, including Ldb19/Art1 (1), Ecm21/Art2 (4), Aly2/Art3 (3), Ygr068/Art5 (1), Aly1/Art6 (1), Rog3/Art7 (2), Csr2/Art8 (3), Rim8/Art9 (1), and Ylr392c/Art10 (1), may also be negatively regulated in manner similar to that we documented for Rod1/Art4 because they all contain consensus Ypk1 phospho-acceptor motifs(number indicated in parentheses) that are highly conserved among the sensu stricto species or have been detected as phosphorylated in phospho-proteomic studies, or both.  
The second mechanism by which Ypk1-mediated phosphorylation impedes endocytosis is via inhibition of Fpk1 and Fpk2. Based on the stringent phospho-acceptor site preference we defined for Fpk1 [52], we examined its potential substrates and validated in vitro and in vivo that, aside from the flippases, a bona fide target of Fpk1 is yet another protein kinase, Akl1 [26]. Although considerably longer at its C-terminal end, Akl1 (1108 residues) is closely related in its kinase domain to protein kinases Ark1 (638 residues) and Prk1 (810 residues), which had been shown to modulate the dynamics of actin patch-mediated endocytosis [182]. We found that Akl1 phosphorylates and blocks the function of several actin patch-associated proteins [26]. We demonstrated that Fpk1 phosphorylates Akl1 at two conserved C-terminal sites, which both Ark1 and Prk1 lack, and that these modifications inhibit Akl1 [26]. Thus, under normal growth conditions, Fpk1-mediated inhibition of Akl1 prevents it from interfering with endocytosis. However, under conditions that upregulate TORC2-Ypk1 signaling, Fpk1 is inhibited by Ypk1-dependent phosphorylation, alleviating the Fpk1-mediated inhibition of Akl1, allowing Akl1 to phosphorylate and block the action of proteins needed for endocytic patch function [26].  
So, overall, under stressful conditions that threaten PM integrity and activate TORC2-Ypk1 signaling (including sphingolipid limitation, heat shock, hypotonic conditions), the cell will not further compromise PM function by endocytic removal of PM proteins because Ypk1 action blocks \(\alpha\)-arrestin function and prevents Fpk1 from inhibiting Akl1. Conversely, upon hypertonic shock, where TORC2-Ypk1 signaling is shut off rapidly, PM proteins can be removed as part of the clearance of the \"excess\" membrane created by cell shrinkage because \(\alpha\)-arrestns remain fully functional and Fpk1 is able to prevent Akl1 from acting. Thus, by these mechanisms, TORC2-Ypk1 signaling adjusts the rate of endocytosis to meet the needs of the cell.