# The TORC2-Dependent Signaling Network in the Yeast _Saccharomyces cerevisiae_
## 4 Structure, Function, and Regulation of Ypk1
### C-Terminal Phosphorylation by TORC2
The C-terminal end of _Saccharomyces cerevisiae_ (See) Ypk1 (top line) was aligned with the corresponding segment of Ypk1 orthologs from sensu stricto Saccharomyces species _S.__paradoxus_ (Spa), _S.__kudriazvezii_ (Sku), _S.__mikatae_ (Smi), _S.__arbricola_ (Sar), and _S.__bayanus_ (Sba), more divergent species _Vanderzualtzyma_ _polspyora_ (Vpo), _Zyogosaccharomyces cerevisiae_ (Zro), _Candida glabrata_ (Cgl), _S.__castelli_ (Sca), _S.__kluyveri_ (Skl), _Ashyya gossypii_ (Ago), _Kluveromyces vadtii_ (Kwa), _Lachanca thermotolerans_ (Lth), and _Kluveromyces lactis_ ([PERSON]), SeeYpk2, _Schizosaccharomyces pombe_ ([PERSON]), and _Homo sapiens_ (Hs) SGK1 (bottom line). Identities (white letters on black boxes) and standard conservative substitutions (bold letters on grey boxes) shared between SeeYpk1 and another ortholog are indicated. One-residue gaps (hyphens) and insertions of the indicated length (parentheses) were introduced to maximize alignment of the most distant orthologs. Period (.) indicates the end of the open-reading-frame. The proximal seven-residue insert in HsSGK1 contains two additional potential mTOR2 sites (KSHDSWL). Sequence sources can be found in [39]. (Tor2-Lst8) of TORC2 via its direct physical association with an ancillary subunit, Slm1 (and its paralog Slm2) [119; 120]. In marked contrast, in both animal cells [121; 122] and fission yeast [123; 124], there is compelling evidence that the Avo1 orthologs (mSIN1 and Sin1, respectively) in TORC2 bind and are required for phosphorylation of the Ypk1 orthologs in these organisms (SGK1 and Gad8, respectively). Likewise, others have shown that _S. cerevisiae_ Ypk2 also seems to interact with TORC2 via binding to Avo1 [125]. Moreover, based on cryo-EM, cross-linking MS, and other approaches, the current model of yeast TORC2 structure [15; 21] suggests that Slm1 and Slm2 are peripheral subunits that dock quite far from the catalytic center of the complex (via their interaction with the non-essential subunits Bit61 and/or its paralog Bit2, as well as Avo2 [119; 126]). By contrast, the structural models show that Avo1 is located in close proximity to the active site of the Tor2-Lst8 complex. Furthermore, convincing data show that a sequence shared by Avo1 with both _S. pombe_ Sin1 and mammalian mSIN1, designated the \"conserved region in the middle\" (CRIM), is a discrete domain that adopts a stable ubiquitin-like fold with a prominent acidic loop and is necessary and sufficient for binding of Gad8 and SGK1, respectively [127]. For example, CRIM\({}^{\text{Sin1}}\) fused to a different TORC2 subunit permits Gad8 hydrophobic motif phosphorylation in an _S. pombe sin1_\(\Delta\) mutant. Therefore, it is likely that Ypk1 is recognized by TORC2 by binding to the CRIM element in Avo1. However, it is incontrovertible that, normally, Slm1 and Slm2 are required for TORC2 function, including Ypk1 phosphorylation at its C-terminal sites [26; 28; 29]. So, some essential role, other than substrate delivery, needs to be invoked to explain how these proteins contribute to Ypk1 stimulation. Aside from its CRIM domain, Avo1, which is tightly Tor2-bound, has a C-terminal PtdIns4,5P2-specific PH domain [70; 93] that is reportedly necessary for efficient PM localization of TORC2 [27]. Likewise, both Slm1 (and Slm2) have C-terminal PH domains [128; 129], which have been shown to be specific for binding PtdIns4,5P2 [70; 93; 128]. It is possible, therefore, that Slm1 and Slm2 help reinforce stable PM recruitment of TORC2 and its positioning near Pkh1- and Pkh2-bound eisosomes, which, as discussed above, are located at PM sites enriched for PtdIns4,5P2. Alternatively, or in addition, Slm1 and Slm2 might help localize TORC2 nearby ER-PM junctions, which also control phosphoinositide metabolism and vice-versa [130]; in fact, several of the demonstrated substrates of Ypk1 are endoplasmic reticulum (ER)-localized proteins, as discussed below.