# The TORC2-Dependent Signaling Network in the Yeast _Saccharomyces cerevisiae_
## 6 Structure, Function and Regulation of Pkc1
### Interaction with Rho1-GTP  
The _S. cerevisiae_ genome encodes five members of the Rho sub-family of Ras-related small GTPases (and Cdc42 is also closely related). Single deletions of each of the other four _RHO_ genes are tolerated, whereas a _rho1\(\Delta\)_ mutation is lethal (218; 219). Based on the phenotypes of a variety of conditional alleles, Rho1 has roles in the establishment of cell polarity, in organization of the actin cytoskeleton, and in bud morphogenesis. Rho1 (209 residues) contains a C-terminal CaaX box, which is subject to geranylgeranylation on the S of the Cys residue, followed by proteolytic cleavage on the C-side of the prenylated Cys and methylation of the now-exposed carboxyl group of the prenylated Cys, providing a lipophilic anchor to the PM (220). Moreover, aside from a single Glu residue, the C-terminal portion of Rho1 is composed exclusively of residues with neutral and positively-charged side chains (-RASLMGKSKTNGKAKKNTTEKKKKKCVLL), facilitating electrostatic interaction with the head groups of negatively-charged phospholipids (221). At the PM, Rho1 is prominently localized at sites of growth, such as the incipient budding site, the bud tip, and the bud neck during cytokinesis (222; 223). The guanine nucleotide exchange factors (GEFs) responsible for production of the GTP-bound state of Rho1 are Rom1 and its paralog Rom2 and a less-related Rho1 GEF Tus1. All three of these GEFs have been linked to activation of Pkc1 and the cell wall integrity pathway under its control (214; 224; 225). Rho1-GTP is downregulated by the GTP-activating proteins (GAPs) Sac7 and its paralog Bag7, Lrg1 and Bem2, as well as by the guanine nucleotide dissociation inhibitor Rdil, which extracts prenylated Rho1 from the membrane. In addition to direct interaction with and activation of Pkc1, the \(\beta\)(1\(\rightarrow\) 3)-glucan synthase Fks1 and its paralog Gsc2/Fks2 both bind and are activated by Rho1-GTP (226; 227). The interplay between the Rho1 GEFs and GAPs, whose activities are, in turn, dictated by their state of phosphorylation (228), determines when and where Rho1-GTP will be available to activate its effectors. Thus, active Rho1 can promote distinct signaling outputs under different conditions.  
In any event, crystal structures and other biochemical evidence document that Rho1-GTP associates exclusively and highly specifically with the \(\alpha\)-helical Hr1 domains in all PKN members (229; 230), including yeast Pkc1 (231). Indeed, it was found that Pkc1 co-immunoprecipitationotides with Rho1 from yeast cell extracts, that recombinant Rho1 associates with Pkc1 in vitro in a GTP-dependent manner, and that the binding of Rho1-GTP is a prerequisite for Pkc1 activity to respond to stimulation by PtdSer (57), as discussed further in the next section. These findings suggest that interaction of the tandem N-terminal Hr1 domains in Pkc1 with Rho1-GTP (Figure 2), aside from tethering Pkc1 to the PM, also enhances its ability to be responsive to its lipid modulators, presumably by causing conformational changes that expose the necessary binding sites and/or by bringing the enzyme to the membrane where they reside.