Budding yeast Rho1 guanosine triphosphatase (GTPase) plays an essential role in polarized cell growth by regulating cell wall glucan synthesis and actin

Budding yeast Rho1 GTPase is an essential regulator of both cell growth and stress response. Active Rho1 serves as an essential regulatory subunit of the β-1,3-glucan synthase complex that has two redundant catalytic subunits, Fks1 and Fks2 (Fig. 1 A; Drgonová et al., 1996; Mazur and Baginsky, 1996; Qadota et al., 1996). In addition, Rho1 regulates the formin Bni1 (Kohno et al., 1996; Tolliday et al., 2002; Dong et al., 2003) and the exocyst subunit Sec3 (Guo et al., 2001), both of which are essential for polarized cell growth and morphogenesis. Rho1 is also essential for the cell wall integrity (CWI) stress response pathway, where it binds and activates Pkc1 (Nonaka et al., 1995; Kamada et al., 1996; Schmitz et al., 2002). Pkc1 terminates polarized cell growth in part by inducing proteasomal degradation of both Bni1 and Sec3 (Kono et al., 2012). Pkc1 also activates a MAPK cascade that leads to the activation of Mpk1/Slt2 to control the transcription of genes important for stress resistance (Fig. 1 A; Lee and Levin, 1992; Irie et al., 1993; Lee et al., 1993; Martín et al., 1993; Kamada et al., 1995; Nonaka et al., 1995; Schmitz et al., 2002). The spatiotemporal regulation of Rho GTPase signaling is governed by the tight control of key regulators that directly affect the GTPase activation cycle, such as guanine nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs). Compared with GEFs, regulation of Rho1 GAP activity is not well understood. There are two major Rho1 GAPs: Lrg1 and Sac7 (Lorberg et al., 2001; Watanabe et al., 2001). Rho1 GAPs have selectivity in downregulating specific Rho1 targets. Specifically, lrg1Δ cells have increased glucan synthesis without affecting Pkc1–Mpk1 activity (Lorberg et al., 2001; Watanabe et al., 2001), and conversely, sac7Δ cells have hyperactivation of Pkc1–Mpk1 without affecting glucan synthesis (Martín et al., 2000; Watanabe et al., 2001; Schmidt et al., 2002). Thus, Lrg1 selectively antagonizes the Rho1–Fks1/Fks2 glucan synthase complex, whereas Sac7 preferentially antagonizes the Rho1–Pkc1 stress response pathway. In a genetic screen, we identified the regulatory B-subunit of the protein phosphatase 2A (PP2A), Cdc55, and its cortical anchoring proteins Zds1/Zds2 as novel regulators of Rho1 signaling. We show that PP2ACdc55 controls the outcome of Rho1 activation without affecting global Rho1 activity. Cortical Zds1/Zds2–PP2ACdc55 promotes cell wall glucan synthesis through inhibition of the Rho1 GAP, Lrg1. The cortical localization of Zds1/Zds2 and Cdc55 depend on Rho1 activity, which suggests a positive feedback loop that maintains cell wall synthesis throughout bud growth. We also show that Rho1 activation of the Pkc1-Mpk1 cascade results in the loss of cortical Cdc55 and Zds1. Collectively, these data suggest that Rho1 activation of polarized cell growth or stress response signaling is maintained by a mutual antagonism between the Rho1– PP2ACdc55 and Rho1–Pkc1 pathways. Budding yeast Rho1 guanosine triphosphatase (GTPase) plays an essential role in polarized cell growth by regulating cell wall glucan synthesis and actin organization. Upon cell wall damage, Rho1 blocks polarized cell growth and repairs the wounds by activating the cell wall integrity (CWI) Pkc1–mitogen-activated protein kinase (MAPK) pathway. A fundamental question is how active Rho1 promotes distinct signaling outputs under different conditions. Here we identified the Zds1/Zds2–protein phosphatase 2ACdc55 (PP2ACdc55) complex as a novel Rho1 effector that regulates Rho1 signaling specificity. Zds1/Zds2–PP2ACdc55 promotes polarized growth and cell wall synthesis by inhibiting Rho1 GTPaseactivating protein (GAP) Lrg1 but inhibits CWI pathway by stabilizing another Rho1 GAP, Sac7, suggesting that active Rho1 is biased toward cell growth over stress response. Conversely, upon cell wall damage, Pkc1–Mpk1 activity inhibits cortical PP2ACdc55, ensuring that Rho1 preferentially activates the CWI pathway for cell wall repair. We propose that PP2ACdc55 specifies Rho1 signaling output and that reciprocal antagonism between Rho1–PP2ACdc55 and Rho1–Pkc1 explains how only one signaling pathway is robustly activated at a time. Zds1/Zds2–PP2ACdc55 complex specifies signaling output from Rho1 GTPase