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. Author manuscript; available in PMC: 2013 Mar 30.
Published in final edited form as: Mol Cell. 2012 Mar 22;45(6):743–753. doi: 10.1016/j.molcel.2012.01.028

The TOR Complex 1 Is a Direct Target of Rho1 GTPase

Gonghong Yan 1, Yumei Lai 1, Yu Jiang 1,*
PMCID: PMC3334367  NIHMSID: NIHMS361183  PMID: 22445487

Summary

The TOR complex 1 (TORC1) in yeast is regulated by various stress conditions. However, the underlying mechanism is poorly understood. In this study, we show that stresses affect TORC1 function through Rho1, a member of Rho-family GTPases. Upon activation by stresses, Rho1 binds directly to Kog1, a unique component of TORC1, resulting in downregulation of TORC1 activity and disruption of its membrane association. The binding also triggers the release and activation of the Tap42-2A phosphatase, a major effector of TORC1 that resides on the complex. Rapamycin and caffeine also induce Rho1 activation. While the two agents inhibit TOR directly, their effects on TORC1 signaling are largely dependent on Rho1 activation. Our findings demonstrate that TORC1 acts both upstream and downstream of Rho1 GTPase, unveiling a mechanism that integrates stress and nutrient signals to coordinate Rho1-mediated spatial expansion and TORC1-dependent mass increase.

Keywords: Rho1, Tor, TORC1, Rapmycin, Tap42, Phosphatase, Stress

Introduction

The target of rapamycin, TOR, is a protein kinase that plays a central role in cell metabolism and growth in eukaryotic cells (Wullschleger et al., 2006). It exists in two distinct multi-protein complexes called the TOR complex 1 (TORC1) and 2 (TORC2) (Loewith et al., 2002). Macrolide drug rapamycin, in complex with a cytosol protein, FKBP12, specifically binds to TORC1 and blocks its function, leading to growth arrest (Heitman et al., 1991).

In yeast, two highly-related TOR proteins, Tor1 and Tor2, have been identified (Kunz et al., 1993). Tor2, an essential protein, exists in both TORC1 and TORC2, whereas Tor1, a non-essential one, resides only in TORC1. The two TOR complexes also share Lst8, a WD-40 protein. But TORC1 is marked by the presence of Kog1 and Tco89, while TORC2 by the Avo proteins and Bit61 (Loewith et al., 2002; Reinke et al., 2004). In addition to the core components, a type 2A protein phosphatase, referred to as the Tap42-2A phosphatase, is also associated with TORC1 (Di Como and Jiang, 2006; Yan et al., 2006). This phosphatase is a trimeric complex that contains a catalytic subunit and two regulatory subunits. The catalytic subunit is served alternatively by three phosphatases, including Sit4, Pph21 and Pph22, and Pph3. An essential protein, Tap42, acts as one regulatory subunit, and two partially redundant proteins, Rrd1 or Rrd2, function as the other (Jiang and Broach, 1999; Zheng and Jiang, 2005). Under normal growth conditions, the Tap42-2A phosphatase is associated with membrane structures through its binding with TORC1, which resides on membranes (Aronova et al., 2007; Kunz et al., 2000).

Two major targets of TORC1 have been identified in yeast that relay the activity of TORC1 to many cellular processes, including Sch9 and the Tap42-2A phosphatase (Duvel et al., 2003; Powers, 2007; Urban et al., 2007). Sch9 acts downstream of TORC1 to control ribosome biogenesis, translation initiation, and entry into G0 phase. It is phosphorylated directly by TORC1 at multiple sites at its C-terminus which is required for its activity (Urban et al., 2007). The Tap42-2A phosphatase is the effector of TORC1 in transcription regulation of genes involved in nitrogen metabolism, TCA cycle and stress response (Duvel et al., 2003; Shamji et al., 2000). It has been shown that rapamycin treatment or nitrogen withdrawal induces dissociation of the Tap42-2A phosphatase from TORC1 (Yan et al., 2006). Once being released, the phosphatase dephosphorylates many factors downstream of the TOR pathway, including Gln3, a transcription factor involved in nitrogen metabolism (Beck and Hall, 1999; Bertram et al., 2000; Schmidt et al., 1998).

In addition to causing Sch9 downregulation and the Tap42-2A phosphatase activation, rapamycin and nutrient starvation have been known to activate the cell wall integrity (CWI) pathway (Krause and Gray, 2002; Torres et al., 2002), a major stress response pathway in yeast that controls actin polarization and cell wall expansion in response to various stress conditions, including heat, caffeine, nutrient starvation, cell wall damage and actin perturbation (Levin, 2005). Rho1, a member of Rho family of small GTPases, is the core component of the pathway, which is activated by Rom2, its guanine nucleotide exchange factor (GEF), and several integrin-like cell surface proteins, such as Wsc1 and Mid2 (Ozaki et al., 1996; Philip and Levin, 2001). These cell surface proteins function as stress sensors to activate Rom2 in response to conditions that perturb cell wall integrity (Bickle et al., 1998; Delley and Hall, 1999). Upon activation, Rho1 binds to and stimulates Pkc1, a protein kinase C homologue in yeast, which in turn activates a MAP kinase cascade that comprises Bck1 (MAPKKK), Mkk1/Mkk2 (MAPKK) and Mpk1 (MAPK) (Gustin et al., 1998; Heinisch et al., 1999; Kamada et al., 1996).

In an attempt to understand how the association of the Tap42-2A phosphatase with TORC1 is regulated by rapamycin and nitrogen starvation, we uncover a stress response mechanism that places TORC1 as a direct target of Rho1 GTPase.

Results

Stresses activate the Tap42-2A phosphatase

When yeast cells are treated with rapamycin, many factors downstream of the TOR pathway undergo rapid dephosphorylation, among which is transcription factor Gln3 (Beck and Hall, 1999; Bertram et al., 2000). As previously reported and shown in Fig. 1, the drug-induced dephosphorylation of Gln3 can be visualized by a downshift of Gln3 appeared on SDS PAGE (Fig. 1A). Inactivation of Tap42 prevents the dephosphorylation, suggesting that the Tap42-2A phosphatase is responsible for the process (Fig. S1A). Consistent with previous observations (Cox et al., 2004; Tate and Cooper, 2007), we found that nitrogen starvation and caffeine treatment also induced dephosphorylation of Gln3 (Fig. 1A) and the Tap42-2A phosphatase was required for caffeine-induced Gln3 dephosphorylation (Fig. S1B). Gln3 dephosphorylation was also observed in cells exposed to heat stress or calcofluor white (CFW), a cell wall damaging agent. However, the dephosphorylation was soon masked by another form of phosphorylation (Fig. 1A and S1C). These findings thus suggest that activation of the Tap42-2A phosphatase can be triggered by many diverse environmental stress conditions.

Figure 1. Stresses activate the Tap42-2A phosphatase.

Figure 1

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A. Wild type cells (Y662) expressing Gln3-myc were grown to early log phase at 25°C followed by exposure to the indicated conditions. The expressed Gln3-myc was detected by western blotting and its phosphorylation states were visualized by its mobility shift appeared on SDS PAGE. B. Wild type cells expressing KOG1-HA (Y1032) were grown to early log phase at 25°C and exposed to the indicated conditions. Cells were collected and lysed at various time points after the exposure. Lysates were precipitated with anti-Tap42 antibody or nonspecific IgG control (C2). The levels of Tap42 and Kog1-HA in the cell extract (Extract) and precipitates (α-Tap42 IP) were determined by western blotting. Untagged wild type cells were used as a negative control (C1) (also see Fig. S1D for co-IP Tap42 with HA-Tor2). C. Y1032 cells were grown to early log phase at 25°C in YPD and shifted to nitrogen-free medium followed by addition of ammonia sulfate (AS) (right panel) or vehicle control (left panel). Cells were collected and processed as in B. D. Y1010 cells were transformed with TOR1S1972R (TOR1-1) or tor1S1972R D2294P (t or1-1 kd). The transformed cells were treated with rapamycin, collected and lysed at various time points after the exposure. Lysates were precipitated with anti-Tap42 antibody or nonspecific IgG control (C2) and the levels of the precipitated proteins were assayed as in B. Untagged wild type cells were used as a negative control (C1). E. Y1032 cells transformed with GLN3-myc plasmid were shifted from 25 to 38°C for 2 hr followed by rapamycin treatment. Cells were collected and lysed in the absence of detergent at indicated time points after the temperature shift. Clarified extracts were fractionated into the soluble and membrane fractions (P100) or precipitated with anti-Tap42 antibody after treating with Triton X-100. The phosphorylation states of Gln3-myc in the cell extracts (top panel), the levels of Tap42 and Kog1-HA in the precipitates (α-Tap42 IP) and the extracts (Ext) were determined by western blotting. F. Wild type cells expressing HA-TOR1 (Y990) were treated as in E and the distribution of HA-Tor1 in the soluble (S100) and membrane (P100) fractions of cell extracts (Ext) were determined by western blotting. Also see Supplemental Fig. S1G for the distribution of Kog1 under stress conditions.

Stresses transiently release Tap42 from TORC1

Previously, we showed that rapamycin induces activation of the Tap42-2A phosphatase by releasing it from TORC1 (Yan et al., 2006). We thus determined whether the above stress conditions were able to trigger the release of the phosphatase. Accordingly, wild type yeast cells with the genomic copy of KOG1 tagged with a triple HA tag at its 3′ end (KOG1-HA) were exposed to various stress conditions and the association of Tap42 with TORC1 in the stressed cells was examined by co-immunoprecipitation of Tap42 and Kog1-HA. As shown in Fig. 1B, we found that stress conditions, including nitrogen starvation, heat, caffeine or CFW treatment caused a rapid dissociation of Tap42 from Kog1-HA, which correlated with Gln3 dephosphorylation induced by these conditions (Fig. 1A). Similar results were obtained when the association of Tap42 with TORC1 was examined by co-immunoprecipitation of Tap42 with Tor2, which also exists in TORC1 (Fig. S1D). These findings suggest that the Tap42-2A phosphatase was released from TORC1 and activated in response to the stress conditions.

The stress-induced dissociation of Tap42 from TORC1 was a transient process. In cells stressed with rapamycin, heat, caffeine or CFW treatment, we found that the released Tap42 was re-associated with TORC1 within 60 min despite the persistent insults (Figs. 1B and S1D). However, in cells deprived of nitrogen, Tap42 was unable to do so unless the starved cells were re-fed with nitrogen (Fig. 1C). In addition, we found that the re-association required new protein synthesis, as it was blocked by translation inhibitor cycloheximide (Fig. S1E). These observations demonstrate that the association of Tap42 with TORC1 is regulated by stress conditions and nitrogen availability.

Intriguingly, when the rapamycin resistant TOR1-1 gene (TOR1S1972R) was introduced into yeast cells expressing HA-TOR2, the association of Tap42 with HA-Tor2 became resistant to rapamycin (Fig. 1D). Because the rapamycin resistant Tor1 does not co-exist with Tor2 in the same TOR complex, the fact that it prevents the drug-induced dissociation of Tap42 from Tor2 suggests that the dissociation is not caused directly by binding of the drug to Tor2. Furthermore, we found that a kinase-dead version of the TOR1-1 gene, TOR1S1972R D2294P (tor1-1 kd), was unable to prevent the drug-induced Tap42 release from Tor2 (Fig. 1D), which suggests that an active rapamycin-resistant TORC1 is required for preventing Tap42 release. As expected, the effect of TOR1-1 is specific for rapamycin, since it failed to block Tap42 release induced by other stresses (Fig. S1F). Altogether, these observations indicate that the release of the Tap42-2A phosphatase is triggered indirectly by rapamycin-induced inactivation of TORC1.

Re-association with TORC1 resets the Tap42-2A phosphatase for subsequent stress responses

The re-association of Tap42 with TORC1 indicates that the stressed cells are able to reset the phosphatase for subsequent stress response. To confirm this notion, we examined whether recurred stresses were able to release and activate the Tap42-2A phosphatase repetitively. Accordingly, yeast cells were exposed to heat stress for 2 hr followed by treating with rapamycin. As shown in Fig. 1E, Tap42 was released from Kog1-HA within 10 min of the heat treatment but re-associated after 60 min, during which time, Gln3 was dephosphorylated and rephosphorylated. Following the re-association, Tap42 was again released by rapamycin treatment and the release coincided with another round of dephosphorylation of Gln3. Thus, the recycle of Tap42 back to TORC1 allows the reset of the Tap42-2A phosphatase for response to additional insults.

Stresses disrupt the membrane association of TORC1

We have previously shown that the Tap42-2A phosphatase associates with membranes through its binding with membrane-bound TORC1 and that rapamycin releases the phosphatase from membranes by disrupting its association with TORC1 (Yan et al., 2006). Interestingly, we found that Tap42 re-associated with TORC1 after cells were shifted to 38°C for 60 min, but its membrane association, as measured by its presence in the membrane fraction of cell extract was not fully restored until 30 min later (Fig. 1E). This observation suggests that the heat stress not only frees the Tap42-2A phosphatase from TORC1 but also disrupts the membrane association of TORC1. It also shows that Tap42 re-associates with TORC1 prior to its membrane re-attachment. To further confirm this finding, we examined the distribution of Tor1, which exists only in TORC1, in the soluble and membrane fractions of cell extracts from cells stressed with elevated temperature and rapamycin treatment. As shown in Fig. 1F, we found that Tor1 was released from the membrane fraction into the soluble fraction 10 min after the cells were shifted from 25 to 38°C and did not became membrane bound until 90 min later. The subsequent rapamycin treatment again disrupted the membrane association. Analysis of Kog1 distribution under various stress conditions revealed a similar distribution pattern (Fig. S1G). However, in cells starved for nitrogen, the released Kog1 was unable to re-associate with membranes (Fig. S1G). Nevertheless, the stresses did not appear to compromise the integrity of TORC1, since we found that the association of Tor1 with Kog1 was unaffected in cells exposed to heat stress (Fig. S1H). These findings demonstrate that the membrane association of TORC1 is sensitive to stress conditions.

The cell wall integrity pathway controls Tap42 release

For yeast cells, rapamycin, nitrogen starvation, heat, caffeine and CFW represent five different stress conditions. The fact that all these conditions trigger Tap42 release from TORC1 suggests the existence of a common stress response mechanism upon which these diverse signals converge. One pathway known to respond to these conditions is the cell wall integrity (CWI) pathway, a major stress response pathway in yeast. We thus examined whether the pathway was required for rapamycin-induced Tap42 release. As mentioned in the introduction, the CWI pathway is composed of a series of signaling modules, with Rho1 GTPase sits at the center (Levin, 2005). We found that rapamycin released Tap42 from TORC1 in cells deleted for the downstream components, including Pkc1 and Mpk1, but not in cells deleted for the upstream regulators of Rho1, including Wsc1/Mid2 and Rom2 (Fig. 2A). These observations indicate that Rho1 may mediate the drug-induced Tap42 release. To test this notion, we utilized two temperature sensitive (Ts−) alleles of RHO1, rho1-1 and rho1-4, and inactivated Rho1 by incubating the mutant cells at the nonpermissive temperature (38°C). As shown in Fig. 2B, upon shifting the mutant cells from 25 to 38°C, Tap42 dissociated from Kog1-HA within 10 min and re-associated after 60 min. After incubation at the nonpermissive temperature for two hours, which inactivated the mutant Rho1 protein, addition of rapamycin into the culture medium failed to induce Tap42 release in the cells. In contrast, the drug was capable of releasing Tap42 in wild type cells subjected to the same treatments. The inability for rapamycin to release Tap42 in the mutant cells was unlikely to be an indirect consequence of the temperature-induced growth arrest, as we found that in another Ts− mutant, cdc25-1, rapamycin was able to release Tap42 when the mutant cells were arrested by an elevated temperature (Fig. 2B).

Figure 2. Rho1-dependent release of Tap42 from TORC1 and phosphatase activation.

Figure 2

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A. Wild type (Y1461), wsc1 mid2 (1488), rom2 (Y1478), pkc1 (Y1489) and mpk1 (Y1477) mutant cells expressing KOG1-HA were grown at 25°C to early log phase and treated with rapamycin. Cells were collected and lysed at the indicated time points after the addition of the drug. Cell extracts were precipitated with anti-Tap42 antibody. The amounts of Tap42 and co-purified Kog1-HA in the precipitates were determined by western blotting. B. Wild type (Y1492), rho1-1 (Y1480), rho1-4 (Y1491) and cdc25-1 mutant (Y1490) cells were grown at 25°C to early log phase and shifted to 38°C. Upon incubation for 2 hr, cells were treated with rapamycin, collected at the indicated time points after the temperature shift and processed as in A. C. Wild type and mutant cells expressing GLN3-myc were treated with rapamycin for indicated times. The phosphorylation states of Gln3-myc were analyzed by western blotting. The input controls for A and B are shown in Supplemental Fig. S2. Also see Fig. S2C for the effects of caffeine treatment and nitrogen starvation.

Consistent with the requirement of the upstream regulators of Rho1 for Tap42 release, deletion of these regulators also blocked the dephosphorylation of Gln3 induced by rapamycin, nitrogen starvation and caffeine treatment. In contrast, deletion of the downstream targets of Rho1 had no effect on the stress-induced dephosphorylation (Figs. 2C and S2C). Altogether, the above observations demonstrate that stress-induced release and activation of the Tap42-2A phosphatase are mediated by the cell wall integrity pathway, likely through Rho1 GTPase.

Rho1 transiently associates with TORC1 in response to stresses

The action mechanism of most small GTPases involves direct binding to their targets (Hall, 1993). We thus determined whether rapamycin was able to induce binding of Rho1 to TORC1 by analyzing its association with Kog1 through co-immunoprecipitation. As shown in Fig. 3A, we found that the drug induced a transient binding of Rho1 with Kog1, which occurred within 10 min of the treatment but disappeared after 30 min. Under the same experimental conditions, we failed to detect association of Rho1 with Avo1, a TORC2 specific component (Fig. 3B), suggesting that Rho1 specifically targets TORC1. Like rapamycin treatment, we found that other stress conditions, including heat, nitrogen starvation, caffeine and CFW, all triggered a brief association of Rho1 with Kog1 (Fig. S3A–D). These observations indicate that TORC1 is a target of Rho1 under stress conditions.

Figure 3. Stress-induced association of Rho1 with TORC1.

Figure 3

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Wild type cells expressing Myc-RHO1 with either KOG1-HA (A) or AVO1-HA (B) were grown at 25°C to early log phase and treated with rapamycin. Cells were collected and lysed at the indicated time points after the addition of the drug. Lysates were precipitates with anti-myc antibody. The amounts of myc-Rho1 and co-purified Kog1-HA or Avo1-HA in the precipitates were determined by western blotting. C. Wild type cells expressing KOG1-HA were transformed with single-copy vector expressing different myc tagged rho1 mutants. Transformed cells were treated with rapamycin and the association of Kog1-HA with the Rho1 mutant proteins was assayed by co-immunoprecipitation. Also see Supplemental Figs. S3 and S4 for the association of Rho1 with Kog1 and Pkc1 under different stress conditions.

Next, we determined whether the binding with TORC1 required Rho1 activation and whether it was mediated through the effector domain of Rho1. Accordingly, we examined the association of Kog1 with Rho1 mutants defective for either activation or effector domain in cells treated with rapamycin. As shown in Fig. 3C, we found that while rapamycin induced the binding of wild type Rho1 with Kog1, it failed to do so for an inactive mutant of Rho1, Rho1T24N. In contrast, an active mutant of Rho1, Rho1Q68L, exhibited a constitutive association with Kog1 regardless whether the drug was present or not. Also, we found that point mutations within the effector domain of Rho1 abolished its ability to bind with Kog1. Hence, the binding with Kog1 requires Rho1 activation and is mediated through its effector domain, which supports the notion that TORC1 is an effector of Rho1. Consistent with this notion, we found that the mutant Rho1 protein of the Ts− rho1-4 allele was unable to bind Kog1 upon heat inactivation (Fig. S3E). To further verify that Rho1 is activated by rapamycin and other stress conditions, we examined the Rho1 activity in the cells subjected to various stress treatments. Because Rho1 binds to Pkc1, a well-established effector of Rho1, only in its GTP-bound and active form (Kamada et al., 1996), we assayed Rho1 activity based on its ability to associate with Pkc1. The results showed that rapamycin, heat, nitrogen starvation, CFW and caffeine treatments all induced a transient binding of Rho1 with Pkc1 (Fig. S4A–E), which mirrored that between Rho1 and Kog1 under the same conditions (Fig. S3), suggesting that the binding of Rho1 with Kog1, like that with Pkc1, requires its activation. Consistent with this view, we found a concurrent binding and dissociation of Rho1 with Pkc1 and Kog1 in cells stressed with rapamycin (Fig. S4F). Taken together, the above results suggest that stress-induced activation of Rho1 promotes its association with TORC1.

While rapamycin triggers Rho1 binding to Kog1 in wild type cells, it failed to do so in cells expressing the rapamycin resistant TOR1-1 gene (Fig. S5A). This failure coincides with the inability of the drug to induce Tap42 release in the same cells ((Fig. 1D), indicating a causal connection between Rho1 binding and Tap42 release. In line with this view, we found that binding of Rho1 with TORC1 concurred with the release of Tap42 (Fig. S5B).

Rho1 associates with TORC1 through direct binding with Kog1

Since Rho1 associates specifically with TORC1 but not TORC2, it is plausible that the association is mediated by a direct binding of Rho1 to Kog1, the unique component of TORC1. To test this notion, we examined the binding between purified recombinant Rho1 and Kog1 in vitro. Because Kog1 is a big protein, expressing the full length protein in bacterial cells was proven to be difficult. Therefore, we divided the KOG1 gene into three fragments corresponding to the three functional regions of the gene product, including the N-terminal RNC domain (amino acids 1–637) that is highly conserved in the counterparts of Kog1 in other organisms, the middle region containing heat motifs (amino acids 638–1157) and the C-terminal WD domains (amino acids 1158–1557). These gene fragments were fused with the GST sequence and expressed in bacterial cells. The GST-fusion proteins were purified and their ability for binding with purified recombinant Rho1 was examined in an in vitro binding assay. As shown in Fig. 4A, we found that only the RNC domain was able to interact with recombinant Rho1. Importantly, the interaction was greatly enhanced when Rho1 was loaded with GTP. This GTP-dependent binding of Rho1 with Kog1 is consistent with the observation that the association of Rho1 with TORC1 occurs in cells only when Rho1 is active (Fig. 3C).

Figure 4. Rho1 binds directly to Kog1.

Figure 4

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A. Glutathione beads loaded with recombinant GST or GST fused Kog1 fragments (GST−) were incubated with His tagged recombinant Rho1 (His-Rho1) loaded with either GTPγS (T) or GDP (D). Following the incubation, the amounts of GST fused proteins (GST−) and His-Rho1 in the reaction mixtures (input) and those bound to the beads (GST Pull down) were determined by western blotting using anti-GST and His antibodies. B. Glutathione beads loaded with recombinant GST or GST fused Kog1 fragments (GST−) were incubated with His tagged recombinant Tap42 (His-Tap42). The amounts of GST fused proteins (GST−) and His-Tap42 in the reaction mixtures (input) and those associated with the beads (GST Pull down) were determined by western blotting. C. Glutathione beads loaded with recombinant GST-RNC were pre-incubated with His tagged recombinant Tap42 (His-Tap42) before addition of the indicated recombinant small GTPases (His-GTPases) loaded with either GTPγS (T) or GDP (D). The amounts of GST-RNC, His-Tap42 and His tagged small GTPases in the reaction mixtures (input) and those associated with the beads (GST Pull down) were determined by western blotting.

Binding of Rho1 causes the release of Tap42 from Kog1

Because the Tap42-2A phosphatase also associates with TORC1, we further tested whether Tap42 was able to interact directly with Kog1 using the same in vitro binding assay. We found that like Rho1, recombinant Tap42 was able to interact with the RNC domain but not the other regions of Kog1 (Fig. 4B). This finding indicates that the Tap42-2A phosphatase associates with TORC1 through a direct binding between Tap42 and Kog1.

The finding that both Rho1 and Tap42 associate with Kog1 by direct binding to the RNC domain raises a possibility that Rho1 and Tap42 may compete for Kog1 binding. To test this possibility, we examined the ability of Rho1 to dislodge Tap42 from the RNC domain. As shown in Fig. 4C, we found that GTP-loaded Rho1 interacted strongly with the RNC domain and effectively reduced the binding of Tap42, whereas GDP-loaded Rho1 was largely inactive. Interestingly, Rho2, a close homolog of Rho1, also displayed a limited ability to bind with the RNC domain and to displace Tap42 when loaded with GTP. In contrast, two other related small GTPases, Cdc42 and Ras1, were completely inactive for the binding and displacement. These findings demonstrate a specific action of Rho1 on the association of Tap42 with the RNC domain. Consistent with this in vitro finding, we found that over-expressing RHO1Q68L, a dominant active mutant, alone was able to induce the release of the Tap42-2A phosphatase in unstressed cells. The Tap42 release activity of Rho1Q68L appeared to depend on its ability to interact with TORC1, as we found that the mutant protein was unable to displace Tap42 from TORC1 when its effector domain was defective (Fig. S6). Taken together, the above findings support a mechanistic theme in yeast cells that Rho1, upon activation, binds to Kog1 and releases the Tap42-2A phosphatase from TORC1.

Rho1 inhibits TORC1 kinase activity

We next determined whether the binding of Rho1 affected TORC1 kinase activity using an in vitro kinase assay. TORC1 was purified by immunoprecipitation of Kog1-HA from cells expressing HA-tagged KOG1 and its kinase activity toward GST-4E-BP1 substrate was assayed in the presence or absence of recombinant Rho1. We found that recombinant Rho1 inhibited TORC1 kinase activity in a GTP-dependent manner (Fig. 5A). The inhibitory effect was also Rho1 specific, as we found that two other small GTPases, Cdc42 and Ras1, lacked such an activity. Although GTP-loaded Rho2 also reduced TORC1 kinase activity, the reduction was not statistically significant under the experimental condition. Consistent with the negative effect of Rho1 in vitro, we found that TORC1 purified from cells overexpressing the active mutant RHO1Q68L displayed a reduced kinase activity in comparison with that from cells overexpressing a wild type RHO1 (Fig. S7).

Figure 5. Active Rho1 inhibits TORC1.

Figure 5

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A. TORC1 was purified from cells expressing Kog1-HA (Y1032) by immunoprecipitation with anti-HA antibody. The kinase activity of the purified TORC1 was measured using recombinant GST-4E-BP1 as substrate. The reactions were carried out in the presence (+) or absence (−) of the indicated agents. Bacterially expressed small GTPases were purified and loaded with either GTPγS (T) or GDP (D). Phosphorylation of 4E-BP1 was determined by western blotting use anti-phospho-4E-BP1 (T37/46) antibody and quantified. B. TORC1 was purified and assayed for kinase activity in the presence of indicated agents as in A. Data shown are the mean ± S.D. of values from three independent experiments (bar graphs).

To determine whether the inhibitory effect of Rho1 depends on its direct interaction with Kog1, we further tested the effect of several Rho1 mutants on TORC1 activity that bear point mutations in the effector domain. We found that while the wild type Rho1 inhibited TORC1 kinase activity when loaded with GTP, the mutants lacked the ability (Fig. 5B). Since these Rho1 mutants are incapable of Kog1 binding (Fig. 3C), this result indicates that the ability to directly interact with Kog1 is required for the inhibitory effect of Rho1 on TORC1 kinase activity.

The CWI pathway is required for rapamycin-induced dephosphorylation of Sch9

Sch9 is a key effector of TORC1 that functions separately from Tap42 to mediate TORC1 signaling (Huber et al., 2009; Urban et al., 2007). Inhibition of TORC1 by rapamycin causes dephosphorylation of Sch9, which can be visualized by downshifts of the C-terminal fragments on SDS PAGE (Urban et al., 2007). To determine the inhibitory effect of Rho1 activation on TORC1 activity in cells, we tested whether CFW, which activates Rho1 by perturbing cell wall integrity, was able to induce Sch9 dephosphorylation. As shown in Fig. 6A, we found that CFW induced Sch9 dephosphorylation in wild type and mpk1 deletion cells, but was ineffective in cells lacking both WSC1 and MID2 or in cells without ROM2. The requirement for the upstream components of Rho1 in the CWI pathway for Sch9-dephosphorylation supports the notion that Rho1 activation is required for the effect of CWF. Despite the fact that rapamycin and caffeine bind directly to TORC1 and inhibit its activity, their effect on Sch9-dephosphorylation appeared to be dependent on Rho1 activation, as we found that the two agents failed to induce the dephosphorylation in cells lacking both WSC1 and MID2 or in cells without ROM2, but was capable in cells deleted for MPK1 (Fig. 6B and C). Similarly, nitrogen starvation also was unable to induce Sch9-dephosphorylation in cells defective for the upstream components of Rho1 (Fig. 6D). These observations indicate that stress-induced Sch9 dephosphorylation requires Rho1 activation, even when the stress acts directly on TORC1. In addition, we found that deletion of genes encoding the phosphatase catalytic subunits that are known to associate with Tap42, including SIT4, PPH21/PPH22 and PPH3, had no obvious effect on rapamycin-induced Sch9 dephosphorylation (Fig. 6E), suggesting the dephosphorylation is independent of the Tap42-2A phosphatase. However, it is worth noting that in the absence of rapamycin treatment, the phosphorylation state of Sch9 in the sit4 mutant cells appeared to be different from that in the wild type controls (Fig. 6E). Taken together, the above findings demonstrate that Rho1 inhibits the Sch9-mediated branch of the TORC1 signaling pathway in yeast cells.

Figure 6. The CWI pathway is required for stress-induced Sch9 dephosphorylation.

Figure 6

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Exponentially growing wild type and mutants cells expressing HA tagged SCH9 were exposed to CFW (A), rapamycin (B), caffeine (C) or nitrogen starvation (D). Aliquots of cells were removed and lysed at the indicated time points following the treatments. The phosphorylation levels of the C-terminal portion of Sch9-HA in the lysates were determined by NTCB treatment and western blotting with anti-HA antibody. E. Exponentially growing wild type and phosphatase mutant cells expressing HA tagged SCH9 were treated with rapamycin and the Sch9 phosphorylation analyzed.

The role of TORC1 in stress-response

Since rapamycin binds and inhibits TORC1 directly, the fact that the drug triggers Rho1 activation suggests that TORC1, in addition to being a target of Rho1, is also an upstream regulator of Rho1. To test this notion, we examined the role of TORC1 in the stress-induced activation of the cell wall integrity pathway. Accordingly, we employed a Ts− allele of KOG1, kog1-105, which has been shown to be defective for TORC1 function when the mutant cells are grown at 38°C (Nakashima et al., 2008). Upon inactivation of TORC1 by shifting the mutant cells to 38°C for 2 hr, the cells were exposed to various stress conditions and the activity of the cell wall integrity pathway was monitored based on the phosphorylation levels of Mpk1, the end kinase of the pathway. Consistent with the findings we showed previously (Guo et al., 2009), an elevated temperature caused a transient increase in Mpk1 phosphorylation in wild type cells, which was reduced to the basal level within 2 hr (Fig. 7A). However, we found that a subsequent nitrogen starvation induced another round of Mpk1 phosphorylation in the cells. In the kog1-105 mutant cells, as in the wild type cells, the elevated temperature also caused a transient increase in Mpk1 phosphorylation. However, after 2 hr incubation at 38°C, which inactivated TORC1, the subsequent nitrogen starvation failed to increase Mpk1 phosphorylation. The inability to respond to nitrogen starvation was unlikely to be a consequence of the temperature-induced grown cessation, as we found that in another Ts− mutant, cdc25-1, the nitrogen starvation was still capable of promoting Mpk1 phosphorylation when the growth was arrested by the elevated temperature (Fig. 7A). Similarly, when TORC1 was inactivated in the mutant cells by the temperature shift, Mpk1 phosphorylation was unable to be stimulated by subsequent caffeine treatment (Fig. 7B). These observations indicate that signals of nitrogen starvation and caffeine treatment are sensed and transmitted by TORC1. The requirement of TORC1 for the effect of caffeine is consistent with the fact that the drug binds directly to TORC1 (Kuranda et al., 2006; Reinke et al., 2006). While the cell wall integrity pathway became unresponsive to nitrogen starvation and caffeine treatment in the kog1 mutant cells upon heat-induced inactivation of TORC1, it remained sensitive to the treatment of calcofluor white (CFW), a dye that binds directly to cell surface and perturbs cell wall integrity (Fig. 7C). This finding suggests that damages in cell wall are sensed independent of TORC1.

Figure 7. The role of TORC1 in the stress-activated MAPK pathway.

Figure 7

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Wild type (YYK410), kog1-105 (YYK409) and cdc25-1 (JBY703) mutant cells were grown to early log phase at 25°C and shifted to 38°C followed by nitrogen starvation (A), caffeine (B) or CFW treatment (C). Cells were collected at the indicate time points after the temperature shift. The phosphorylation levels of Mpk1 in the cells were examined by western blotting using anti-phospho-MAPK antibody. The levels of Tpd3 served as loading controls.

Collectively, these results demonstrate that TORC1 plays a key role in stress responses by acting at both upstream and downstream of Rho1 GTPase.

Discussion

We have previously shown that the Tap42-2A phosphatase associates with TORC1 under normal growth conditions but is released and activated upon rapamycin treatment (Yan et al., 2006). In this study, we show that binding of rapamycin to TORC1 does not trigger the release directly. Instead, the drug acts upon TORC1 to induce activation of Rho1 GTPase, which in turn binds to TORC1 and releases the phosphatase.

Rho1 imparts stress signals to TORC1

As a key component of in a major stress response pathway in yeast, Rho1 responds to various environmental stresses. Thereby, its ability to bind and regulate TORC1 renders the complex amendable to environmental conditions. The unique component in TORC1, Kog1, serves as the direct target of Rho1, which is also the docking site for Tap42, allowing the Tap42-2A phosphatase to associate with TORC1. The fact that both Rho1 and Tap42 bind to the same region in Kog1 explains why Rho1 is able to dislodge the Tap42-2A phosphatase from TORC1. Rho1 may simply compete with Tap42 for Kog1 binding. However, since Rho1 binding also downregulates TORC1 kinase activity and perturbs its membrane association (Figs. 1 and 5), it is likely that the binding also alters the conformation of Kog1 and hence the overall function of TORC1.

Upon activation, Rho1 impacts on the two key branches of TORC1 signaling by inducing Sch9 dephosphorylation and release of the Tap42-2A phosphatase. Since the TORC1-dependent phosphorylation is required for Sch9 activity (Urban et al., 2007), the Rho1-induced dephosphorylation is likely to affect Sch9 function in ribosome biogenesis and translation initiation. On the other hand, the release of the Tap42-2A phosphatase causes activation of the phosphatase, which as demonstrated in previous studies (Duvel et al., 2003; Shamji et al., 2000), sets off changes in transcription of genes involved in nitrogen metabolism, TCA cycle and stress response. Therefore, the impact of stress conditions on TORC1 function is profound and enduring, and is likely to play a key role in the cell’s accommodation to the adverse conditions. In addition, our finding that Rho1 is required for rapamycin-induced release and activation of the Tap42-2A phosphatase indicates that the initial transcriptional changes induced by the drug are mediated through Rho1 and hence similar to those induced by other stress conditions, such as heat and cell wall damages. This mechanism may explain why environmental stresses and rapamycin have similar effects on Msn2 and Msn4, two stress responsive transcription factors (Beck and Hall, 1999; Santhanam et al., 2004).

Although Rho1-induced Sch9 dephosphorylation coincides with the release of the Tap42-2A phosphatase, we find that none of the phosphatase known to associate with Tap42 is required for the dephosphorylation (Fig. 6E). This observation is consistent with the notion that Sch9 function is independent of Tap42 (Huber et al., 2009). In this regard, the recent finding that Glc7. a type 1 protein phosphatase, is an effector of TORC1 may represent a potential mechanism responsible for Rho1-induced Sch9 dephosphorylation (Tatchell et al., 2011).

Resetting TORC1 and the Tap42-2A phosphatase

While the binding of Rho1 to TORC1 triggers the release of the Tap42-2A phosphatase, the process is transient. Following Rho1 dissociation, the phosphatase re-assembles on TORC1. It appears that the transient association of Rho1 with TORC1 permits a timely recycle of the Tap42-2A phosphatase and reset of TORC1 for additional adverse environmental conditions. Nevertheless, dissociation of Rho1 from TORC1 alone is not sufficient for the reset of TORC1. Additional requirements include nitrogen and new protein synthesis. These requirements suggest that restoration of TORC1 function in a cell following its exposure to stress conditions is a complicated process involving more steps than simply dissociation of Rho1 from TORC1. How nitrogen availability imparts the restoration process is unclear. Previous studies have identified two vacuolar membrane associated complexes, namely the Ego/GSE and Vps complexes, that are required for yeast cells to exit from rapamycin-induced growth arrest (Dubouloz et al., 2005; Zurita-Martinez et al., 2007). Both complexes have been shown to play critical roles in amino acids and nitrogen mediated TORC1 activation (Binda et al., 2009; Dubouloz et al., 2005; Gao and Kaiser, 2006). Given the fact that TORC1 has been found to associate with vacuolar membranes (Cardenas and Heitman, 1995), the possibility that the Ego/GSE and Vps complexes are involved in the nitrogen-dependent restoration of TORC1 function may warrant a future study.

TORC1 signals to Rho1

The finding that Rho1 is activated by rapamycin places the small GTPase downstream of TORC1. Because the drug activates Rho1 in normal cells but not in those expressing rapamycin-resistant TOR1-1, it is conceivable that the activation is triggered by TORC1 inactivation. The reduced TORC1 activity may initiate a distress signal, which as inferred by our analysis of rapamycin-induced and Rho1-dependent release of the Tap42-phopshatases (Fig. 2), acts through the upstream regulators of Rho1, including the cell surface proteins, Wsc1, Mid2 and its guanine nucleotide exchange factor Rom2, leading to Rho1 activation. However, as in other stress conditions, the nature of the distress signal and the mechanism by which the reduced TORC1 activity activates the cell surface sensors remains unknown. Interestingly, TORC1 has been previously found to co-exist with Wsc1 in a unique membrane fraction (Aronova et al., 2007), which supports a notion that TORC1 may associate with Wsc1 or other cell sensor proteins to regulate their function. It is thus possible that the sensor proteins are phosphorylated by TORC1 and a reduction in TORC1-dependent phosphorylation increase their activity, which leads to Rho1 activation. Alternatively, TORC1 may act through Rom2 or its upstream regulators, such as Stt4 PI-4 kinase (Audhya and Emr, 2002), in which case, the requirement of Wsc1 and Mid2 for TORC1-mediated Rho1 activation reflects their role in targeting Rom2 to the plasma membrane. Consistent with this view, Rom2 activity are found to be reduced in a tor2 mutant with defective TORC1 function (Bickle et al., 1998). However, there is no evidence indicating that Rom2 or Rho1 itself is phosphorylated directly by TORC1. Further study is required to delineate the molecular details underlying this connection.

Rho1 GTPase and TORC1 are two key factors in yeast involved, respectively, in polarized cell expansion and cell mass increase. The mutual regulation of these two factors is thus likely to be a pivotal mechanism whereby yeast cells coordinate their spatial expansion with mass increase. In addition, we find that overexpression of RhoA, the mammalian ortholog of Rho1, also suppresses mTORC1 signaling activity in mammalian cells, indicating that the Rho GTPase-dependent regulation of TORC1 may be an evolutionally conserved mechanism (data not shown). Our findings thus uncover a conserved signaling mechanism that integrates Rho GTPase-mediated spatial control with TORC1-dependent growth regulation.

Experimental Procedures

Media

Yeast cells were normally grown in YP medium or synthetic complete media lacking an appropriate amino acid for selection. All media contain 2% glucose as the carbon source unless indicated otherwise. For nitrogen starvation, yeast cells grown in YP medium were collected with ice-filled tubes and resuspended into nitrogen starvation medium containing 2% glucose and 1 × yeast nitrogen base without ammonium sulfate and amino acids. Ammonium sulfate at final concentration of 50 mg/ml, together with appropriate amino acids for auxotrophic strains, was used for nitrogen re-addition.

Co-immunoprecipitation

Yeast cells were grown overnight at 25°C to early log phase followed by exposure to various stress treatments. At the indicated time points after a treatment, an aliquot of cells (1×109) was transferred to a centrifuge tube filled with ice and collected by centrifugation at 500 g for 5 min. For co-immunoprecipitation of Myc-Rho1 with other proteins, cells were lysed with glass beads in EDTA-free buffer and processed as described before (Guo et al., 2009). Co-immunoprecipitation of Tap42 with epitope tagged Tor2 and Kog1 was performed as previous reported (Yan et al., 2006).

In vitro kinase assay

The kinase assay was performed as describe previously with modifications (Bai et al., 2007). Exponential growing Y1032 (KOG1-HA) cells were lysed with glass beads in buffer containing 50 mM HEPES, pH7.4, 100 mM NaCl, 0.7% CHAPS, 1 mM EDTA, 1 mM DTT and 1x protease inhibitor cocktail (Roche). Lysate (15 mg) was incubated with anti-HA antibody (10 μg) at 4°C for 2 hr followed by addition of Protein A beads (200 μl). Beads were washed 2x with wash buffer containing 50 mM HEPES, pH7.4, 300 mM NaCl, 0.7% CHAPS and 1 mM DTT and 2x with kinase buffer containing 50 mM HEPES, pH7.4, 50 mM NaCl, 5mM MgCl2 and 1 mM DTT. After the final wash, beads were divided into aliquots of 15 μl each and preincubated with 2 μg of indicated small GTPases loaded with either GTPγS or GDP in 30 μl of kinase buffer at 30°C for 10 min. Purified GST-4E-BP1 (2 μg) and ATP (2 mM) in 30 μl of kinase buffer were then added to the reactions. To demonstrate that the kinase activity associated with the purified beads was elicited by TORC1, GST-FKBP12 (5 μg) and rapamycin (1 μM) were included as a control. Upon further incubation for 20 min, the kinase reactions were terminated by adding 1 vol of 2x SDS sample buffer and immediately boiled for 5 min. The reaction mixtures were subjected to SDS PAGE followed by western blotting using anti-phospho-4E-BP1 antibody for phosphorylation levels and anti-HA antibody for levels of purified Kog1-HA. Phosphorylation level was quantified by densitometry.

Additional information is provided in the Supplemental Materials.

Supplementary Material

01

Highlights.

  • Active Rho1 inhibits TORC1 activity by binding to Kog1

  • Inhibition of TORC1 activates Rho1

  • Tap42 associates with TORC1 through direct binding with Kog1

  • Rho1 activation triggers the release of Tap42-2A phosphatase from TORC1

  • Nitrogen is required for the re-association of the Tap42-2A phosphatase with TORC1

Acknowledgments

We appreciate Yoshikazu Ohya, Yoshiaki Kamada, David Levin and James Broach for providing yeast strains and plasmids. We thank members of our laboratory for comments and stimulating discussion during the course of this study. This work was supported by grant GM068832 and CA129821 from the National Institutes of Health (to YJ).

Footnotes

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