The yeast Gα subunit Gpa2p and its coupled receptor Gpr1p function in a signaling pathway that is required for the transition to pseudohyphal and invasive growth. A two-hybrid screen using a constitutively active allele of GPA2 identified the KRH1 gene as encoding a potential binding partner of Gpa2p. Strains containing deletions of KRH1 and its homolog KRH2 were hyper-invasive and displayed a high level of expression of FLO11, a gene involved in pseudohyphal and invasive growth. Therefore, KRH1 and KRH2 encode negative regulators of the invasive growth pathway. Cells containing krh1Δ krh2Δ mutations also displayed increased sensitivity to heat shock and decreased sporulation efficiency, indicating that Krh1p and Krh2p regulate multiple processes controlled by the cAMP/PKA pathway. The krh1Δ krh2Δ mutations suppressed the effect of a gpa2Δ mutation on FLO11 expression and eliminated the effect of a constitutively active GPA2 allele on induction of FLO11 and heat shock sensitivity, suggesting that Krh1p and Krh2p act downstream of Gpa2p. The Sch9p kinase was not required for the signal generated by deletion of KRH1 and KRH2; however, the cAMP-dependent kinase Tpk2p was required for generation of this signal. These results support a model in which activation of Gpa2p relieves the inhibition exerted by Krh1p and Krh2p on components of the cAMP/PKA signaling pathway.
The GPA2 gene of the yeast Saccharomyces cerevisiae encodes a protein that is homologous to heterotrimeric G proteinα -subunits (Nakafuku et al., 1988). The function of GPA2 is required for pseudohyphal and invasive growth in response to changes in nutrient conditions (Kübler et al., 1997; Lorenz and Heitman, 1997; Lorenz et al., 2000). Nutrient limitation causes both haploid and diploid cells to undergo morhphological changes, although the nutritional condition that induces the transition is different in each case. Whereas nitrogen limitation causes diploid cells to form pseudohyphae, glucose limitation causes haploid cells to undergo invasive growth (Cullen and Sprague, 2000; Gimeno et al., 1992). Pseudohyphae formation and invasive growth are regulated by both the MAPK and cAMP/PKA signaling pathways (Gancedo, 2001; Levin and Errede, 1995; Madhani and Fink, 1998). However, little is known about the proteins involved in signal transduction through Gpa2p.
One component of the Gpa2p pathway that has been identified is the G protein-coupled receptor that interacts with the Gpa2p α-subunit. This receptor, called Gpr1p, was identified by a two-hybrid screen using Gpa2p as the bait (Kraakman et al., 1999; Xue et al., 1998; Yun et al., 1997). The presence of this receptor on the cell surface suggests that it recognizes an extracellular ligand (Xue et al., 1998). However, the ligand that activates Gpr1p has not been identified as yet. Like GPA2, GPR1 is required for the switch to pseudohyphal and invasive forms of growth (Lorenz et al., 2000; Tamaki et al., 2000). A constitutively active allele of GPA2 suppresses the filamentous growth defect conferred by a gpr1Δ mutation, in agreement with the idea that Gpr1p couples to Gpa2p and initiates signaling through the Gpa2p pathway (Lorenz et al., 2000).
The signaling pathway that functions downstream of Gpr1p and Gpa2p is not well understood. One possibility that has been proposed is that Gpa2p acts in an analogous manner to the mammalian α-subunit Gαs, which directly activates adenylyl cyclase. This potential mechanism would constitute a second way of activating adenylyl cyclase in yeast, in addition to the known mechanism of direct activation of adenylyl cyclase by Ras proteins (Broach, 1991; Thevelein and de Winde, 1999). Production of cAMP by adenylyl cyclase activates the cAMP-dependent kinases Tpk1p, Tpk2p and Tpk3p. These kinases phosphorylate substrates that regulate metabolism, growth and filament formation (Borges-Walmsley and Walmsley, 2000; Madhani and Fink, 1998).
cAMP-dependent kinase is involved in many cellular processes, and the three different forms of this kinase that are present in yeast are not equivalent for all functions. Although TPK1, TPK2 or TPK3 can provide the essential function that is revealed when all three genes are mutated (Toda et al., 1987b), they appear to play different roles with respect to filamentous growth. Whereas Tpk2p is required for filamentous growth, Tpk1p and Tpk3p have either no effect or a small inhibitory effect on this process (Pan and Heitman, 1999; Robertson and Fink, 1998). Activation of the cAMP-dependent kinases occurs by binding of cAMP to a regulatory subunit, Bcy1p, which is presumed to bind to all three forms of the kinase. It is therefore likely that the different cAMP-dependent kinases are subject to additional types of regulation that affect their involvement in different cellular processes.
Several observations suggest that there is a relationship between Gpa2p-mediated signaling and cAMP-dependent signaling. First, overexpression of GPA2 augments the rapid increase in cAMP levels that occurs when glucose is added to glucose-starved cells (Nakafuku et al., 1988). Second, the defect in pseudohyphal growth conferred by a gpa2Δ or gpr1Δ mutation is reversed by the addition of cAMP (Kübler et al., 1997; Lorenz and Heitman, 1997; Lorenz et al., 2000; Tamaki et al., 2000). Finally, a gpa2Δ or gpr1Δ mutation eliminates the glucose-induced increase in cAMP levels under certain conditions (Colombo et al., 1998; Kraakman et al., 1999; Lorenz et al., 2000; Yun et al., 1998). Gpa2p has been shown to function independently of Ras, which would be consistent with the possibility that Gpa2p directly activates adenylyl cyclase (Colombo et al., 1998; Xue et al., 1998). However, direct activation of adenylyl cyclase by Gpa2p has not been demonstrated experimentally as yet.
Another candidate for a downstream component of the Gpa2p pathway is the kinase Sch9p. The SCH9 gene was isolated based on the fact that its overexpression suppresses the growth defect conferred by mutations in genes that encode components of the cAMP signaling pathway (Toda et al., 1988). Sch9p appears to function in a separate pathway from Ras, adenylyl cyclase and the cAMP-dependent kinases (Hartley et al., 1994; Toda et al., 1988). The possibility that Sch9p acts downstream of Gpa2p was raised by the finding that an sch9Δ mutation eliminates the sensitivity to heat shock conferred by a constitutively active version of Gpa2p (Xue et al., 1998). However, Sch9p and Gpa2p do not appear to function in a linear signaling pathway because sch9Δ mutant cells have a different phenotype from gpa2Δ mutant cells. Cells containing an sch9Δ mutation display a significant growth defect (Toda et al., 1988) and are defective for induction of trehalase activity in response to nitrogen addition (Crauwels et al., 1997). Cells containing a gpa2Δ mutation do not have a growth defect (Nakafuku et al., 1988) and respond normally to nitrogen addition (Kraakman et al., 1999). Moreover, double mutant sch9Δ gpa2Δ cells display a much more severe growth defect than single mutant sch9Δ cells, indicating that the functions of these two genes cannot be completely overlapping (Kraakman et al., 1999; Lorenz et al., 2000).
In summary, the Gpa2p α-subunit cannot be placed in the context of a simple, linear signaling pathway because all potential downstream components of the Gpa2p pathway respond to multiple inputs. Here, we report the identification of two novel genes, KRH1 and KRH2, encoding proteins that play a role in the Gpa2p pathway. The KRH1 and KRH2 gene products act downstream of Gpa2p and function by exerting a negative regulatory effect on haploid invasive growth.
Materials and Methods
Construction of plasmids pGBT9-GPA2 and pG2CT-112.2 was described previously (Xue et al., 1998). Plasmid pG2CT-T9.2 was constructed by changing the arginine at position 273 in the GPA2 sequence of pGBT9-GPA2 to an alanine, as described for pG2CT-112.2.
Construction of plasmids YEpSCH9 and YEpADH-SCH9 was described previously (Toda et al., 1988). Plasmid pS9CLON.111 was made by cloning a 7.6 kb BamHI fragment from YEpSCH9 into the BamHI site of YEplac111. Plasmid pS9-7K.Bs was made by cloning a 7.0 kb BamHI-KpnI fragment from pS9CLON.111 into the BamHI-KpnI sites of pBluescript. Plasmid pURA3.Bs was made by cloning a 1.2 kb XbaI fragment containing the URA3 gene from pAC100-2 into the SpeI site of pBluescript. To construct a URA3 disruption of SCH9, a 1.0 kb EcoRI-NsiI fragment from pURA3.Bs was cloned into the EcoRI-PstI sites of pS9-7K.Bs to produce pS9-7K::URA3.Bs.
Construction of plasmid YEpTPK2 was described previously (Toda et al., 1987b). Plasmid YEpTPK2.2 was made by cloning a 2.0 kb BglII fragment from YEpTPK2 into the BamHI site of YEp351.
The KRH1 gene was cloned by amplifying a 1.2 kb fragment from yeast genomic DNA by polymerase chain reaction (PCR) using primers 5TH14-1 [5′-CGCTGCAGTGATTCATTGGCAGGTCC-3′ (genomic sequences are underlined in all primers)], which contains a flanking PstI site (shown in bold), and 3TH14-1 (5′-CGGTCCGTTAATTTGGATCC-3′), which contains an internal BamHI site present in the genomic DNA site (shown in bold). This fragment was cloned into the PstI/BamHI sites of pUC19 to create pUC19.Th14. To construct a HIS3 disruption of KRH1, a 1.8 kb HincII-SmaI fragment from pUC18-HIS3 was cloned into the HincII sites of KRH1 to produce Th14::HIS3. Plasmid YEp181-FLKRH1 was cloned by PCR using yeast genomic DNA as a template and contains sequences from the EcoRI site at nucleotide -755 (where +1 is the A of the start codon) to nucleotide +2778.
The KRH2 gene was cloned by amplifying a 0.8 kb fragment from yeast genomic DNA by PCR using primers oYOR1.5 (5′-CCGAGCTCGTATGGTATGGTGCCCATCAC-3′), which contains a flanking SacI site (shown in bold), and oYOR4 (5′-CCTTAGGTCTACCGTCAAAAGC-3′). This fragment was cloned into the SacI/HindIII sites of pUC19, using the introduced SacI site and an internal HindIII site, to produce pYOR37.1. A 1.1 kb fragment was amplified from yeast genomic DNA by PCR using primers oYOR5 (5′-CGAGTGTAATGCCAAGTGCCA-3′) and oYOR6 (5′-CCAAGCTTAATTGCATCATCCTCTAAATA-3′), which contains a flanking HindIII site (shown in bold). This fragment was cloned into the HindIII site of pYOR37.1, using the introduced HindIII site and an internal HindIII site, to produce pYOR37.12. To construct a URA3 disruption of KRH2, a 1.2 kb XbaI fragment from pAC100-2 was cloned into the XbaI sites of pYOR37.12 to produce pYOR37.12U. Plasmid YEp112-KRH2 was cloned by PCR using yeast genomic DNA as a template and contains sequences from the EcoRI site at nucleotide -766 (where +1 is the A of the start codon) to nucleotide +2701.
The plasmid used for isolating the FLO11 probe was made by amplifying a 0.5 kb fragment from pYSL12 (Lo and Dranginis, 1998) by PCR using primers oFLO3, 5′-GGGGATCCGTAACTCCTGCCACTAATGC-3′, which contains a flanking BamHI site (shown in bold), and oFLO4, 5′-CCACATAAAGTTTCCAAGAACCTTG-3′. The fragment was cloned into the BamHI/XhoI sites of pBluescript, using the introduced BamHI site and an internal XhoI site, to produce pFLO11CT.Bs. The plasmid used for isolating the ACT1 probe was made by amplifying a 0.9 kb fragment from yeast genomic DNA by PCR using primers GMS288 (5′-CCTCGTGCTGTCTTCCCATCTATC-3′) and GMS289 (5′-GCATTCTTTCGGCAATACCTGG-3′). The fragment was cloned into plasmid PCR2.1-TOPO to produce pActin.21.
Strain construction and media
Strains used in this study are listed in Table 1. The gpa2::TRP1 allele was made by transformation of cells with the 1.4 kb BamHI fragment from pgpa2-1::TRP1 (Xue et al., 1998). The sch9::URA3 allele was made by transformation of cells with a 3.8 kb HindIII fragment from plasmid pS9-7K::URA3.Bs. The tpk2::HIS3 allele was made by transformation of cells with a 3.6 kb EcoRI fragment from plasmid ptpk2::HIS3 (Toda et al., 1987b). The tpk2::TRP1 allele was made by transformation of a tpk2::HIS3 strain with a 3.8 kb SmaI/XhoI fragment from marker swap plasmid pHT6 (Cross, 1997). The krh2::URA3 allele was made by transformation of cells with the 1.6 kb SacI/HindIII fragment from pYOR37.12U. The krh2::TRP1 allele was made by transformation of a krh2::URA3 strain with a 3.6 kb SmaI fragment from marker swap plasmid pUT11 (Cross, 1997). The krh1::HIS3 allele was made by transformation of cells with the 2.3 kb SacI fragment from Th14::HIS3. The flo11::lacZ-HIS3 allele was made by transformation of cells with BstEII-digested plasmid pMUC1-lacZ. All strain constructions involving transformations were confirmed by Southern blot.
Strains were grown on YEPD (2% glucose), and strains under selection were grown on synthetic dropout media, as described (Guthrie and Fink, 1991).
Two-hybrid screen and yeast methods
pG2CT-T9.2 was transformed into reporter strain HF7c (Clontech) and the resulting strain was individually transformed with each of three yeast genomic DNA fusion libraries, Y2HL-C1, Y2HL-C2 and Y2HL-C3 (James et al., 1996). Transformation mixtures were plated on medium lacking histidine, and positive transformants were retested for β-galactosidase expression by incubation in the presence of 0.3 mg/ml X-gal. Plasmid TH14, which encodes a fusion to KRH1 at codon 531, was isolated in this screen.
Heat shock assays, sporulation assays, and yeast RNA extraction were performed as described previously (Xue et al., 1998). Invasive growth assays were performed according to Kuchin et al. (Kuchin et al., 2002). Yeast transformations were performed by the lithium acetate method using standard procedures (Guthrie and Fink, 1991).
Total RNA was isolated from cells grown to logarithmic phase. RNA was electrophoresed in a 0.9% agarose formaldehyde-containing gel. Following electrophoresis, the gel was incubated in 0.05 M NaOH, 0.15 M NaCl for 20 minutes and neutralized in 0.1 M Tris-HCl (pH 7.5), 0.15 M NaCl for 30 minutes. RNA was transferred to a nylon GeneScreen membrane (NEN) by applying a pressure of 80 mmHG with the Posiblot Pressure Blotter (Stratagene) for 2.5 hours using 1.5 M NaCl, 0.15 M sodium citrate as the transfer buffer. The RNA was UV crosslinked to the membrane using a Stratalinker UV box. Prehybridization was carried out at 42°C in a buffer containing 50% formamide, 0.75 M NaCl, 75 mM sodium citrate, 50 mM sodium phosphate (pH 6.5), 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin, 0.1% SDS, and 0.1 mg/ml of denatured salmon testes DNA (Sigma). Hybridization was carried out at 42°C in a buffer containing 50% formamide, 0.75 M NaCl, 75 mM sodium citrate, 50 mM sodium phosphate (pH 6.5), 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin, 12.5% dextran sulfate, and 0.1 mg/ml of denatured salmon testes DNA. Blots were washed four times for 15 minutes at 65°C with 0.3 M NaCl, 30 mM sodium citrate, 0.5% SDS and exposed to a phosphor storage screen and scanned with a PhosphorImager (Molecular Dynamics) or exposed to film. The probes used were gel-purified DNA restriction fragments 32P-labeled by random primer labeling using a Prime-It kit (Stratagene). The fragments used were a 0.5 kb BamHI/XhoI fragment from plasmid pFLO11CT.Bs for the FLO11 probe and a 0.9 kb EcoRI fragment from plasmid pActin.21 for the ACT1 probe.
Cells were viewed using Nomarski optics for differential interference contrast microscopy on a Zeiss Axiophot microscope. They were photographed with a 100× objective. Plates were viewed using a Zeiss Axioplan 2 microscope and photographed with a 2.5× objective.
Identification of KRH1
To identify proteins that interact with the active form of the Gpa2pα -subunit, a two-hybrid screen was performed using as bait the constitutively active form of Gpa2p encoded by the GPA2R273A allele. This allele encodes a protein that is predicted to exist predominantly in the GTP-bound state as a result of its impaired GTPase activity (Freissmuth and Gilman, 1989). The two-hybrid screen resulted in the isolation of a plasmid encoding amino acids 531-740 of open reading frame YAL056W, which encodes a protein of 880 amino acids. This gene had been assigned the name KRH1 (for kelch repeat homologue; see Discussion) in the Saccharomyces Genome Database. Expression of the C-terminal region of Krh1p fused to the Gal4p activation domain produced a positive signal forβ -galactosidase activity when present in cells expressing either wild-type or constitutively active Gpa2p fused to the Gal4p DNA binding domain (Table 2, plasmids Th14 and pGBT9-GPA2 or pG2CT-T9.2). Moreover, the GPA2R273A construct gave a two- to threefold higher level of β-galactosidase activity than the wild-type GPA2 construct. This result suggests that there is a slight preference for interaction of Krh1p with the GTP-bound form of Gpa2p. In contrast, a construct containing the entire open reading frame of KRH1 fused to the GAL4 activation domain did not generate a positive signal for β-galactosidase activity when expressed with either the GPA2 or GPA2R273A constructs (Table 2, plasmids pGPE2-424.2 and pGBT9-GPA2 or pG2CT-T9.2). Therefore, if Krh1p binds to Gpa2p under physiological conditions, this interaction may be subject to additional regulatory mechanisms that expose the C-terminal binding site on Krh1p.
KRH1 and KRH2 encode negative regulators of FLO11 expression and invasive growth
A search of the Saccharomyces Genome Database revealed that a gene with high homology to KRH1 is present in the yeast genome. The protein encoded by this gene, which is called KRH2, is 35% identical to Krh1p. If Krh1p and Krh2p function in the Gpa2p signaling pathway, then deletion of the KRH1 and KRH2 genes would be expected to affect cellular processes that require GPA2. Such processes include the ability of cells to undergo the transition to pseudohyphal or invasive growth (Kübler et al., 1997; Lorenz and Heitman, 1997). The involvement of the KRH1 and KRH2 gene products in invasive growth was investigated in a strain of the Σ1278b background, which contains an intact signaling pathway for this process (Kron, 1997).
Haploid invasive growth correlates with induction of the FLO11 gene, which encodes a flocculin that is required for both pseudohyphal and invasive growth (Lo and Dranginis, 1998). To test whether the KRH1 and KRH2 genes are involved in the signaling pathway that results in invasive growth, the expression of FLO11 was determined in strains containing deletions of these genes. Under conditions of log phase growth, the abundance of FLO11 RNA was low but detectable in wild-type cells (Fig. 1A, lane 1). In cells containing a gpa2Δ mutation, the level of FLO11 RNA was greatly decreased relative to wild-type cells (lane 5), as described previously (Lorenz et al., 2000; Tamaki et al., 2000). In contrast, individual deletions of KRH1 and KRH2, or double deletion of both KRH1 and KRH2 resulted in an increase in FLO11 RNA abundance over the level seen in wild-type cells (lanes 2-4). Quantification of the normalized results from several experiments showed that the level of FLO11 RNA in krh1Δ krh2Δ cells is three- to fourfold higher than in wild-type cells. Because deleting the genes causes activation of the signaling pathway, these results indicate that KRH1 and KRH2 act as negative regulators of signaling. One possible interpretation of these findings is that Krh1p and Krh2p inhibit signaling of a component that acts downstream of Gpa2p under conditions that promote turning the pathway off. Alternatively, Krh1p and Krh2p could be required for modification or localization of the Gpa2p protein. To distinguish between these possibilities, the effect of the krh1Δ and krh2Δ mutations was determined in a strain lacking Gpa2p. In triple mutant krh1Δ krh2Δ gpa2Δ cells, the level of FLO11 RNA was substantially higher than that seen in gpa2Δ cells (lanes 5 and 6). A comparison of krh1Δ krh2Δ cells with krh1Δ krh2Δ gpa2Δ cells revealed that the presence of the gpa2Δ mutation in a krh1Δ krh2Δ background causes an approximately two- to threefold decrease in FLO11 RNA abundance (lanes 4 and 6). However, the level of FLO11 RNA was more than tenfold higher in krh1Δ krh2Δ gpa2Δ cells than in gpa2Δ cells, indicating that the predominant effect of the krh1Δ krh2Δ mutations is to suppress the phenotype of the gpa2Δ mutation.
Induction of the FLO11 gene generally correlates with an increase in invasive growth in haploid cells and pseudohyphal growth in diploid cells. To test whether the increased level of FLO11 expression in krh1Δ krh2Δ cells has physiological consequences, the ability of wild-type and krh1Δ krh2Δ strains to undergo haploid invasive growth was determined. krh1Δ, krh2Δ and krh1Δ krh2Δ cells showed a significant increase in their ability to invade solid medium when compared with wild-type cells (Fig. 1B). The most pronounced effect was seen with krh1Δ krh2Δ double mutant cells. Whereas gpa2Δ cells did not invade the medium to a significant degree, krh1Δ krh2Δ gpa2Δ cells displayed substantial invasive growth. Therefore, the krh1Δ krh2Δ mutations suppress the defect in invasive growth conferred by a gpa2Δ mutation. Deletion of the FLO11 gene eliminated the increase in invasiveness conferred by the krh1Δ krh2Δ mutations, consistent with previously identified characteristics of invasive growth (Fig. 1B).
In addition to the phenotype displayed by krh1Δ krh2Δ cells on solid medium, krh1Δ krh2Δ cells grown in rich liquid medium for two days displayed an altered morphology. In liquid medium, krh1Δ krh2Δ cells formed chains of elongated cells that appear similar to cells undergoing the early stages of pseudohyphal growth (Fig. 1C). Depletion of glucose from the medium, which occurs under these conditions, is known to promote invasive growth in haploid cells (Cullen and Sprague, 2000). Introduction of a gpa2Δ mutation into a krh1Δ krh2Δ strain had no effect on the altered morphology of cells grown to saturation in liquid medium (data not shown). Given that pseudohyphal formation normally requires growth on solid medium, and that krh1Δ krh2Δ cells overcome this requirement, it can be concluded that the KRH1 and KRH2 gene products exert a strong negative effect on filamentous forms of growth in wild-type cells.
Another morphological phenotype conferred by the krh1Δ krh2Δ mutations involves the macroscopic appearance of patches of cells. Deletion of the KRH1 and KRH2 genes causes cell cultures incubated on solid medium to grow up off the plate in extended sheets (Fig. 1D). This macroscopic phenotype is reminiscent of that seen when S. cerevisiae is grown under conditions of fungal biofilm formation (Reynolds and Fink, 2001). Biofilm formation in S. cerevisiae requires FLO11 (Reynolds and Fink, 2001), suggesting that the phenotype of krh1Δ krh2Δ mutants may be the result of increased cell adhesion due to higher levels of FLO11 expression.
KRH1 and KRH2 negatively regulate processes controlled by the cAMP/PKA pathway
Gpa2p activity also has effects in strains that are not capable of undergoing pseudohyphal or invasive growth. For example, cells of a non-filamentous strain that contain a constitutively active allele of GPA2 display a decrease in sporulation efficiency and an increase in heat shock sensitivity (Xue et al., 1998). Increased sensitivity to heat shock is a phenotype associated with cells that have increased activity of growth control pathways, including both the cAMP/PKA pathway and other redundant pathways (Cameron et al., 1988; Toda et al., 1987a). To determine whether the KRH1 and KRH2 genes play a role in heat shock sensitivity, survival following a heat shock was measured for wild-type, krh1Δ, krh2Δ and krh1Δ krh2Δ cells in a W303 background. Whereas 48% of wild-type cells in stationary phase survived after a 50°C heat shock, only 1.0% of krh1Δ krh2Δ cells survived after this treatment (Fig. 2A). Single krh1Δ and krh2Δ mutants displayed intermediate survival levels of 2.8% and 22.4%, respectively. This finding confirms that the KRH1 and KRH2 gene products are negative regulators of the signaling pathway because an increase in heat shock sensitivity occurs either when constitutively active GPA2 is present (Xue et al., 1998) or when the KRH1 and KRH2 genes are deleted.
The effect of krh1Δ krh2Δ mutations on the ability of cells to sporulate was also examined. Cells containing intact KRH1 and KRH2 genes displayed 51% sporulation efficiency (Fig. 2B). krh1Δ, krh2Δ and krh1Δ krh2Δ cells all showed a decrease in sporulation efficiency. The largest effect was seen in krh1Δ krh2Δ cells, which had a sporulation efficiency of 6%. Single krh1Δ and krh2Δ mutants displayed intermediate levels of sporulation of 26% and 35%, respectively. Therefore, deletion of the KRH1 and KRH2 genes results in multiple physiological changes that are associated with increased activation of the cAMP/PKA pathway.
The interaction between Krh1p and Gpa2p revealed by the two-hybrid assay raised the possibility that Krh1p and Krh2p are proteins that interact with Gα subunits in general. Therefore, it was possible that these proteins also interact with Gpa1p, the Gα subunit that mediates the pheromone response pathway. krh1Δ, krh2Δ and krh1Δ krh2Δ cells were tested for their ability to undergo cell cycle arrest in response to pheromone by measuring the density of cell growth in an area surrounding a filter disk containing α-factor (halo assay). No differences were detected in this assay among wild-type, krh1Δ, krh2Δ or krh1Δ krh2Δ cells (data not shown). Therefore, the function of KRH1 and KRH2 appears to be specific to the Gpa2p pathway.
KRH1 and KRH2 act downstream of GPA2
If Krh1p and Krh2p couple Gpa2p to downstream components, then deletion of the KRH1 and KRH2 genes would be expected to eliminate the effects of a constitutively active form of Gpa2p. To test this idea, the effect of Gpa2p activation on FLO11 RNA abundance was determined by isolating RNA from strains containing either vector or the constitutively active GPA2R273A allele. In wild-type cells, FLO11 RNA levels were significantly higher in cells containing the GPA2R273A allele than in cells containing vector alone (Fig. 3A, lanes 1,2). These findings are in agreement with previous results that indicate that GPA2 is involved in the transition to filamentous growth (Kübler et al., 1997; Lorenz and Heitman, 1997). The presence of the GPA2R273A allele had no effect on FLO11 RNA abundance in the krh1Δ krh2Δ strain (lanes 3 and 4). One interpretation of this result is that transmission of the signal generated by activated Gpa2p is largely prevented by deletion of KRH1 and KRH2.
To determine whether the KRH1 and KRH2 genes are required for the effect of Gpa2p activation on heat shock sensitivity, survival following a heat shock was measured in wild-type and krh1Δ krh2Δ strains. Heat shock treatment of wild-type cells containing the constitutively active GPA2R273A allele caused a decrease in survival of approximately 70-fold compared with vector-containing cells (Fig. 3B), confirming previous results (Xue et al., 1998). However, krh1Δ krh2Δ cells containing either vector or the GPA2R273A plasmid displayed the same survival levels (Fig. 3B). Elimination of the effects of the GPA2R273A allele by the krh1Δ krh2Δ mutations is consistent with a model in which Krh1p and Krh2p act downstream of the Gpa2p α-subunit.
Signal generated by lack of KRH1 and KRH2 does not require SCH9
Previous results from our laboratory suggested that the Sch9p kinase acts downstream of Gpa2p (Xue et al., 1998). It was therefore of interest to test whether deletion of the SCH9 gene blocks the signal resulting from deletion of the KRH1 and KRH2 genes. If Krh1p and Krh2p act immediately downstream of Gpa2p, then components that act further downstream in the same pathway would be expected to eliminate the signal generated by the absence of the negative regulators. To determine the effect of an sch9Δ mutation on the phenotype conferred by the krh1Δ and krh2Δ mutations, the heat shock sensitivity of strains containing different mutations was measured. Cells containing an sch9Δ mutation did not display a heat shock sensitive phenotype (Fig. 4A), in agreement with previous results (Xue et al., 1998). Heat shock treatment of the triple mutant krh1Δ krh2Δ sch9Δ strain caused a large decrease in survival, similar to the effect seen in a krh1Δ krh2Δ strain (Fig. 4A). Therefore, the sch9Δ mutation does not eliminate the phenotype caused by deletion of the KRH1 and KRH2 genes. This finding suggests that Sch9p does not act downstream in a pathway that is negatively regulated by Krh1p and Krh2p.
The data presented above are consistent with the idea that Krh1p and Krh2p act downstream of Gpa2p to negatively regulate a signaling pathway that does not require Sch9p. If this were the case, then krh1Δ krh2Δ mutations would be expected to suppress phenotypes present in gpa2Δ sch9Δ double mutants by activating the downstream pathway. To test this possibility, strains were constructed that contained either deletions of KRH1 and KRH2, deletions of GPA2 and SCH9, or all four mutations together. krh1Δ krh2Δ mutant strains did not display an apparent growth defect (Fig. 4B). gpa2Δ sch9Δ mutant strains displayed a severe growth defect and did not produce visible colonies after 2 days of growth, as described previously (Kraakman et al., 1999; Lorenz et al., 2000). Strains containing all four mutations showed substantial suppression of the growth defect seen in the gpa2Δ sch9Δ strain. These results indicate that loss of KRH1 and KRH2 results in activation of a pathway that partially compensates for the lack of SCH9.
Signal generated by lack of KRH1 and KRH2 or activation of Gpa2p requires TPK2
Activation of the RAS/cAMP pathway suppresses the growth defect conferred by an sch9Δ mutation (Hartley et al., 1994; Toda et al., 1988). Although the Ras proteins function independently of Gpa2p (Colombo et al., 1998; Xue et al., 1998), suppression of the sch9Δ growth defect by krh1Δ krh2Δ mutations could be explained by activation of downstream components of the RAS/cAMP pathway under these conditions. One candidate for a downstream component that is activated in krh1Δ krh2Δ cells is the cAMP-dependent protein kinase Tpk2p. In yeast, cAMP-dependent protein kinase is encoded by three genes, TPK1, TPK2 and TPK3 (Toda et al., 1987b). However, only the TPK2 gene product positively regulates FLO11 transcription (Pan and Heitman, 1999; Robertson and Fink, 1998). To determine whether TPK2 is required for the signal generated by krh1Δ krh2Δ mutations, the basal level of FLO11 RNA was measured in cells containing different combinations of mutations. FLO11 RNA was present at a basal level in wild-type cells, but was undetectable in cells containing a tpk2Δ mutation (Fig. 5A, lanes 1,2), as described previously (Pan and Heitman, 1999; Robertson and Fink, 1998). FLO11 RNA was also undetectable in krh1Δ krh2Δ tpk2Δ triple mutant cells (lane 4), demonstrating that TPK2 is required for the signal generated in krh1Δ krh2Δ cells. These results imply that the signal transmitted through Krh1p and Krh2p inhibits the activity of the Tpk2p kinase, either directly or indirectly.
Krh1p and Krh2p appear to act downstream of Gpa2p and upstream of the cAMP-dependent protein kinase Tpk2p. To investigate further the ordering of the signaling pathway, the requirement for TPK2 in signaling through activated Gpa2p was determined. Wild-type cells carrying the GPA2R273A allele contained significantly more FLO11 RNA than cells carrying vector alone (Fig. 5B, lanes 1,2), as described above. However, no detectable FLO11 RNA was present in tpk2Δ mutant cells carrying either vector or the GPA2R273A allele (lanes 3,4), indicating that TPK2 acts downstream of GPA2.
The effect of a tpk2Δ mutation on the altered cell morphology conferred by the krh1Δ krh2Δ mutations was also examined. Whereas krh1Δ krh2Δ mutants grown to saturation in rich medium formed chains of elongated cells, krh1Δ krh2Δ tpk2Δ mutants formed clusters of more rounded cells (Fig. 5C). Therefore, the tpk2Δ mutation largely suppresses the cell morphology phenotype of krh1Δ krh2Δ cells. In addition, krh1Δ krh2Δ tpk2Δ cells displayed significantly less haploid invasive growth than krh1Δ krh2Δ cells when the plate washing was done after 3 days of growth (data not shown).
The effect of TPK2 overexpression was determined in both wild-type and krh1Δ krh2Δ strains. In wild-type cells, TPK2 overexpression resulted in a large increase in the abundance of FLO11 RNA (Fig. 5D, lanes 1,2). However, TPK2 overexpression had no effect on the high level of FLO11 RNA present in cells containing deletions of the KRH1 and KRH2 genes (lanes 3,4).
Cells containing krh1Δ krh2Δ mutations display phenotypes also seen in cells containing a constitutively active allele of GPA2. These phenotypes include increased expression of FLO11, increased invasive growth, decreased survival following heat shock, and decreased efficiency of sporulation. Moreover, deletion of the TPK2 gene eliminates the effects of both krh1Δ krh2Δ mutations and constitutively active GPA2. These observations, coupled with the finding that KRH1 was identified in a two-hybrid screen with GPA2, strongly suggest that Krh1p, Krh2p and the Gpa2p α-subunit are involved in the same signaling process. To investigate the relationship between KRH1, KRH2 and GPA2, the phenotype of cells containing mutations in all three genes were studied. Whereas gpa2Δ mutant cells did not invade the agar and expressed a very low level of FLO11 RNA, krh1Δ krh2Δ gpa2Δ mutant cells displayed increased invasive growth and a high level of FLO11 RNA. Similarly, krh1Δ krh2Δ mutations suppressed the growth defect of gpa2Δ sch9Δ mutant cells. The finding that krh1Δ krh2Δ mutations suppress a gpa2Δ mutation indicates that the negative regulatory function of Krh1p and Krh2p is likely to act on a downstream component of the Gpa2p signaling pathway rather than on Gpa2p itself. One model to explain these results is that binding of Krh1p and Krh2p to Gpa2p prevents them from inhibiting the activity of a downstream component. This idea is supported by results showing that krh1Δ krh2Δ mutations eliminate the effects of a constitutively active allele of GPA2. In this case, the link between Gpa2p and downstream components of the pathway would be missing due to the absence of the KRH1 and KRH2 gene products. An observation not explained by this model is the finding that krh1Δ krh2Δ gpa2Δ cells express two- to threefold less FLO11 RNA than krh1Δ krh2Δ cells. This situation could occur if signaling through Gpa2p is partially independent of Krh1p and Krh2p under some circumstances. Alternatively, Krh1p and Krh2p could have multiple negative regulatory functions and could exert their effects by inhibiting Gpa2p signaling as well as inhibiting another component in the pathway that acts upstream of Tpk2p. Nevertheless, several experiments show that deletion of KRH1 and KRH2 activates signaling in cells that lack Gpa2p and prevents signaling by constitutively active Gpa2p. These observations are most consistent with a model in which Krh1p and Krh2p mediate signaling from Gpa2p to downstream components.
The association between GPA2 mutant phenotypes and cAMP signaling has led to the suggestion that yeast adenylyl cyclase is directly activated by the Gα subunit Gpa2p. This situation would be analogous to the direct activation of mammalian adenylyl cyclase by the Gα subunit Gαs. In contrast to this idea, we have shown that the novel proteins Krh1p and Krh2p appear to act immediately downstream of Gpa2p. Moreover, Krh1p and Krh2p require the cAMP-dependent kinase Tpk2p for their signaling function, in agreement with previous observations that show an association between GPA2 and cAMP signaling. Therefore, it seems likely that the effect of Gpa2p on cAMP signaling is mediated by Krh1p and Krh2p. However, overexpression of TPK2 had no effect in cells containing krh1Δ krh2Δ mutations. Therefore, further experiments will be needed to determine whether this result is due to maximal activation of the pathway in krh1Δ krh2Δ cells or to the existence of a complex relationship between Krh1p, Krh2p and Tpk2p.
Krh1p and Krh2p do not display any obvious sequence identity to other known proteins, but they do contain six repeats that display some similarity to kelch repeats (Fig. 6). Kelch repeats are segments of about 50 amino acids that contain a characteristic double glycine motif (Adams et al., 2000). The double glycine is situated C-terminal to four hydrophobic residues and N-terminal to conserved tyrosine and tryptophan residues that are separated from it by spacer regions. The repeats in Krh1p and Krh2p also contain a double glycine motif situated C-terminal to four hydrophobic residues. However, only one of these repeats (Fig. 6, repeat 3) contains the conserved tyrosine and tryptophan residues, and the spacer region between these residues and the double glycine repeat is smaller than that seen in the consensus for kelch repeats. The other repeats are missing either the tyrosine or tryptophan at the appropriate position. Therefore, they can be thought of as variant kelch repeats. The crystal structure of a protein containing seven kelch repeats has revealed that each repeat forms a four-strandedβ -sheet, resulting in a protein that consists of a seven-bladedβ -propeller (Ito et al., 1994). This 3D structure is similar to that of G proteinβ -subunits, which contain seven WD domains that form a seven-bladedβ -propeller (Sondek et al., 1996; Wall et al., 1995). These observations raise the interesting possibility that Krh1p and Krh2p bind to Gpa2p in a manner similar to that by which Gβ subunits bind to Gα subunits. Given that Gpa2p is a positive regulator of the signaling pathway and that Krh1p and Krh2p are negative regulators, it would be predicted that binding of Gpa2p to Krh1p and Krh2p blocks their ability to inhibit downstream steps in the pathway.
The KRH1 gene was identified previously by a mutation that causes cells to continue to grow when incubated in alkaline sporulation medium (Ohkuni and Yamashita, 2000). This medium contains acetate as a carbon source and is limiting for nitrogen, a nutritional condition that induces sporulation in wild-type cells. Previous results have shown that mutational activation of Gpa2p inhibits sporulation to a significant degree (Xue et al., 1998). The finding that a loss-of-function mutation in the KRH1 gene causes cells to continue to divide under conditions that induce sporulation is consistent with our observation that the sporulation efficiency of krh1Δ krh2Δ mutants is very low.
The KRH2 gene was isolated previously as one of 29 genes that encode proteins that interact with the nuclear export factor Crm1p in a two-hybrid assay (Jensen et al., 2000). Although KRH2 was not characterized further in that study, interaction with Crm1p could indicate that Krh2p is capable of nucleocytoplasmic shuttling. It is therefore of interest to note that other proteins involved in cAMP signaling, such as the catalytic and regulatory subunits of cAMP-dependent kinase, localize differentially to the nucleus or cytoplasm in response to different physiological conditions (Griffioen et al., 2000). Differential localization of Krh2p could limit inhibition of the signaling pathway to a particular cellular compartment, providing an additional level of regulation to the pathway.
Although it is likely that Krh1p and Krh2p function by transmitting a signal from Gpa2p to downstream components, the pathway that relays this signal to its ultimate physiological targets is not clearly understood. For example, work presented here demonstrates that the Sch9p kinase is not required for the increase in FLO11 RNA that is observed in cells lacking KRH1 and KRH2. However, we have shown previously that SCH9 is required for the increase in heat shock sensitivity conferred by a constitutively active allele of GPA2 (Xue et al., 1998). Other experiments exploring the relationship between SCH9 and GPA2 showed that SCH9 is required for the increase in trehalase activity observed when a nitrogen source is added to cells starved for nitrogen, but that GPA2 is not required for this process (Crauwels et al., 1997; Kraakman et al., 1999). Similarly, GPA2 is required for the increase in cAMP observed when glucose is added to cells starved for glucose, but SCH9 is not required for this process (Colombo et al., 1998; Crauwels et al., 1997). Moreover, double mutant sch9Δ gpa2Δ cells have a much more severe growth defect than either of the single mutants, suggesting that GPA2 and SCH9 function, at least in part, in different pathways (Kraakman et al., 1999; Lorenz et al., 2000). One interpretation of the relationship between SCH9 and GPA2 is that the Sch9p kinase is required only for a subset of the phenotypes associated with activation of Gpa2p. In that case, it would suggest that there are branchpoints in the signaling pathway downstream of Gpa2p. Alternatively, it is possible that Sch9p is required only for signaling through the Gpa2p pathway under particular growth conditions. Such a result would suggest that Gpa2p couples to different downstream components depending on growth conditions.
The uncovering of a novel class of signaling molecules that act downstream of a Gα subunit could have implications for G protein signaling in a wide variety of eukaryotic organisms. A homologue of KRH2 is present in the genome of the yeast Kluyveromyces lactis (Ozier-Kalogeropoulos et al., 1998), and potential homologues also exist in multicellular organisms. It is likely that these KRH1 and KRH2 homologues couple G proteins to downstream signaling components in other organisms. Therefore, further studies of the function of Krh1p and Krh2p in signaling through the Gpa2p pathway have the potential to contribute to the understanding of general mechanisms of G protein signaling.
This work was supported by Grant-In-Aid 9951029T from the American Heart Association, Heritage Affiliate and by grant GM60332 from the National Institutes of Health. We thank P. James and E. Craig for the yeast two-hybrid libraries and C. Denis, A. Dranginis, D. van Dyk, I. Pretorius, S. Garrett, S. Palecek, S. Kron, L. Robertson and G. Fink for plasmids and strains used in this work. We thank K. Ray for identification of the repeat sequences in KRH1 and KRH2 and R. Kelso and L. Cooley for helpful comments on the repeat sequences. We also thank Alina Toma for excellent technical assistance. Microscopy was performed in the Mount Sinai Microscopy Shared Resources Facility with the help of Scott Henderson.
- Accepted November 11, 2002.
- © The Company of Biologists Limited 2003