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• W2093434319 abstract "Article1 December 1997free access Yeast pseudohyphal growth is regulated by GPA2, a G protein α homolog Michael C. Lorenz Michael C. Lorenz Departments of Genetics and Pharmacology and the Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC, 27710 USA Search for more papers by this author Joseph Heitman Corresponding Author Joseph Heitman Departments of Genetics and Pharmacology and the Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC, 27710 USA Search for more papers by this author Michael C. Lorenz Michael C. Lorenz Departments of Genetics and Pharmacology and the Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC, 27710 USA Search for more papers by this author Joseph Heitman Corresponding Author Joseph Heitman Departments of Genetics and Pharmacology and the Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC, 27710 USA Search for more papers by this author Author Information Michael C. Lorenz1 and Joseph Heitman 1 1Departments of Genetics and Pharmacology and the Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC, 27710 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:7008-7018https://doi.org/10.1093/emboj/16.23.7008 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Pseudohyphal differentiation, a filamentous growth form of the budding yeast Saccharomyces cerevisiae, is induced by nitrogen starvation. The mechanisms by which nitrogen limitation regulates this process are currently unknown. We have found that GPA2, one of the two heterotrimeric G protein α subunit homologs in yeast, regulates pseudohyphal differentiation. Δgpa2/Δgpa2 mutant strains have a defect in pseudohyphal growth. In contrast, a constitutively active allele of GPA2 stimulates filamentation, even on nitrogen-rich media. Moreover, a dominant negative GPA2 allele inhibits filamentation of wild-type strains. Several findings, including epistasis analysis and reporter gene studies, indicate that GPA2 does not regulate the MAP kinase cascade known to regulate filamentous growth. Previous studies have implicated GPA2 in the control of intracellular cAMP levels; we find that expression of the dominant RAS2Gly19Val mutant or exogenous cAMP suppresses the Δgpa2 pseudohyphal defect. cAMP also stimulates filamentation in strains lacking the cAMP phosphodiesterase PDE2, even in the absence of nitrogen starvation. Our findings suggest that GPA2 is an element of the nitrogen sensing machinery that regulates pseudohyphal differentiation by modulating cAMP levels. Introduction In response to severe nitrogen starvation, diploid cells of the budding yeast Saccharomyces cerevisiae undergo a dimorphic transition known as pseudohyphal differentiation (Gimeno et al., 1992). During pseudohyphal growth, cells adopt a unipolar budding pattern, elongate and can invade the growth substrate. In addition, cells remain attached following cytokinesis, resulting in chains of cells reminiscent of fungal hyphae. It has been suggested that this mode of growth may allow this non-motile species to forage for nutrients under adverse conditions (Gimeno et al., 1992; Kron et al., 1994). Pseudohyphal differentiation is regulated, in part, by elements of the MAP kinase cascade which also controls mating, namely the protein kinases STE20, STE11 and STE7 and the transcription factor STE12 (Liu et al., 1993). A heterodimer composed of STE12 and TEC1 binds to a filamentation response element (FRE), which may regulate expression of genes necessary for pseudohyphal differentiation (Laloux et al., 1994; Mösch et al., 1996; Madhani and Fink, 1997). A reporter gene under the control of an FRE [FG(TyA)::lacZ] is induced under nitrogen starvation conditions in a STE12- and TEC1-dependent manner (Gavrias et al., 1996; Mösch et al., 1996; Madhani and Fink, 1997). The proximal elements that regulate mating, the pheromone receptors (STE2 and STE3) and the heterotrimeric guanine nucleotide binding protein (GPA1, STE4 and STE18), are not expressed in diploids and play no role in pseudohyphal differentiation (Liu et al., 1993). The mechanisms by which nitrogen starvation activates the MAP kinase cascade or triggers filamentous growth are not known, but the parallels between the signalling pathways in haploid and diploid cells led us to consider whether pseudohyphal growth might also be G protein regulated. Heterotrimeric G proteins mediate a vast array of signalling processes in all eukaryotes, including mating response and development in fungi, vision in mammals and metazoans, and neurotransmitter and hormone action in mammals (reviewed in Neer, 1995; Borkovich, 1996). These regulatory complexes typically serve as a bridge between transmembrane receptors and effectors, including adenylate cyclase, phospholipases or protein kinases. A ligand-bound receptor can either activate or inhibit effectors via the G protein. The activity of the G protein is controlled by a guanine nucleotide cycle in which an activated receptor promotes GDP to GTP exchange on the α subunit of the G protein. GTP binding stimulates dissociation of the α subunit from the βγ complex, and either the free α or free βγ dimer, or in some cases both, regulate downstream effectors. The intrinsic GTPase activity of the α subunit hydrolyzes GTP to GDP, promoting reassociation of the heterotrimer and attenuation of signalling. The best characterized Gα subunit in S.cerevisiae, GPA1 (also known as SCG1), regulates mating response by binding to the pheromone receptors STE2 (α-factor receptor) and STE3 (a-factor receptor; reviewed in Kurjan, 1993; Bardwell et al., 1994). In this pathway, the free βγ subunit transmits the signal to a conserved MAP kinase cascade through the interaction of a large multimeric complex (Leeuw et al., 1995; Akada et al., 1996). Haploid Δgpa1 mutants are inviable due to constitutive activation of the pheromone response pathway and subsequent cell cycle arrest (Miyajima et al., 1987). G proteins also mediate mating response in the fission yeast Schizosaccharomyces pombe. In this organism two distinct Gγ subunits regulate the two environmental signals required for mating: the presence of pheromone is signalled by Gpa1 (Obara et al., 1991) and nitrogen starvation is signalled by Gpa2 (Isshiki et al., 1992). Morphogenesis triggered by environmental stimuli is regulated by G protein action in the fungi Coprinus congregatus (Kozak et al., 1995) and Neurospora crassa (Ivey et al., 1996). The yeast genome project has identified only one protein other than GPA1 likely to encode a Gα subunit, GPA2, which was first isolated on the basis of its homology to a rat brain Gαs isoform (Nakafuku et al., 1988). Addition of glucose to glucose-starved yeast cells induces a rapid but transient increase in cAMP levels. Expression of GPA2 from a high copy plasmid enhances this rise in cAMP (Nakafuku et al., 1988; Papasavvas et al., 1992). The glucose-stimulated cAMP pulse is partially inhibited in the presence of mating pheromone; this inhibition requires GPA2 and appears to involve interactions between GPA2 and RAS2 (Arkinstall et al., 1991; Papasavvas et al., 1992). Yeast Ras modulates adenylate cyclase activity and cAMP levels (Toda et al., 1985; Field et al., 1988), and overexpression of GPA2 suppresses the growth defect of a temperature-sensitive ras2 mutant (Nakafuku et al., 1988). High levels of cAMP activate the cAMP-dependent protein kinase (protein kinase A, PKA); among its many functions, PKA mediates cellular responses to nutrient stress, heat shock and oxidative damage (reviewed in Broach and Deschenes, 1990). GPA2 and S.pombe Gpa2 are highly homologous (42% identity). The fission yeast Gpa2 protein is known to regulate mating in response to nitrogen starvation; S.pombe gpa2− mutant cells mate under nutrient-rich conditions unlike wild-type gpa2+ cells (Isshiki et al., 1992). This phenotype is shared by S.pombe mutants lacking adenylate cyclase (Maeda et al., 1990), and Gpa2 regulates cAMP levels in fission yeast (Isshiki et al., 1992). Ras also regulates filamentous growth in budding yeast, and activated mutants of yeast RAS2 stimulate pseudohyphal differentiation (Gimeno et al., 1992). It was initially proposed that RAS2 would regulate pseudohyphal differentiation by modulating cAMP levels (Gimeno et al., 1992), a suggestion supported by the finding that overproduction of the cAMP phosphodiesterase PDE2 inhibits pseudohyphal growth in wild-type strains and blocks hyperfilamentation induced by the dominant active RAS2Gly19Val mutant (Ward et al., 1995). Other work, however, has suggested that Ras acts upstream of the MAP kinase cascade (Mösch et al., 1996; Madhani and Fink, 1997). The interactions observed between RAS2, GPA2 and cAMP led us to test whether GPA2 is involved in filamentous growth. We report here that Δgpa2/Δgpa2 mutant strains have a defect in filamentous growth. Moreover, a dominant active GPA2 allele stimulates pseudohyphal differentiation, even on nitrogen-rich media. Epistasis analysis and reporter gene experiments, however, indicate that GPA2 does not act upstream of the MAP kinase cascade. We also find that cAMP stimulates filamentation, and both RAS2Gly19Val and cAMP suppress the pseudohyphal defect conferred by the Δgpa2 mutation. We propose that GPA2 is an element of the nitrogen sensing machinery which signals pseudohyphal differentiation under starvation conditions via a signalling pathway involving cAMP. Results The GPA2 Gα protein regulates pseudohyphal growth Elements upstream of the pheromone responsive MAP kinase cascade which sense nitrogen starvation during pseudohyphal growth have not yet been identified. In fission yeast, the Gα subunit Gpa2 regulates mating in response to nitrogen limitation; this G protein is distinct from the pheromone-stimulated Gpa1 protein (Obara et al., 1991; Isshiki et al., 1992). These studies led us to test whether nitrogen sensing in budding yeast might also be G protein-mediated. Previous work found no role for S.cerevisiae GPA1 in pseudohyphal differentiation (Liu et al., 1993), thus we focused on GPA2, which has been implicated in the regulation of cAMP levels and Ras functions (Nakafuku et al., 1988). We replaced the GPA2 open reading frame with a gene encoding resistance to G418 in the Σ1278b background (Grenson et al., 1966) commonly used for analysis of filamentous growth (Gimeno et al., 1992). Cells lacking GPA2 (gpa2-Δ1) were viable and had no obvious defects in growth, mating or sporulation (data not shown). A homozygous Δgpa2/Δgpa2 diploid strain, however, exhibited a defect in pseudohyphal differentiation when grown on low nitrogen (50 μM ammonium sulfate) SLAD medium (Figure 1). Δgpa2/Δgpa2 mutant strains filament weakly, as do strains with mutations of MAP kinase elements (with the exception of Δste20 mutant strains, which do not filament at all; Liu et al., 1993). Expression of wild-type GPA2 complemented and restored pseudohyphal growth in Δgpa2/Δgpa2 mutant strains (data not shown). The pseudohyphal defect conferred by the Δgpa2 mutation was also observed when cells were grown in the presence of limiting concentrations of proline, glutamine or urea (data not shown), indicating that GPA2 is a general regulator of filamentation. Figure 1.GPA2 regulates pseudohyphal growth. Diploid strains of the Σ1278b background with the indicated genotypes (wild-type, MLY61; Δgpa2/Δgpa2, MLY132a/α; Δste11/Δste11, HLY506) were incubated on nitrogen limiting SLAD medium for 4 days at 30°C. Download figure Download PowerPoint A dominant active GPA2 allele enhances pseudohyphal growth Several mutations have been described in other Gα subunits that perturb proper function of the G protein and have been invaluable in dissecting G protein function. One such mutation, in which valine replaces the second glycine in the highly conserved GXGXXG motif, has been shown to reduce the GTPase activity of Gαs and the small G protein Ras (Graziano and Gilman, 1989; Masters et al., 1989), thus promoting the GTP-bound and active forms of these proteins. The human H-rasGly12Val mutation confers a transforming phenotype (reviewed in Lowy and Willumsen, 1993). In yeast, RAS2Gly19Val constitutively activates adenylate cyclase, raises intracellular cAMP levels (Toda et al., 1985) and promotes pseudohyphal differentiation (Gimeno et al., 1992). The corresponding mutation in GPA1, Gly50Val, increases basal activation of the pheromone response pathway, leading to a growth defect and impaired adaptation to prolonged pheromone exposure (Kurjan et al., 1991). To characterize further the function of GPA2, we constructed an allele with the analogous mutation, Gly132Val (GPA2-2), under the control of a galactose-inducible promoter in a low-copy, centromeric plasmid. Expression of the GPA2Gly132Val allele on nitrogen limiting medium containing galactose greatly enhanced pseudohyphal differentiation (Figure 2A). This stimulation was not observed with expression of the wild-type allele or when expression was repressed with glucose (Figure 2A). In addition, this activated allele did not promote filamentous growth when expressed in a haploid strain (data not shown). Strains expressing the GPA2Gly132Val allele grew more slowly than wild-type strains, and formed smaller colonies. This is not unusual in hyperfilamentous strains, as overexpression of STE12 confers a similar phenotype (Liu et al., 1993, and data not shown). Figure 2.A dominant GPA2 allele stimulates pseudohyphal growth. (A) Wild-type diploid strain MLY61 with a control plasmid (vector), or plasmids expressing GPA2 (pML180) or GPA2Gly132Val (pML160) under repressing (glucose, left) or inducing (galactose, right) conditions were incubated on nitrogen limiting medium (SLAD or SLARG) for 4 days at 30°C. The colonies were photographed at 25× magnification. (B) The strains and GPA2 plasmids indicated in (A) were incubated on nitrogen-rich medium containing 5 mM ammonium sulfate for 2 days at 30°C under repressing (glucose, left) or inducing (galactose, right) conditions. The colonies were photographed at 50× magnification. Download figure Download PowerPoint If GPA2 is indeed a component of the nitrogen sensor, the GPA2Gly132Val allele might induce filamentation under nitrogen-rich conditions in which this developmental pathway is normally repressed. Consistent with this hypothesis, expression of the GPA2Gly132Val mutation stimulated filamentous growth on media containing 5 mM ammonium sulfate (Figure 2B), a 100-fold excess over standard pseudohyphal media (SLAD; 50 μM ammonium sulfate). Filamentation also occured, albeit to a lesser extent, on the synthetic medium YNB, which contains an even higher nitrogen concentration (38 mM ammonium sulfate; data not shown). Thus, constitutive activation of GPA2 relieves the requirement for nitrogen starvation to induce pseudohyphal growth. A dominant negative GPA2 allele inhibits pseudohyphal growth We next introduced a mutation in the hinge region of GPA2 which would be predicted to result in a dominant negative allele. The corresponding mutation in other Gα subunits prevents conformational changes normally induced by GTP binding that are required for βγ release and signalling (Miller et al., 1988), resulting in a dominant negative phenotype. This mutation in yeast GPA1 complements the lethality of the Δgpa1 mutation, but confers a semidominant sterile phenotype (Kurjan et al., 1991). The corresponding mutant allele, GPA2Gly299Ala (GPA2-3), again under the control of a galactose-inducible promoter, did not complement the pseudohyphal defect of Δgpa2 mutant strains, as expected (data not shown). Expression of GPA2Gly299Ala inhibited pseudohyphal differentiation in wild-type diploid strains (Figure 3A). Thus, two mutations predicted to alter GPA2 function both have significant effects on filamentous growth and demonstrate that pseudohyphal differentiation is G protein regulated. Figure 3.A dominant negative GPA2 allele inhibits pseudohyphal growth. Wild-type diploid strain MLY61 expressing GPA2 (pML180) or GPA2Gly299Ala (pML179) under repressing (glucose, left) or inducing (galactose, right) conditions on nitrogen limiting media was incubated for 4 days at 30°C. Download figure Download PowerPoint β and γ subunits remain to be identified In several G protein mediated signalling pathways, both the α and the βγ subunits positively contribute to signalling. The residual filamentation observed in Δgpa2 mutants could be indicative of a role for a βγ complex in signalling, although it is clear that the pheromone-responsive βγ subunits (STE4 and STE18) do not regulate pseudohyphal growth (Liu et al., 1993). Using the fungal β proteins STE4 (S.cerevisiae) and Gpb1 (S.pombe) in BLAST searches, we identified eight candidate β genes in the S.cerevisiae genome; gene disruption experiments did not reveal a role for any of these genes in pseudohyphal differentiation (see Materials and methods). STE18 is the only known fungal γ subunit (Whiteway et al., 1989); none of the three candidate γ subunits we identified affected pseudohyphal growth (see Materials and methods). Other methods will be necessary to identify these components. GPA2 does not regulate the MAP kinase cascade Previous work has identified several genes and mutant alleles that promote filamentous growth (Gimeno et al., 1992; Liu et al., 1993; Gimeno and Fink, 1994), and these allowed us to test the point at which GPA2 acts by epistasis analysis. Both the dominant STE11-4 allele and overexpression of STE12 stimulate pseudohyphal differentiation when expressed in wild-type strains and suppress mutations of upstream components (Liu et al., 1993). If GPA2 were to act solely upstream of the MAP kinase cascade, expression of these alleles should suppress the pseudohyphal defect conferred by the Δgpa2 mutation. To our surprise, neither STE11-4 nor increased STE12 expression resulted in filamentation in a Δgpa2/Δgpa2 mutant strain (Figure 4A). These observations suggest that GPA2 does not act upstream of the MAP kinase elements that regulate filamentous growth. Likewise, overexpression of PHD1, which induces hyperfilamentation in other strains (Gimeno et al., 1992; Gimeno and Fink, 1994), had no effect in Δgpa2/Δgpa2 strains. In contrast, expression of the dominant RAS2Gly12Val mutant did suppress the pseudohyphal defects conferred by the Δgpa2 mutation. This finding is consistent with a role for GPA2 in regulating cAMP levels, as has been suggested (Nakafuku et al., 1988). Others have proposed that RAS2 lies solely upstream of the MAP kinase elements (Mösch et al., 1996); however, we also find that RAS2Gly12Val suppresses the pseudohyphal defects of the Δste7, Δste11 and Δste12 mutants, findings which do not support this model. RAS2 may instead have multiple roles in regulating filamentous growth, as has recently been suggested (Mösch and Fink, 1997). Figure 4.GPA2 functions independently of the MAP kinase pathway. (A) Diploid wild-type (MLY61) or Δgpa2/Δgpa2 (MLY132a/α) strains expressing RAS2Gly19Val, STE11-4 or PHD1 were incubated on SLAD medium for 4 days at 30°C. The colonies were photographed at 25× magnification. (B) Diploid wild-type (L3566), Δste12/Δste12 (HLY506) or Δste20/Δste20 (HLY492) strains expressing GPA2 (pML180) or GPA2Gly132Val (pML160) were incubated on SLARG medium for 4 days at 30°C. The colonies were photographed at 50× magnification. Download figure Download PowerPoint The dominant active GPA2Gly132Val allele allowed us to address the relationship between GPA2 and the MAP kinase elements by another means. The GPA2Gly132Val allele was expressed in diploid strains carrying homozygous deletions of STE20, STE11, STE7 and STE12. None of the Δste mutations blocked the ability of GPA2Gly132Val to stimulate pseudohyphal differentiation (Figure 4B). Moreover, as in wild-type STE+ strains, GPA2Gly132Val also allowed pseudohyphal growth of Δste mutant strains on nitrogen-rich medium (data not shown). Filamentation in the Δste20/Δste20 strain expressing GPA2Gly132Val was somewhat reduced compared with wild-type or other Δste mutants (Figure 4B), which is consistent with previous observations that the Δste20 mutant phenotype is more severe than that of other Δste mutants (Liu et al., 1993). These observations again support the conclusion that GPA2 does not regulate pseudohyphal differentiation via the MAP kinase cascade. GPA2 alleles do not affect expression of a MAP kinase-regulated reporter gene To address an alternate explanation for these epistasis results, namely that GPA2 might regulate both the MAP kinase cascade and another pathway, we employed a reporter gene previously found to respond to MAP kinase activation under nitrogen starvation conditions (Laloux et al., 1994; Mösch et al., 1996). This reporter, FG(TyA):: lacZ, was first identified as a control element for the transposable element Ty1, and includes binding sites for TEC1 and STE12. It is induced upon nitrogen starvation but not in response to mating pheromone (Gavrias et al., 1996; Mösch et al., 1996; Madhani and Fink, 1997). Expression of the dominant active GPA2 allele had no affect on the activity of this reporter gene (Table I), and pseudohyphal growth induced by GPA2Gly132Val on nitrogen-rich media (see Figure 2C) was not accompanied by an increase in reporter activity (Table I). Conversely, the dominant-negative GPA2Gly299Ala allele did not prevent induction of FG(TyA)::lacZ under low nitrogen conditions, despite inhibiting pseudohyphal growth (Table I). In addition, alleles which induce expression of this reporter, such as STE11-4 (Mösch et al., 1996), were not affected by deletion of GPA2 (Table II), even though STE11-4 does not suppress the pseudohyphal defects of Δgpa2 mutant strains. Furthermore, the GPA2Gly132Val allele, while it suppressed the filamentation defect of Δste mutants, did not rescue reporter activity in these strains (Table III). Thus, the phenotypes conferred by mutant GPA2 alleles occur in the absence of any detectable difference in activation of the MAP kinase cascade, indicating that GPA2 does not regulate this pathway. Moreover, the activity of the FG(TyA)::lacZ reporter gene and the morphological response of filamentation can be separated under some conditions. Table 1. Activation of a FG(TyA)::lacZ reporter by GPA2 alleles Plasmid Relative β-galactosidase activity 50 μM NH4+ 5 mM NH4+ Vector 6.2 1.0 GPA2Gly132Val 4.9 0.5 GPA2Gly299Ala 4.7 2.6 FG(TyA)::lacZ expression was assayed by monitoring β-galactosidase activity in wild-type cells (MLY97) expressing pIL30-LEU2 and the indicated GPA2 allele after incubation on solid SLARG medium for 48 h at 30°C essentially as described (Mösch et al., 1996). Activities were normalized to protein concentration and are reported as relative to the vector control on high nitrogen media. Values are the average of two independent transformants, each tested in duplicate. Table 2. FG(TyA)::lacZ induction in GPA2 versus Δgpa2 strains Construct Relative β-galactosidase activity GPA2/GPA2 Δgpa2/Δgpa2 50 μM NH4+ 5 mM NH4+ 50 μM NH4+ 5 mM NH4+ Control 1.9 1.0 2.3 0.5 RAS2wt 2.1 0.7 2.9 0.7 RAS2Gly19Val 6.8 0.4 5.0 0.7 STE11-4 26.7 4.0 10.3 1.5 PHD1 3.6 1.3 3.2 1.1 FG(TyA)::lacZ expression was assayed in wild-type (MLY97) or Δgpa2/Δgpa2 (MLY212a/α) cells by coexpressing pIL30-LEU2 with the indicated allele. After 48 h on solid media containing 50 μM or 5 mM ammonium sulfate, β-galactosidase activity was measured as described (see Materials and methods). Activities were normalized to protein concentration and are reported as relative to the vector control in wild-type cells on high nitrogen media. Values are the average of two independent transformants assayed in duplicate. Table 3. Effects of GPA2 alleles on FG(TyA)::lacZ expression in Δste strains GPA2 allele Relative β-galactosidase activity Wild-type Δste20/Δste20 Δste12/Δste12 50 μM 5 mM 50 μM 5 mM 50 μM 5 mM None 11.8 1.0 2.8 0.2 3.1 0.1 GPA2wt 20.0 0.7 6.2 0.1 3.4 0.2 GPA2Gly132Val 13.3 1.8 2.1 0.2 1.5 <0.1 FG(TyA)::lacZ expression was assayed in wild-type (MLY97), Δste20/Δste20 (MLY219a/α) and Δste12/Δste12 (MLY216a/α) strains coexpressing pIL30-LEU2 and the indicated GPA2 allele. Cells were incubated for 48 h at 30°C on solid media containing 50 μM or 5 mM ammonium sulfate with 0.5% galactose and 2% raffinose. Activities were normalized to protein concentrations and are reported as relative to the vector control in wild-type cells on high nitrogen media. Values are the average of two independent transformants, each assayed in duplicate. Pseudohyphal growth is regulated by cAMP Earlier reports have suggested a connection between GPA2, Ras and cAMP metabolism (Nakafuku et al., 1988; Papasavvas et al., 1992). Our findings that GPA2 regulates filamentation and that the Δgpa2 mutation is suppressed by RAS2Gly19Val led us to test whether cAMP might also regulate this dimorphic transition. The efficacy of exogenous cAMP in yeast is greatly enhanced by mutations in the high affinity cAMP phosphodiesterase PDE2. Addition of 1 mM cAMP to nitrogen-limiting media stimulated filamentation of diploid Δpde2/Δpde2 mutant strains, and filamentation was even more dramatic at 10 mM cAMP (Figure 5A). At 10 mM cAMP, even wild-type (PDE2+) strains exhibit enhanced pseudohyphal growth (Figure 5A), and pseudohyphal differentiation also occured on nitrogen-rich media (5 mM NH4+; Figure 6). These effects of cAMP were not observed with either AMP or cGMP (data not shown), indicating a specific role for the second messenger cAMP in regulating pseudohyphal differentiation. Figure 5.cAMP stimulates pseudohyphal differentiation. (A) Homozygous diploid strains with genotypes: wild-type (MLY61), Δpde2/Δpde2 (MLY162a/α) or Δgpa2/Δgpa2 Δpde2/Δpde2 (MLY171a/α) were incubated on SLAD medium containing the indicated concentrations of cAMP for 4 days at 30°C. (B) Strains of genotypes Δpde2 MATα (MLY173), Δste7 Δpde2 MATa (MLY174) and Δste12 Δpde2 MATa (MLY175) expressing a plasmid-borne copy of the opposite MAT locus were incubated on SLAD medium with or without 10 mM cAMP for 4 days at 30°C. Download figure Download PowerPoint Figure 6.cAMP allows pseudohyphal growth on nitrogen-rich media. Homozygous diploid wild-type (MLY61) and Δpde2/Δpde2 strains (MLY162a/α) were incubated on media containing 5 mM ammonium sulfate without (top) or with (bottom) 10 mM cAMP for 8 days at 30°C. Download figure Download PowerPoint If the role of GPA2 is to regulate intracellular cAMP levels, as has been previously suggested (Nakafuku et al., 1988; Papasavvas et al., 1992), the pseudohyphal defects of Δgpa2 mutant strains might be suppressed by exogenous cAMP. To test this hypothesis, we constructed a homozygous diploid strain lacking both GPA2 and PDE2. As shown in Figure 5A, 100 μM cAMP restored filamentation to approximately wild-type levels in a Δgpa2/Δgpa2 Δpde2/Δpde2 mutant strain. The effects of cAMP are not mediated by the MAP kinase pathway, as cAMP also stimulated filamentation in Δste7 Δpde2 and Δste12 Δpde2 mutant strains (Figure 5B), as did expression of the dominant active GPA2Gly132Val allele (Figure 4B and data not shown). Surprisingly, cAMP actually repressed expression of the FG(TyA)::lacZ reporter under nitrogen limiting conditions (Table IV). This is another example in which increased filamentous growth, as assayed morphologically, was not accompanied by increased expression of this reporter. These findings provide support for a model in which GPA2 regulates pseudohyphal differentiation by stimulating cAMP production and regulating a signalling pathway independent of the MAP kinase cascade. Table 4. Effects of cAMP on FG(TyA)::lacZ expression Genotype cAMP (mM) Relative β-galactosidase activity 50 μM NH4+ 5 mM NH4+ Δpde2 STE+ 0 4.8 1.0 1 0.6 1.2 10 <0.1 1.1 Δpde2 Δste7 0 1.8 0.5 1 0.1 1.2 10 <0.1 0.7 Δpde2 Δste12 0 0.8 0.4 1 0.2 0.5 10 <0.1 <0.1 Δpde2 (MLY213), Δpde2 Δste7 (MLY214) and Δpde2 Δste12 (MLY215) strains were incubated on solid media containing 50 μM or 5 mM ammonium sulfate and the indicated concentration of cAMP for 48 h at 30°C. Values are reported as relative to the Δpde2 STE+ strain on high nitrogen media lacking cAMP. Each determination was the average of two independent transformants, each assayed in duplicate. Discussion Pseudohyphal differentiation is regulated by the Gα subunit GPA2 We find that cells lacking the Gα homolog GPA2 have a defect in pseudohyphal development. A constitutively active mutant allele, GPA2Gly132Val, which is predicted to have decreased GTPase activity, stimulates pseudohyphal growth; remarkably, this occurs even under nitrogen-rich conditions. These findings suggest that GPA2 is a component of the nitrogen sensing machinery. A second mutation, GPA2Gly299Ala, which is predicted to prevent βγ release and interaction with signalling effectors, results in a dominant-negative GPA2 allele that inhibits filamentation in wild-type strains. We propose that GPA2 detects nitrogen starvation conditions through its interaction with an as yet unknown receptor (see below) and stimulates filamentous growth. Our data also indicate that GPA2 plays a positive role in signa" @default.
• W2093434319 created "2016-06-24" @default.
• W2093434319 creator A5052159588 @default.
• W2093434319 creator A5076041024 @default.
• W2093434319 date "1997-12-01" @default.
• W2093434319 modified "2023-10-03" @default.
• W2093434319 title "Yeast pseudohyphal growth is regulated by GPA2, a G protein alpha homolog" @default.
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