Pseudohyphal differentiation in the budding yeast Saccharomyces cerevisiae is induced in diploid cells in response to nitrogen starvation and abundant fermentable carbon source. Filamentous growth requires at least two signaling pathways: the pheromone responsive MAP kinase cascade and the Gpa2p-cAMP-PKA signaling pathway. Recent studies have established a physical and functional link between the Gα protein Gpa2 and the G protein-coupled receptor homolog Gpr1. We report here that the Gpr1 receptor is required for filamentous and haploid invasive growth and regulates expression of the cell surface flocculin Flo11. Epistasis analysis supports a model in which the Gpr1 receptor regulates pseudohyphal growth via the Gpa2p-cAMP-PKA pathway and independently of both the MAP kinase cascade and the PKA related kinase Sch9. Genetic and physiological studies indicate that the Gpr1 receptor is activated by glucose and other structurally related sugars. Because expression of the GPR1 gene is known to be induced by nitrogen starvation, the Gpr1 receptor may serve as a dual sensor of abundant carbon source (sugar ligand) and nitrogen starvation. In summary, our studies reveal a novel G protein-coupled receptor senses nutrients and regulates the dimorphic transition to filamentous growth via a Gα protein-cAMP-PKA signal transduction cascade.
ALL organisms employ signaling mechanisms to regulate transcription and translation, cell-cycle progression, and development on the basis of changes in the extracellular environment. In one specific example, mating in yeast, cell-cell communication is mediated by secreted peptide pheromones that stimulate physiological responses leading to mating. In the budding yeast Saccharomyces cerevisiae, pheromone induces cell-cycle arrest, stimulates transcription, and causes alterations in cell morphology (Sprague and Thorner 1992). In other yeast or fungi, such as Schizosaccharomyces pombe, Ustilago maydis, and Cryptococcus neoformans, conjugation requires both pheromone and specific nutritional conditions. Thus, multiple signaling events are coordinated to regulate developmental processes.
In S. cerevisiae, nutritional signals also regulate pseudohyphal differentiation, a filamentous growth form induced upon nitrogen starvation (Gimenoet al. 1992). Similar to pheromone-induced mating, filamentous differentiation involves changes in gene transcription, cell cycle progression, and morphology, although unlike mating, pseudohyphal differentiation occurs only in diploid cells. Pseudohyphal cells are elongated and cylindrical compared to vegetative cells, employ a unipolar (rather than bipolar) budding pattern, and invade the growth substrate (reviewed in Kron 1997).
The regulation of pseudohyphal differentiation is complex, involving at least two separate, but interconnected, signaling pathways. One is the pheromone responsive MAP kinase cascade including the Ste20, Ste11, Ste7, and Kss1 protein kinases and the Ste12 and Tec1 transcription factors (Liuet al. 1993; Cooket al. 1997; Madhaniet al. 1997). The pheromone receptors and the Gpa1/Ste4/Ste18 G protein, upstream elements in the mating response, do not regulate filamentous growth (Liuet al. 1993). The second pathway involves the Gα protein Gpa2, which functions to regulate cAMP levels (Nakafukuet al. 1988; Kübleret al. 1997; Lorenz and Heitman 1997; Colomboet al. 1998). cAMP then modulates the activity of the cAMP-dependent protein kinase (PKA); Tpk2p, one of the three catalytic subunits of PKA, specifically controls the filamentation response (Robertson and Fink 1998; Pan and Heitman 1999). One additional component that is involved in nitrogen sensing is the Mep2 ammonium permease, which is required for pseudohyphal differentiation when ammonium is the sole source of nitrogen and may serve as a receptor for ammonium ions (Lorenz and Heitman 1998a).
There are several links between the MAP kinase and Gpa2p-cAMP signaling pathways. First, Ras2p functions at a branchpoint and serves to regulate filamentous growth by activating both the MAP kinase and the cAMP-PKA signaling cascades (Gimenoet al. 1992; Mösch et al. 1996, 1999; Kübleret al. 1997; Lorenz and Heitman 1997; Robertson and Fink 1998; Pan and Heitman 1999). Second, recent findings reveal that both the MAP kinase and the cAMP-PKA signaling pathways converge to coordinately regulate expression of the cell surface flocculin Flo11, which is required for haploid invasive growth and diploid pseudohyphal differentiation (Lambrechtset al. 1996; Lo and Dranginis 1998; Pan and Heitman 1999; Ruppet al. 1999).
The MAP kinase cascade that regulates filamentous growth has a dual function and also regulates mating in response to pheromone. Similarly, the cAMP-PKA pathway has a second function in addition to filamentation. PKA activity is essential in yeast, and cells with elevated PKA activity are hypersensitive to stresses such as heat shock, are unable to sporulate, and have diminished levels of storage carbohydrates such as glycogen (reviewed in Broach 1991). Furthermore, the PKA pathway responds to carbon source availability; in particular, readdition of glucose to starved cells triggers a rapid and transient increase in cAMP that requires Gpa2p, Ras2p, and adenylyl cyclase (Colomboet al. 1998; Yunet al. 1998). Some of the defects associated with loss of PKA activity can be suppressed by overexpression of Sch9p, a PKA-related kinase (Todaet al. 1988). Deletion of the SCH9 gene blocks the heat shock sensitive phenotype conferred by dominant active Gpa2p mutants (Xueet al. 1998). Thus, the Sch9 kinase also impinges on the PKA signaling cascade.
Heterotrimeric G proteins are regulated by seven transmembrane domain receptors of the β-adrenergic receptor family. Signaling via these receptor-G protein modules regulates a diverse array of processes in all eukaryotes, including neurotransmission in animals from nematodes to mammals, cell growth and differentiation, chemotaxis both in immune function and in microorganisms such as Dictyostelium discoideum, vesicle trafficking, and sensory transduction (see, for example, Reed 1992; Neer 1995; Parent and Devreotes 1996). As primary sensors of extracellular signals, G protein-coupled receptors bind to a wide variety of ligands, including amino acids and analogues, peptides, photons, lipids, and volatile compounds. Defects in receptor-G protein signaling are the cause of a number of human diseases, including visual defects like retinitis pigmentosa and color blindness; hormone signaling dys-functions such as hyper- or hypothyroidism and abnormal sexual development resulting from mutations in receptors for luteinizing hormone, follicle stimulating hormone, and thyroid stimulating hormone; and diabetes (see Spiegel 1997).
It is clearly of significant importance to understand the mechanisms by which heterotrimeric G proteins regulate signaling events and our understanding of these processes have benefited from studies in fungi, in particular pheromone signaling in S. cerevisiae and S. pombe. The molecular mechanisms of G protein-coupled receptor signaling have been functionally conserved, and heterologous expression of many different G protein-coupled receptors in yeast results in ligand-dependent activation of the pheromone response pathway (Kinget al. 1990); thus the fungal system is a valuble tool for probing G protein signaling. A number of Gα subunits have been recently cloned from other fungal species, including Ustilago maydis, Cryptococcus neoformans, Candida albicans, and Cryphonectria parasitica, among others. Despite the proliferation of Gα proteins, only a few G protein-coupled receptors are known (reviewed in Bölker 1998). With only a few exceptions, namely G protein-coupled receptor homologs in S. cerevisiae (Gpr1p) and S. pombe (Git3), all of these fungal G protein-coupled receptors bind pheromones during mating responses. The Gpr1 receptor was recently identified in a two-hybrid screen via its ability to interact with the Gα protein Gpa2 (Yunet al. 1997; Xueet al. 1998) and subsequently implicated in regulating Gpa2p function (Xueet al. 1998); both gpa2Δ ras2Δ and gpr1Δ ras2Δ double mutant strains have a similar cAMP-remediated synthetic growth defect (Kübleret al. 1997; Lorenz and Heitman 1997; Xueet al. 1998).
In this article, we demonstrate that the G protein-coupled receptor homolog Gpr1 is required for pseudohyphal differentiation and regulates a signal transduction cascade involving Gpa2p, adenylyl cyclase, and PKA. The Gpr1 receptor is also required for haploid invasive growth and regulates expression of the cell surface flocculin Flo11, which is required for invasive and pseudohyphal growth. We present genetic and physiological evidence that the Gpr1 receptor is activated by glucose and other structurally related fermentable sugars. Signaling via the Gpr1p-Gpa2p-cAMP pathway is independent of the MAP kinase signaling cascade. We also present genetic evidence that the Gpr1p-Gpa2p-cAMP-PKA signaling pathway regulates vegetative growth in parallel with an Sch9p-dependent signaling pathway that does not play a primary role in regulating filamentous growth. In summary, our studies suggest that the Gpr1p signaling cascade serves as a dual sensor of carbon abundance and nitrogen deprivation in which the Gpr1 receptor is transcriptionally induced by nitrogen starvation and then senses the presence of fermentable sugars by a ligand-receptor interaction that regulates cAMP production.
MATERIALS AND METHODS
Yeast strains, media, and genetic methods: Standard media and other genetic methods were as described in Sherman (1991). Low ammonium media (SLAD; 50 μM ammonium sulfate, 2% glucose, 2% Bacto-agar) for induction of pseudohyphal differentiation has been described (Gimenoet al. 1992) as has SLARG media (50 μm ammonium sulfate, 2% raffinose, 0.5% galactose, 2% Bacto-agar) for induction of pGal-GPA2 alleles (Lorenz and Heitman 1997) and SLADG media (50 μm ammonium sulfate, 2% galactose, 0.13% glucose, 2% Bacto-agar) for induction of the pGAL-STE12 allele (Liuet al. 1993).
Yeast transformations were done via the lithium acetate/polyethylene glycol protocol of Schiestl et al. (1993). Strains used in these studies are listed in Table 1. The wild-type strains of the R1278b background, MLY40 (ura3-52 MATα), MLY41 (ura3-52 MATa), and MLY61 (ura3-52/ura3-52 MATa/α) have been described, as have the gpa2Δ and ste12Δ strains, MLY132a/α (gpa2Δ::G418/gpa2Δ::G418 ura3-52/ura3-52 MATa/α) and HLY352 (ste12Δ::LEU2/ste12Δ:: LEU2 leu2Δ::hisG/leu2Δ::hisG ura3-52/ura3-52 MATa/α), respectively (Liuet al. 1993; Lorenz and Heitman 1997). The gpr1Δ::G418/gpr1Δ::G418 homozygous diploid strain was contructed by deleting the GPR1 gene in haploid strains of the R1278b background MLY40 (MATα) and MLY41 (MATa) using a PCR disruption protocol (Wachet al. 1994). Disruptants were confirmed by PCR and mated to produce the homozygous diploid strain MLY232a/α. A similar process was used to construct the sch9Δ::G418/sch9Δ::G418 strain MLY265a/α. Haploid strains with single gene mutations were crossed, sporulated, and dissected to produce double mutant strains. Tetrads in which there were two G418-resistant and two G418-sensitive segregants were chosen and the presence of both mutations was confirmed by PCR analysis of genomic DNA. The hygromycin B resistance gene cassette (provided by Alan Goldstein and John McCusker) was used to delete one allele of the SCH9 gene in diploid strains MLY61a/α, MLY132a/α, MLY187a/α, and MLY232a/α. The resulting strains, XPY163a/α, XPY164a/α, XPY165a/α, and XPY166a/α, were sporulated and dissected on YPD medium and then replica-plated to YPD medium containing G418 or hygromycin B.
Plasmids: Plasmids used in this study include pML160 and pML180 [CEN URA3 GPA2-2 and GPA2, respectively; Lorenz and Heitman (1997)], pMW1 and pMW2 [CEN URA3 RAS2 and RAS2Val19, respectively; Ward et al. (1995)]; pSL1509 [CEN URA3 STE11-4; Stevenson et al. (1992)]: pNC252 [CEN URA3 pGAL-STE12; Liu et al. (1993)]; pCG38 [2μ URA3 PHD1; Gimeno and Fink (1994)]; p2.5-2 [2μ URA3 TEC1; Lorenz and Heitman (1998b)]; and YEplac195 [2μ URA3; Gietz and Sugino (1988)]. The 2μ-GPR1 plasmid pML224 was constructed as follows. The GPR1 cassette from pGPR1-112.2 (Xueet al. 1998) was excised with SacI-PstI and inserted into those sites in YEplac195. This plasmid did not complement the pseudohyphal defect of a gpr1Δ/gpr1Δ strain due to insufficient sequences at the 3′ end of the gene. We PCR-amplified a fragment from an internal SalI site to ~500 bp 3′ to the GPR1 stop codon, appending an artificial SphI site. This fragment was used to replace the incomplete 3′ end in YEplac195, creating the 2μ-URA3-GPR1 plasmid pML224. This plasmid complements the pseudohyphal growth defect conferred by the gpr1Δ mutation.
The SCH9 gene, including the open reading frame and 600 bp each of the 5′- and 3′-untranslated regions, was PCR-amplified using genomic DNA of strain MLY40α as template. A PCR product of 3.7 kb was cloned into the 2μ plasmid YEplac195. The resulting plasmid, pXP77, complements the vegetative and filamentous growth defect conferred by the sch9 mutation.
Photomicroscopy: Whole colony photographs were taken using a Nikon Eclipse E400 microscope using a ×10 objective and ×2.5 trinocular adaptor (×25 magnification) connected to a Nikon N70 camera. Colonies were photographed directly on solid medium after 4 days of incubation at 30°, unless otherwise indicated.
Northern blot analysis: Haploid yeast strains were incubated in YPD liquid medium overnight and then transferred to fresh YPD medium and incubated to an OD600 of 1.0. Cells were washed with ice water and total RNA was isolated by using acid phenol, separated by electrophoresis, and transferred overnight by capillary action to nylon membranes (VWR). DNA fragments to be used as probes (a 300-bp PCR product derived from the 5′ region of the FLO11 open reading frame and a 500-bp 5′ fragment of the ACT1 open reading frame) were gel-purified and radiolabeled by random priming (Boehringer Mannheim, Indianapolis). Hybridization and washing were performed as described (Sambrooket al. 1989).
Invasion assays: Haploid strains were grown as patches on YPD medium and incubated at 30° for 4 days. The plate was photographed and then washed with running water, and remaining invaded cells were photographed.
cAMP assays: Cells of isogenic wild-type (MLY41a), gpr1 mutant (MLY232a), and gpa2 mutant (MLY132a) haploid strains were grown in YPD medium for 2 days at 30° with shaking and were collected by centrifugation. After washing twice with water and once with buffer (10 mm MES, pH 6.0, 0.1 mm EDTA), cells were resuspended in buffer and incubated for 2 hr to induce glucose starvation prior to the readdition of different sugars, as indicated, to a final concentration of 2%. At various time points, 0.5-ml aliquots were removed and transferred to 2-ml screw-cap tubes containing 0.3 ml of glass beads and 0.5 ml of ice-cold water and were frozen in liquid nitrogen. The samples were then homogenized with a bead beater at 4° and crude cell extracts were lyophilized. Concentrations of intracellular cAMP were determined using a cAMP enzyme immunoassay kit (Amersham, Arlington Heights, IL) and samples were normalized to total cell weight. Samples were analyzed in duplicate and the values were averaged. Three independent experiments were conducted with similar results.
Detection of the Gpr1-GFP hybrids: The Gpr1-GFP fusion protein was localized as described (Xueet al. 1998), using a trp1 derivative of strain YJM836 × YJM837 (kindly provided by J. McCusker). Cells expressing the Gpr1-GFP fusion protein were grown either at 30° in liquid synthetic medium or at room temperature on SLAD solid medium and viewed using either the fluorescein isothiocyanate (FITC) filter for fluorescence microscopy or phase contrast optics using a Zeiss Axiophot microscope.
The G protein-coupled receptor Gpr1 regulates filamentous growth: The G protein-coupled receptor homolog Gpr1 was identified in a two-hybrid screen with the Gα protein Gpa2 (Yunet al. 1997; Xueet al. 1998). gpr1 or gpa2 mutations confer a synthetic growth defect in conjunction with mutations in the small G protein Ras2 (Xueet al. 1998). These findings indicate that Gpr1p and Gpa2p are physically and functionally related. Because the Gα protein Gpa2 is required for pseudohyphal differentiation (Kübleret al. 1997; Lorenz and Heitman 1997), we tested whether the Gpr1 receptor has a similar role.
The GPR1 open reading frame was precisely deleted in the R1278b strain background commonly used for studies on filamentous growth. When incubated on nitrogen limiting SLAD medium, diploid gpr1Δ/gpr1Δ mutant strains exhibited a significant defect in filamentous growth (Figure 1). Expression of the wild-type GPR1 gene from a plasmid complemented the gpr1Δ/gpr1Δ filamentous growth defect (Figure 1). The gpr1Δ mutation conferred a pseudohyphal defect on media containing a variety of limiting nitrogen sources, including ammonium (Figure 1), glutamine, proline, aspartate, asparagine, and serine (data not shown).
The defect in filamentous growth in gpr1 and gpa2 mutant strains was not absolute, as some modest filaments were still observed in the null mutants. gpr1 gpa2 double mutant strains exhibited a filamentation defect similar to the gpa2 and gpr1 single mutant strains (data not shown), consistent with the interpretation that Gpr1p and Gpa2p function in a linear signaling cascade.
Gpr1p and Gpa2p regulate haploid invasive growth and FLO11 gene expression: Haploid strains of S. cerevisiae undergo a dimorphic transition to invasive growth that shares several features with pseudohyphal differentiation of diploid S. cerevisiae strains (Roberts and Fink 1994). First, the MAP kinase cascade components Ste-20p, Ste11p, Ste7p, and Ste12p regulate both haploid invasive growth and diploid pseudohyphal growth. Second, haploid invasive cells are elongated, invade the agar, and switch budding pattern from axial to unipolar budding. Because the Gpr1 receptor and Gpa2 Gα protein regulate pseudohyphal differentiation of diploid strains, we addressed whether these proteins also regulate haploid invasive growth.
As shown in Figure 2A, gpr1 and gpa2 haploid mutant strains exhibited a marked defect in agar invasive growth. The agar invasion defect of the gpr1 and gpa2 mutant strains was comparable to that observed with mutant strains lacking components of the MAP kinase cascade (data not shown), to a gpr1 gpa2 double mutant strain (Figure 2A), and to a mutant strain lacking the PKA catalytic subunit Tpk2 (Figure 2A). Thus, Gpr1p and Gpa2p are required for haploid invasive growth.
The PKA and MAP kinase signaling cascades regulate haploid invasive growth and diploid filamentous growth, in part by regulating expression of the cell surface flocculin Flo11 (Lo and Dranginis 1998; Pan and Heitman 1999; Ruppet al. 1999). We therefore addressed whether the Gpr1 receptor and the Gα protein Gpa2 regulate FLO11 gene expression. RNA was isolated from haploid strains grown in YPD rich medium and analyzed by Northern blot analysis with probes directed to the FLO11 gene, and also to the ACT1 gene as a loading control. FLO11 expression was readily detected in the wild-type strain, but little or no FLO11 expression was observed in isogenic mutant strains lacking Gpr1, Gpa2, or both Gpr1 and Gpa2 (Figure 2B). The defect in FLO11 expression in the gpr1 and gpa2 mutant strains was comparable to that observed in mutant strains lacking both Gpr1p and Gpa2p (Figure 2B), consistent with a model in which the two function in a linear pathway. The defect in FLO11 expression in the gpr1 and gpa2 mutant strains was somewhat less severe than the defect observed in a mutant strain of the Tpk2 PKA catalytic subunit, suggesting that some activation of PKA can occur in gpr1 and gpa2 mutant strains, possibly via Ras2 activation of adenylyl cyclase (Figure 2B; Pan and Heitman 1999). In conclusion, the Gpr1 receptor and the coupled Gα subunit Gpa2 are required for haploid invasive growth and regulate expression of the cell surface flocculin Flo11.
Gpr1p receptor signals via a pathway involving Gpa2p, Ras2p, and cAMP: Given the connection between the functions of Gpr1p and Gpa2p and the similar phenotypes conferred by the gpr1 and gpa2 mutations (Figures 1 and 2), we tested by epistasis analysis whether the Gpr1 receptor functions upstream of the Gpa2 Gα protein and other components of the cAMP-PKA signaling cascade. Expression of the dominant active GPA2 allele containing a Gly132Val mutation in the GTPase active site enhances pseudohyphal differentiation (Lorenz and Heitman 1997). Expression of the dominant active GPA2 allele suppressed the filamentation defect of gpr1Δ/gpr1Δ mutant strains (Figure 3A), providing genetic evidence that the Gα protein Gpa2 functions downstream of the Gpr1 receptor.
In previous studies, we demonstrated that the filamentation defect of gpa2 mutant strains could be suppressed by activation of the cAMP signaling pathway, either by expression of a dominant active RAS2 allele (RAS2Gly19Val) or by the addition of cAMP. If the Gpr1 receptor acts upstream of Gpa2p, then either the RAS2Gly19Val dominant active mutant or cAMP should restore filamentation in a gpr1Δ/gpr1Δ mutant strain. This is indeed the case, as expression of RAS2Gly19Val (Figure 3B) or addition of 1 to 10 mm cAMP restored filamentation in gpr1Δ/gpr1Δ pde2Δ/pde2Δ mutant strains (Figure 3C). These findings provide a further link between the functions of the Gpr1 receptor and Gpa2 Gα protein and suggest a role in regulating the cAMP signaling pathway.
The Gpa2-cAMP-PKA pathway functions, in part, independently from the MAP kinase pathway in regulating pseudohyphal differentiation. The filamentation defect of gpa2/gpa2 mutant strains is suppressed by marked overexpression of STE12, while expression of the dominant active STE11-4 allele does not (Lorenz and Heitman 1997, 1998a,b) (Figure 4). Similar epistasis results were observed in gpr1Δ/gpr1Δ mutant strains (Figure 4). The gpr1 and gpa2 mutations also had no effect on expression of an FRE-lacZ reporter gene that is known to be regulated by the MAP kinase cascade, whereas a ras2 mutation inhibited FRE-lacZ expression (data not shown). Taken together, these findings suggest that Gpr1p and Gpa2p regulate the cAMP signaling cascade and not the MAP kinase pathway.
Gpr1 receptor plays a role in sensing fermentable sugars: Pseudohyphal growth occurs on medium containing not only limiting concentrations of nitrogen source, such as 50 μm ammonium sulfate, but also an abundant level of a fermentable carbon source, such as 2% glucose. In contrast, on a medium with low nitrogen levels and a nonfermentable carbon source, such as acetate, sporulation occurs. Thus, yeast cells must sense both abundant fermentable carbon source and nitrogen deprivation to undergo pseudohyphal differentiation. When glucose is readded to glucose-starved yeast cells, cAMP is rapidly produced and growth ensues. Recent studies reveal that this response to glucose readdition requires Gpr1p, Gpa2p, and Ras2p, suggesting a role in carbon source sensing (Colomboet al. 1998; Jianget al. 1998; Yunet al. 1998; Kraakmanet al. 1999).
We therefore assessed filamentous growth of the isogenic wild-type, gpr1/gpr1, and gpa2/gpa2 mutant strains on synthetic low ammonium (SLA) medium containing different carbon sources (2%). In wild-type cells, the monosaccharides glucose, fructose, and mannose all supported pseudohyphal growth to similar extents (Figure 5A and data not shown). In contrast, the monosaccharide galactose supported growth but only poor filamentation was observed (Figure 5A). The disaccharide sucrose, which is rapidly metabolized to fructose and glucose by invertase, also supported pseudohyphal growth in SLA medium (Figure 5A). Interestingly, the disaccharide maltose dramatically enhances pseudohyphal growth. This finding may be related to the earlier observation that starches, glucose polysaccharides that are metabolized to maltose by starch-utilizing yeast strains, stimulate filamentous growth (Lambrechtset al. 1996; Figure 5A).
Importantly, both gpr1/gpr1 and gpa2/gpa2 mutant strains exhibited defects in pseudohyphal differentiation on medium containing the monosaccharides glucose, mannose, or fructose, or the disaccharide sucrose (Figure 5A and data not shown). These findings suggest a specific role for the Gpr1 receptor/Gpa2 G protein complex in sensing the presence of abundant structurally related fermentable monosaccharides in the growth medium. The defect in gpr1/gpr1 or gpa2/gpa2 mutant cells on glucose medium could be suppressed by the addition of 1% ethanol, which induces hyperfilamentation in diploid yeast strains, further indicating that these mutant cells do not have an intrinsic defect in filamentation (data not shown). The gpr1 and gpa2 mutations had no effect on the robust pseudohyphal filamentation induced by maltose, or on the low level of filamentation observed with galactose, indicating that maltose and galactose are not sensed by the Gpr1p-Gpa2p pathway (Figure 5A). Finally, mutant strains lacking the PKA catalytic subunit Tpk2 exhibited a filamentation defect on glucose, mannose, sucrose, fructose, and maltose (Figure 5A and data not shown), indicating that PKA plays a role in the responses to all of these sugars. These findings suggest that the functions of Gpr1p, Gpa2p, and PKA are coupled in sensing carbon sources.
Finally, we sought to establish whether the role of Gpr1p and Gpa2p in regulating filamentation in response to different carbon sources is correlated with the production of cAMP. To this end, isogenic wild-type, gpr1, and gpa2 mutant strains were starved for carbon source, and glucose, sucrose, galactose, or maltose was added to the cells. As shown in Figure 5B, wild-type cells respond to glucose or sucrose addition by producing cAMP, and this requires both Gpr1p and Gpa2p. In marked contrast, neither galactose nor maltose stimulated cAMP production (Figure 5B). Because maltose dramatically enhances filamentous growth independently of Gpr1p and Gpa2p and does not stimulate cAMP production, another sensing mechanism likely operates to detect maltose and regulate filamentation, which may involve the maltose permease (X. Wang and C. Michels, personal communication).
Taken together, our observations indicate that the Gpr1 receptor/Gpa2 G protein complex regulates the PKA pathway, FLO11 transcription, and filamentous growth based on the availability of glucose and other structurally related saccharides in the extracellular medium. Several models can be envisioned to explain these findings. The first and most parsimonious is that these sugars are the direct ligands of Gpr1p. Alternatively, these saccharides may bind to other cell surface proteins that then interact with Gpr1p. Finally, the Gpr1p ligand could be a compound that is rapidly produced and secreted following glucose readdition. Yeast cells are known to secrete H+, ATP, and the lipid second messengers glycerophosphatidylinositol-4-phosphate (gPI4P) and -4,5 bisphosphate (gPI4,5P2) in response to glucose (Serrano 1983; Hawkinset al. 1993; Brandaoet al. 1994; Boyum and Guidotti 1997). However, we found that altering the pH of SLAD medium with buffer inhibited filamentous growth, whereas ATP and other nucleotides and nucleosides had no effect (data not shown). Finally, gPI4P and gPI4,5P2 did not stimulate filamentous growth or FLO11 gene expression (data not shown). These observations provide support for the model that glucose and structurally related sugars are agonists of the Gpr1 receptor.
Gpr1p does not localize to hyphal projections: Detection of Gpr1p in vegetative cells using a green florescent protein (GFP)-tagged variant localized the protein to the plasma membrane (Xueet al. 1998). We postulated that the Gpr1p receptor might localize to the tips of pseudohyphal filament cells. In this model, Gpr1p would act in the same manner as the pheromone receptors, Ste2p and Ste3p, which are localized to the shmoo tip and stimulate morphogenesis in the direction of the highest pheromone concentration (Jacksonet al. 1991; Segall 1993; Chenevert 1994). In contrast, we found that the uniform plasma membrane staining of the Gpr1-GFP fusion protein was maintained even in elongated cells grown under pseudohyphal inducing conditions (data not shown). The overall level of the Gpr1-GFP fusion protein was, however, lower in filamentous than in vegetative cells (data not shown). These findings suggest that Gpr1p and Gpa2p may not function to regulate the direction of polarized growth.
Gpr1p and Gpa2p signal in parallel with Sch9p during vegetative growth: In previous studies, the Sch9 kinase was implicated as a downstream signaling component of the Gpa2p signal transduction cascade. Namely, expression of a dominant active Gpa2 mutant causes heat shock sensitivity and this effect is blocked by an sch9 mutation, suggesting that Sch9p might be a downstream mediator of Gpa2 signaling (Xueet al. 1998). The Sch9 kinase was originally identified via its ability to suppress lethality of tpk1,2,3 triple mutant strains when overexpressed, indicating a role in parallel with PKA downstream of cAMP (Todaet al. 1988). In addition, the Gpr1 receptor and Gpa2 G protein signal in a pathway that functions in parallel with Ras2p, regulates cAMP production, and is required for vegetative growth. For example, ras2 gpr1 and ras2 gpa2 mutants have a severe synthetic growth defect, and normal growth of these mutant strains is restored by cAMP or a pde2 mutation that increases intracellular cAMP levels (Kübleret al. 1997; Xueet al. 1998).
To address a possible role of Sch9p in signaling downstream of Gpr1p, Gpa2p, and Ras2p, we employed genetic epistasis analysis. We considered three possible models: Sch9p could signal downstream of Gpr1p and Gpa2p, downstream of Ras2p, or in a pathway parallel to both the Ras2p and Gpr1p-Gpa2p pathways. Our epistasis analysis supports this third model. First, an sch9 mutation is synthetically lethal with a ras2 mutation, indicating that Sch9p does not signal solely downstream of Ras2p (Figure 6). Second, an sch9 mutation was also synthetically lethal with either a gpr1 or a gpa2 mutation (Figure 6). This last finding does not support a model in which one pathway consists of a linear Gpr1p-Gpa2p-Sch9p cascade that functions in parallel with Ras2p and is required for vegetative growth on rich medium; it instead supports an alternative model in which Sch9p functions in an independent pathway parallel to Gpr1p and Gpa2p. Regulation of filamentous growth by Gpr1p and
Gpa2p does not require Sch9p: We next addressed whether Sch9p plays a role in the regulation of pseudohyphal differentiation by Gpr1p and Gpa2p. As shown in Figure 7, an sch9/sch9 mutant strain had no defect in filamentous growth on SLAD medium at early time points (12 hr), whereas a filamentation defect was readily apparent in gpr1/gpr1 or gpa2/gpa2 mutant microcolonies. Following 3 days of incubation, a filamentation defect was apparent in the sch9 mutant strain but was not as severe as the defect observed with either gpr1 or gpa2 mutant strains (Figure 7). This filamentation defect of sch9 mutant strains may be in part attributable to a nonspecific effect of the growth defect that is conferred by the sch9 mutation but not by gpr1 or gpa2 mutations (see Figure 6). Finally, overexpression of the Sch9 kinase from a 2μ multicopy plasmid did not enhance or inhibit pseudohyphal differentiation in a wild-type strain (data not shown).
To investigate the upstream elements that regulate Sch9p, we expressed the dominant RAS2 and GPA2 alleles in the sch9/sch9 mutant strain. Deletion of SCH9 had no effect on the ability of the active RAS2Val19 allele to enhance filamentous growth (Figure 8A). Moreover, expression of the dominant active GPA2-2 allele (Gly13-2Val) still stimulated filamentation, though not to wild-type levels, in an sch9/sch9 mutant strain (Figure 8B), indicating that activation of filamentous growth by Gpa2p is not dependent upon Sch9p.
We also investigated whether Sch9p regulates haploid invasive growth or FLO11 gene expression. In contrast to gpr1, gpa2, or tpk2 mutations, the sch9 mutation did not block invasive growth; in fact, sch9 mutant strains were hyperinvasive (Figure 2A). Moreover, gpr1, gpa2, and tpk2 mutations each blocked FLO11 expression, whereas FLO11 expression was enhanced in an sch9 mutant strain (Figure 2B), consistent with the hyperinvasive phenotype.
The findings that gpa2 and sch9 mutations confer opposite phenotypes with respect to haploid invasion and FLO11 expression, that the sch9 mutation does not block hyperfilamentation conferred by the dominant active GPA2 allele, and that sch9 mutations are synthetically lethal when combined with gpr1 or gpa2 mutations, all support a model in which Sch9p is not the direct target of the Gpr1p-Gpa2p signaling cascade. We conclude that Sch9p functions in an independent signaling cascade, parallel to the Gpr1p-Gpa2p-cAMP-PKA signaling pathway, which regulates vegetative growth and responses to heat shock and inhibits haploid invasive growth and FLO11 gene regulation. Further, Sch9p is not directly downstream of Ras2p, because sch9 and ras2 mutations are also synthetically lethal and the RASVal19 allele still stimulates hyperfilamentation in an sch9 mutant strain. Sch9 may regulate diploid filamentous growth, although this interpretation must be qualified because the sch9 mutation confers a marked growth defect whereas gpr1, gpa2, tpk2, and flo11 mutations do not.
The Gpr1 G protein-coupled receptor regulates filamentous growth: Our studies reveal that the G protein-coupled receptor Gpr1 regulates pseudohyphal differentiation in S. cerevisiae (Figure 9). The Gpr1 receptor also regulates invasive growth of haploid strains and is required for expression of the FLO11 gene encoding a cell surface flocculin. The phenotypes of mutant strains lacking the Gpr1 receptor are similar to those of mutant strains lacking the Gα protein Gpa2, and genetic evidence supports a model in which ligand binding to the Gpr1 receptor activates Gpa2p via a direct interaction. The downstream elements of this signaling pathway consist of adenylyl cyclase, which is activated by Gpa2p to produce cAMP (Nakafukuet al. 1988; Colomboet al. 1998), resulting in activation of PKA (Figure 9). Recent studies have revealed that the Tpk2 catalytic subunit of PKA regulates pseudohyphal differentiation via the Sfl1 and Flo8 transcription factors that regulate expression of the cell surface flocculin Flo11 (see Figure 9; Robertson and Fink 1998; Pan and Heitman 1999).
The two-hybrid screen that identified Gpr1p also identified portions of three additional proteins that bind to Gpa2p: Ime2p, YAL056, and YGL121 (Xueet al. 1998). These proteins do not regulate filamentous growth as assayed by deletion analysis (data not shown), and thus these proteins may regulate additional phenotypes of Gpa2p, such as heat shock, sporulation, or glycogen accumulation.
Gpr1p as a receptor for fermentable sugars: Pseudohyphal differentiation occurs in response to two nutritional signals: nitrogen starvation and the presence of abundant fermentable carbon source. The Gpr1 receptor plays a role in sensing both of these extracellular signals. First, we have presented evidence here that Gpr1p plays a role in sensing the presence of glucose or other fermentable sugars, resulting in cAMP production. The simplest model for explaining these findings is that sugar binding to Gpr1p results in activation of the coupled Gα protein Gpa2, and the Gpa2-GTP complex then binds to and activates cAMP production by adenylyl cyclase (see Figure 9). More complex models in which glucose addition to cells results in the production and secretion of an unknown Gpr1 ligand cannot be excluded, and further studies employing biochemical approaches will be required to test the hypothesis that glucose directly binds to Gpr1p. This model is consistent with previous studies demonstrating that glucose stimulates cAMP and that this requires both Gpr1p and Gpa2p (Colomboet al. 1998; Yunet al. 1998; Kraakmanet al. 1999). This glucose sensing signaling pathway also appears to be conserved in both S. pombe and Kluyveromyces lactis (Hoffman and Winston 1990, 1991; Noceroet al. 1994; Saviñón-Tejedaet al. 1996).
The Gpr1p-Gpa2p signaling also plays a role in sensing nitrogen starvation. For example, activation of the Gpr1-coupled Gα protein Gpa2 or the addition of exogenous cAMP can, in part, bypass the requirement for nitrogen starvation for filamentous growth (Lorenz and Heitman 1997). Second, the GPR1 gene is known to be transcriptionally induced in response to nitrogen starvation (Xueet al. 1998). Thus, nitrogen starvation induces the expression of the Gpr1 receptor, increasing the sensitivity of this signaling cascade for the extracellular ligand. By this means, the Gpr1p-Gpa2p signaling pathway serves as a dual sensor of both carbon abundance and nitrogen limitation.
Sch9p signals in a pathway distinct from Gpr1p and Gpa2p: Previous studies suggested that the PKA-related protein kinase Sch9 might participate in a signaling pathway directly downstream of the Gpr1 receptor and Gpa2 Gα protein. Namely, expression of an activated GPA2 allele confers a heat shock sensitive phenotype that can be blocked by an sch9 mutation, whereas the heat shock sensitive phenotype conferred by a dominant active RAS2 mutant allele is not (Xueet al. 1998).
Here we have further analyzed the role of the Sch9 kinase in signaling regulated by Gpr1p and Gpa2p. First, we found by genetic epistasis analysis that sch9 mutations are synthetically lethal with ras2, gpr1, or gpa2 mutations. That sch9 mutations are synthetically lethal with ras2 mutations, as is also the case with gpr1 ras2 and gpa2 ras2 double mutants, could have been interpreted to suggest that Sch9 kinase functions downstream of Gpr1p and Gpa2p. However, the finding that sch9 gpr1 and sch9 gpa2 double mutant strains are also inviable is not consistent with a simple linear pathway consisting of a Gpr1p-Gpa2p-Sch9p cascade that signals in parallel with Ras2p. Second, we find that sch9 mutations confer a modest defect in pseudohyphal growth that is not as severe as gpr1 or gpa2 mutations, and sch9 mutations fail to block hyperfilamentation in response to an activated RAS2 allele and only partially block filamentous growth in response to a dominant activated GPA2 mutant. These effects may be attributable to the growth defect conferred by the sch9 mutation. Finally, gpr1 and gpa2 mutations block haploid invasive growth and FLO11 expression, whereas sch9 mutations enhance invasive growth and increase FLO11 gene expression.
Taken together, these findings suggest that Sch9p does not mediate Gpr1p-Gpa2p signaling for vegetative, invasive, or filamentous growth, but may contribute by signaling in a parallel pathway (outlined in Figure 9). Sch9p is required for heat shock sensitivity induced by activation of Gpa2p, which could either be the result of a direct regulation of Sch9p by the Gpa2p pathway, or reflect an indirect role for the Sch9 kinase in the action of the Gpa2p-regulated target PKA.
Gpr1 receptor homologs in other organisms: Gpr1p is the first G protein-coupled receptor to be identified in fungi that has a ligand other than mating pheromone peptides. Given the importance of nutrients, and in particular carbon and nitrogen sources, a role for Gpr1p as a novel nutrient sensor could be broadly conserved. For example, a protein that shares identity with Gpr1p from S. pombe, Git3, is known to play a role in a cAMP-dependent signal transduction pathway that mediates repression of gene expression in response to glucose (Noceroet al. 1994). Further, the C. albicans genome sequencing project has also identified a Gpr1 homolog.
Other Gpr1 receptor homologs likely remain to be identified in other fungi, because homologs of the Gpr1-coupled Gα protein Gpa2 have been identified and correlated with differentiation in S. pombe, C. neoformans, U. hordei, U. maydis, and Podospora anserina (Isshikiet al. 1992; Alspaughet al. 1997; Lichter and Mills 1997; Regenfelderet al. 1997; Krügeret al. 1998; Loubradouet al. 1999). Previous studies have demonstrated that the C-terminal regions of Gα proteins interact with G protein-coupled receptors. The C-terminal domains of this family of Gα proteins share up to 80% identity. Thus, the Gpr1 receptor likely belongs to a family of conserved receptors that regulate differentiation in numerous fungal species in response to carbon source and via Gα protein-cAMP signaling cascades. Finally, Gpr1 receptor homologs could function in mammals as the sweet receptors of the tongue, which are known to be G protein-coupled, or as part of the glucose sensing apparatus that regulates insulin production by pancreatic β-cells.
Nutrient receptors: The Gpr1 receptor joins a growing family of other novel cell surface proteins that serve as receptors for extracellular nutrients in yeast and fungi. For example, the ammonium permease Mep2 plays a novel role as an ammonium ion sensor required for filamentous differentiation in response to ammonium ion limitation (Lorenz and Heitman 1998a,b). The glucose permease homologs Rgt2 and Snf3 have diverged from other glucose permeases and no longer transport sugar, but instead play a novel signal transduction role, activating a signaling cascade that detects the extracellular concentration of glucose and regulates transcription of glucose permease genes (Liang and Gaber 1996; Özcan et al. 1996, 1998; Jianget al. 1997; Schmidtet al. 1999). A related glucose permease homolog, RCO3, appears to play a similar signaling function in regulating glucose transport and conidiation in Neurospora crassa (Madiet al. 1997). Finally, several recent studies reveal that a novel amino acid permease homolog, Ssy1, has diverged from other amino acid permeases and now plays a novel role as a receptor for extracellular amino acids that regulates expression of amino acid permease genes (Didionet al. 1998; Iraquiet al. 1999; Klassonet al. 1999). Taken together, these studies reveal a diverse and growing family of novel nutrient receptors that play diverse roles in regulating gene expression and differentiation in microorganisms and that could be conserved in multicellular eukaryotes.
Potential role of Gpr1 receptor homologs in virulence of human fungal pathogens: Given the correlation between differentiation and virulence in pathogenic fungi, the understanding of extracellular signaling events is particularly important. If, for example, hyphal or pseudohyphal development in C. albicans is regulated via the Gpr1 receptor homolog, receptor antagonists that inhibit signaling would be potent antifungal agents, as the ability to filament is required for virulence (Loet al. 1997). In the pathogenic basidiomycete C. neoformans, the Gα protein Gpa1p is homologous to the S. cerevisiae Gpa2 protein, plays a conserved role in nutrient sensing, regulates virulence factor expression, and is required for virulence (Alspaughet al. 1997). The receptor binding portions of the C. neoformans Gpa1 and S. cerevisiae Gpa2 proteins share marked identity, and thus a Gpr1 homolog that could be targeted for therapeutic intervention is likely coupled to Gpa1 in this pathogenic fungus. Similar approaches could also be used to combat plant fungal pathogens.
We thank Gerry Fink, Steve Garrett, and John McCusker for strains and plasmids; Danny Lew, Steve Garrett, Charles Hoffmann, Corinne Michels, and Alan Myers for helpful discussions; and Bob Lefkowitz, Marc Caron, and Pat Casey for advice and encouragement. This project was supported by a grant-in-aid from the American Heart Association, New York City affiliate (to J.P.H) and by K01 career development award CA77075 from the National Cancer Institute (to M.E.C.). Joseph Heitman is an associate investigator of the Howard Hughes Medical Institute and a Burroughs Wellcome Scholar in Molecular Pathogenic Mycology.
Communicating editor: M. D. Rose
- Received August 4, 1999.
- Accepted October 29, 1999.
- Copyright © 2000 by the Genetics Society of America