the g protein-coupled receptor gpr1 is a nutrient sensor

Copyright 2000 by the Genetics Society of America

The G Protein-Coupled Receptor Gpr1 Is a Nutrient Sensor That RegulatesPseudohyphal Differentiation in Saccharomyces cerevisiae

Michael C. Lorenz,*,1 Xuewen Pan,*,1 Toshiaki Harashima,*,†,1 Maria E. Cardenas,* Yong Xue,‡

Jeanne P. Hirsch‡ and Joseph Heitman*,†

*Departments of Genetics, Pharmacology and Cancer Biology, Microbiology, and Medicine,†Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710

and ‡Department of Cell Biology and Anatomy, Mount Sinai School of Medicine, New York, New York 10029

Manuscript received August 4, 1999Accepted for publication October 29, 1999

ABSTRACTPseudohyphal differentiation in the budding yeast Saccharomyces cerevisiae is induced in diploid cells in

response to nitrogen starvation and abundant fermentable carbon source. Filamentous growth requiresat least two signaling pathways: the pheromone responsive MAP kinase cascade and the Gpa2p-cAMP-PKAsignaling pathway. Recent studies have established a physical and functional link between the Ga proteinGpa2 and the G protein-coupled receptor homolog Gpr1. We report here that the Gpr1 receptor isrequired for filamentous and haploid invasive growth and regulates expression of the cell surface flocculinFlo11. Epistasis analysis supports a model in which the Gpr1 receptor regulates pseudohyphal growth viathe Gpa2p-cAMP-PKA pathway and independently of both the MAP kinase cascade and the PKA relatedkinase Sch9. Genetic and physiological studies indicate that the Gpr1 receptor is activated by glucose andother structurally related sugars. Because expression of the GPR1 gene is known to be induced by nitrogenstarvation, the Gpr1 receptor may serve as a dual sensor of abundant carbon source (sugar ligand) andnitrogen starvation. In summary, our studies reveal a novel G protein-coupled receptor senses nutrients andregulates the dimorphic transition to filamentous growth via a Ga protein-cAMP-PKA signal transductioncascade.

ALL organisms employ signaling mechanisms to regu- diploid cells. Pseudohyphal cells are elongated and cy-lindrical compared to vegetative cells, employ a unipolarlate transcription and translation, cell-cycle pro-(rather than bipolar) budding pattern, and invade thegression, and development on the basis of changes ingrowth substrate (reviewed in Kron 1997).the extracellular environment. In one specific example,

The regulation of pseudohyphal differentiation ismating in yeast, cell-cell communication is mediated bycomplex, involving at least two separate, but intercon-secreted peptide pheromones that stimulate physiologi-nected, signaling pathways. One is the pheromone re-cal responses leading to mating. In the budding yeastsponsive MAP kinase cascade including the Ste20, Ste11,Saccharomyces cerevisiae, pheromone induces cell-cycle ar-Ste7, and Kss1 protein kinases and the Ste12 and Tec1rest, stimulates transcription, and causes alterations intranscription factors (Liu et al. 1993; Cook et al. 1997;cell morphology (Sprague and Thorner 1992). InMadhani et al. 1997). The pheromone receptors andother yeast or fungi, such as Schizosaccharomyces pombe,the Gpa1/Ste4/Ste18 G protein, upstream elements inUstilago maydis, and Cryptococcus neoformans, conjugationthe mating response, do not regulate filamentousrequires both pheromone and specific nutritional con-growth (Liu et al. 1993). The second pathway involvesditions. Thus, multiple signaling events are coordinatedthe Ga protein Gpa2, which functions to regulate cAMPto regulate developmental processes.levels (Nakafuku et al. 1988; Kubler et al. 1997; LorenzIn S. cerevisiae, nutritional signals also regulate pseu-and Heitman 1997; Colombo et al. 1998). cAMP thendohyphal differentiation, a filamentous growth formmodulates the activity of the cAMP-dependent proteininduced upon nitrogen starvation (Gimeno et al. 1992).kinase (PKA); Tpk2p, one of the three catalytic subunitsSimilar to pheromone-induced mating, filamentous dif-of PKA, specifically controls the filamentation responseferentiation involves changes in gene transcription, cell(Robertson and Fink 1998; Pan and Heitman 1999).cycle progression, and morphology, although unlikeOne additional component that is involved in nitrogenmating, pseudohyphal differentiation occurs only insensing is the Mep2 ammonium permease, which isrequired for pseudohyphal differentiation when ammo-nium is the sole source of nitrogen and may serve as a

Corresponding author: Joseph Heitman, Department of Genetics, Box receptor for ammonium ions (Lorenz and Heitman3546, 322 CARL Bldg., Research Dr., Duke University Medical Ctr.,1998a).Durham, NC 27710. E-mail: [email protected]

1 These three authors contributed equally to this work. There are several links between the MAP kinase and

Genetics 154: 609–622 (February 2000)

610 M. C. Lorenz et al.

Gpa2p-cAMP signaling pathways. First, Ras2p functions regulate signaling events and our understanding ofthese processes have benefited from studies in fungi, inat a branchpoint and serves to regulate filamentous

growth by activating both the MAP kinase and the cAMP- particular pheromone signaling in S. cerevisiae and S.pombe. The molecular mechanisms of G protein-coupledPKA signaling cascades (Gimeno et al. 1992; Mosch et

al. 1996, 1999; Kubler et al. 1997; Lorenz and Heitman receptor signaling have been functionally conserved,and heterologous expression of many different G pro-1997; Robertson and Fink 1998; Pan and Heitman

1999). Second, recent findings reveal that both the MAP tein-coupled receptors in yeast results in ligand-depen-dent activation of the pheromone response pathwaykinase and the cAMP-PKA signaling pathways converge

to coordinately regulate expression of the cell surface (King et al. 1990); thus the fungal system is a valubletool for probing G protein signaling. A number of Gaflocculin Flo11, which is required for haploid invasive

growth and diploid pseudohyphal differentiation (Lam- subunits have been recently cloned from other fungalspecies, including Ustilago maydis, Cryptococcus neo-brechts et al. 1996; Lo and Dranginis 1998; Pan and

Heitman 1999; Rupp et al. 1999). formans, Candida albicans, and Cryphonectria parasitica,among others. Despite the proliferation of Ga proteins,The MAP kinase cascade that regulates filamentous

growth has a dual function and also regulates mating only a few G protein-coupled receptors are known (re-viewed in Bolker 1998). With only a few exceptions,in response to pheromone. Similarly, the cAMP-PKA

pathway has a second function in addition to filamenta- namely G protein-coupled receptor homologs in S. cere-visiae (Gpr1p) and S. pombe (Git3), all of these fungaltion. PKA activity is essential in yeast, and cells with

elevated PKA activity are hypersensitive to stresses such G protein-coupled receptors bind pheromones duringmating responses. The Gpr1 receptor was recently iden-as heat shock, are unable to sporulate, and have dimin-

ished levels of storage carbohydrates such as glycogen tified in a two-hybrid screen via its ability to interactwith the Ga protein Gpa2 (Yun et al. 1997; Xue et al.(reviewed in Broach 1991). Furthermore, the PKA

pathway responds to carbon source availability; in partic- 1998) and subsequently implicated in regulating Gpa2pfunction (Xue et al. 1998); both gpa2D ras2D and gpr1Dular, readdition of glucose to starved cells triggers a

rapid and transient increase in cAMP that requires ras2D double mutant strains have a similar cAMP-reme-diated synthetic growth defect (Kubler et al. 1997;Gpa2p, Ras2p, and adenylyl cyclase (Colombo et al.

1998; Yun et al. 1998). Some of the defects associated Lorenz and Heitman 1997; Xue et al. 1998).In this article, we demonstrate that the G protein-with loss of PKA activity can be suppressed by overex-

pression of Sch9p, a PKA-related kinase (Toda et al. coupled receptor homolog Gpr1 is required for pseu-dohyphal differentiation and regulates a signal trans-1988). Deletion of the SCH9 gene blocks the heat shock

sensitive phenotype conferred by dominant active duction cascade involving Gpa2p, adenylyl cyclase, andPKA. The Gpr1 receptor is also required for haploidGpa2p mutants (Xue et al. 1998). Thus, the Sch9 kinase

also impinges on the PKA signaling cascade. invasive growth and regulates expression of the cell sur-face flocculin Flo11, which is required for invasive andHeterotrimeric G proteins are regulated by seven

transmembrane domain receptors of the b-adrenergic pseudohyphal growth. We present genetic and physio-logical evidence that the Gpr1 receptor is activated byreceptor family. Signaling via these receptor-G protein

modules regulates a diverse array of processes in all glucose and other structurally related fermentable sug-ars. Signaling via the Gpr1p-Gpa2p-cAMP pathway iseukaryotes, including neurotransmission in animals

from nematodes to mammals, cell growth and differenti- independent of the MAP kinase signaling cascade. Wealso present genetic evidence that the Gpr1p-Gpa2p-ation, chemotaxis both in immune function and in mi-

croorganisms such as Dictyostelium discoideum, vesicle traf- cAMP-PKA signaling pathway regulates vegetativegrowth in parallel with an Sch9p-dependent signalingficking, and sensory transduction (see, for example,

Reed 1992; Neer 1995; Parent and Devreotes 1996). pathway that does not play a primary role in regulatingfilamentous growth. In summary, our studies suggestAs primary sensors of extracellular signals, G protein-

coupled receptors bind to a wide variety of ligands, that the Gpr1p signaling cascade serves as a dual sensorincluding amino acids and analogues, peptides, pho- of carbon abundance and nitrogen deprivation in whichtons, lipids, and volatile compounds. Defects in recep- the Gpr1 receptor is transcriptionally induced by nitro-tor-G protein signaling are the cause of a number of gen starvation and then senses the presence of fer-human diseases, including visual defects like retinitis mentable sugars by a ligand-receptor interaction thatpigmentosa and color blindness; hormone signaling dys- regulates cAMP production.functions such as hyper- or hypothyroidism and abnor-mal sexual development resulting from mutations in

MATERIALS AND METHODSreceptors for luteinizing hormone, follicle stimulatinghormone, and thyroid stimulating hormone; and diabe-

Yeast strains, media, and genetic methods: Standard mediates (see Spiegel 1997). and other genetic methods were as described in Sherman

It is clearly of significant importance to understand (1991). Low ammonium media (SLAD; 50 mM ammoniumsulfate, 2% glucose, 2% Bacto-agar) for induction of pseudohy-the mechanisms by which heterotrimeric G proteins

611Gpr1 Receptor Regulates Filamentous Growth

TABLE 1

Yeast strains

Strains Genotype Reference

MLY40a ura3-52 MATa Lorenz and Heitman (1997)MLY41a ura3-52 MATa Lorenz and Heitman (1997)MLY61a/a ura3-52/ura3-52 MATa/a Lorenz and Heitman (1997)MLY132a gpa2D::G418 ura3-52 MATa Lorenz and Heitman (1997)MLY132a gpa2D::G418 ura3-52 MATa Lorenz and Heitman (1997)MLY132a/a gpa2D::G418/gpa2D::G418 ura3-52/ura3-52 MATa/a Lorenz and Heitman (1997)MLY162a/a pde2D::G418/pde2D::G418 ura3-52/ura3-52 MATa/a Lorenz and Heitman (1997)MLY171a/a gpa2D::G418/gpa2D::G418 pde2D::G418/pde2D::G418 ura3-52/ura3-52 This study

MATa/aMLY187a ras2D::G418 ura3-52 MATa This studyMLY187a/a ras2D::G418/ras2D::G418 ura3-52/ura3-52 MATa/a This studyMLY232a gpr1D::G418 ura3-52 MATa This studyMLY232a gpr1D::G418 ura3-52 MATa This studyMLY232a/a gpr1D::G418/gpr1D::G418 ura3-52/ura3-52 MATa/a This studyMLY259a/a gpr1D::G418/gpr1D::G418 pde2D::G418/pde2D::G418 ura3-52/ura3-52 This study

MATa/aMLY265a sch9D::G418 ura3-52 MATa This studyMLY265a/a sch9D::G418/sch9D::G418 ura3-52/ura3-52 MATa/a This studyXPY5a tpk2D::G418 ura3-5 MATa Pan and Heitman (1999)XPY5a/a tpk2D::G418/tpk2D::G418 ura3-52/ura3-52 MATa/a Pan and Heitman (1999)XPY135a gpr1D::G418 gpa2D::G418 ura3-5 MATa This studyXPY135a/a gpr1D::G418/gpr1D::G418 gpa2D::G418/gpa2D::G418 ura3-5/ura3-52 This study

MATa/aXPY163a/a sch9D::HygB/SCH9 ura3-52/ura3-52 MATa/a This studyXPY164a/a sch9D::HygB/SCH9 gpr1D::G418/gpr1D::G418 ura3-52/ura3-52 MATa/a This studyXPY165a/a sch9D::HygB/SCH9 gpa2D::G418/gpa2D::G418 ura3-52/ura3-52 MATa/a This studyXPY166a/a sch9D::HygB/SCH9 ras2D::G418/ras2D::G418 ura3-52/ura3-52 MATa/a This studyHLY352a/a ste12D::LEU2/ste12D::LEU2 ura3-52/ura3-52 MATa/a Liu et al. (1993)

phal differentiation has been described (Gimeno et al. 1992) of the SCH9 gene in diploid strains MLY61a/a, MLY132a/a,MLY187a/a, and MLY232a/a. The resulting strains, XPY163a/as has SLARG media (50 mm ammonium sulfate, 2% raffinose,

0.5% galactose, 2% Bacto-agar) for induction of pGal-GPA2 a, XPY164a/a, XPY165a/a, and XPY166a/a, were sporulatedand dissected on YPD medium and then replica-plated to YPDalleles (Lorenz and Heitman 1997) and SLADG media (50

mm ammonium sulfate, 2% galactose, 0.13% glucose, 2% medium containing G418 or hygromycin B.Plasmids: Plasmids used in this study include pML160 andBacto-agar) for induction of the pGAL-STE12 allele (Liu et al.

1993). Yeast transformations were done via the lithium ace- pML180 [CEN URA3 GPA2-2 and GPA2, respectively; Lorenzand Heitman (1997)], pMW1 and pMW2 [CEN URA3 RAS2tate/polyethylene glycol protocol of Schiestl et al. (1993).

Strains used in these studies are listed in Table 1. The and RAS2Val19, respectively; Ward et al. (1995)]; pSL1509 [CENURA3 STE11-4; Stevenson et al. (1992)]: pNC252 [CEN URA3wild-type strains of the R1278b background, MLY40 (ura3-52

MATa), MLY41 (ura3-52 MATa), and MLY61 (ura3-52/ura3- pGAL-STE12; Liu et al. (1993)]; pCG38 [2m URA3 PHD1;Gimeno and Fink (1994)]; p2.5-2 [2m URA3 TEC1; Lorenz52 MATa/a) have been described, as have the gpa2D and

ste12D strains, MLY132a/a (gpa2D::G418/gpa2D::G418 ura3- and Heitman (1998b)]; and YEplac195 [2m URA3; Gietz andSugino (1988)]. The 2m-GPR1 plasmid pML224 was con-52/ura3-52 MATa/a) and HLY352 (ste12D::LEU2/ste12D::

LEU2 leu2D::hisG/leu2D::hisG ura3-52/ura3-52 MATa/a), re- structed as follows. The GPR1 cassette from pGPR1-112.2 (Xueet al. 1998) was excised with SacI-PstI and inserted into thosespectively (Liu et al. 1993; Lorenz and Heitman 1997). The

gpr1D::G418/gpr1D::G418 homozygous diploid strain was con- sites in YEplac195. This plasmid did not complement the pseu-dohyphal defect of a gpr1D/gpr1D strain due to insufficienttructed by deleting the GPR1 gene in haploid strains of the

R1278b background MLY40 (MATa) and MLY41 (MATa) us- sequences at the 39 end of the gene. We PCR-amplified afragment from an internal SalI site to z500 bp 39 to the GPR1ing a PCR disruption protocol (Wach et al. 1994). Disruptants

were confirmed by PCR and mated to produce the homozy- stop codon, appending an artificial SphI site. This fragmentwas used to replace the incomplete 39 end in YEplac195, creat-gous diploid strain MLY232a/a. A similar process was used

to construct the sch9D::G418/sch9D::G418 strain MLY265a/ ing the 2m-URA3-GPR1 plasmid pML224. This plasmid comple-ments the pseudohyphal growth defect conferred by the gpr1Da. Haploid strains with single gene mutations were crossed,

sporulated, and dissected to produce double mutant strains. mutation.The SCH9 gene, including the open reading frame andTetrads in which there were two G418-resistant and two G418-

sensitive segregants were chosen and the presence of both 600 bp each of the 59- and 39-untranslated regions, was PCR-amplified using genomic DNA of strain MLY40a as template.mutations was confirmed by PCR analysis of genomic DNA.

The hygromycin B resistance gene cassette (provided by Alan A PCR product of 3.7 kb was cloned into the 2m plasmidYEplac195. The resulting plasmid, pXP77, complements theGoldstein and John McCusker) was used to delete one allele


vegetative and filamentous growth defect conferred by thesch9 mutation.

Photomicroscopy: Whole colony photographs were takenusing a Nikon Eclipse E400 microscope using a 310 objectiveand 32.5 trinocular adaptor (325 magnification) connectedto a Nikon N70 camera. Colonies were photographed directlyon solid medium after 4 days of incubation at 308, unlessotherwise indicated.

Northern blot analysis: Haploid yeast strains were incubatedin YPD liquid medium overnight and then transferred to freshYPD medium and incubated to an OD600 of 1.0. Cells werewashed with ice water and total RNA was isolated by usingacid phenol, separated by electrophoresis, and transferredovernight by capillary action to nylon membranes (VWR).DNA fragments to be used as probes (a 300-bp PCR productderived from the 59 region of the FLO11 open reading frameand a 500-bp 59 fragment of the ACT1 open reading frame)were gel-purified and radiolabeled by random priming (Boeh-ringer Mannheim, Indianapolis). Hybridization and washingwere performed as described (Sambrook et al. 1989).

Invasion assays: Haploid strains were grown as patches onYPD medium and incubated at 308 for 4 days. The plate wasphotographed and then washed with running water, and re-maining invaded cells were photographed. Figure 1.—G protein-coupled receptor homolog Gpr1

cAMP assays: Cells of isogenic wild-type (MLY41a), gpr1 regulates filamentous growth. Isogenic diploid wild-typemutant (MLY232a), and gpa2 mutant (MLY132a) haploid (MLY61a/a), gpa2D/gpa2D (MLY132a/a), and gpr1D/gpr1Dstrains were grown in YPD medium for 2 days at 308 with (MLY232a/a) strains, each containing a control URA3 plas-shaking and were collected by centrifugation. After washing mid, and the gpr1D/gpr1D strain (MLY232a/a) containing thetwice with water and once with buffer (10 mm MES, pH 6.0, wild-type GPR1 gene on a plasmid (pML224) were incubated0.1 mm EDTA), cells were resuspended in buffer and incu- on nitrogen limiting SLAD medium for 4 days at 308.bated for 2 hr to induce glucose starvation prior to the readdi-tion of different sugars, as indicated, to a final concentrationof 2%. At various time points, 0.5-ml aliquots were removed

and Heitman 1997), we tested whether the Gpr1 recep-and transferred to 2-ml screw-cap tubes containing 0.3 ml oftor has a similar role.glass beads and 0.5 ml of ice-cold water and were frozen in

The GPR1 open reading frame was precisely deletedliquid nitrogen. The samples were then homogenized with abead beater at 48 and crude cell extracts were lyophilized. in the R1278b strain background commonly used forConcentrations of intracellular cAMP were determined using studies on filamentous growth. When incubated on ni-a cAMP enzyme immunoassay kit (Amersham, Arlington trogen limiting SLAD medium, diploid gpr1D/gpr1DHeights, IL) and samples were normalized to total cell weight.

mutant strains exhibited a significant defect in filamen-Samples were analyzed in duplicate and the values were aver-tous growth (Figure 1). Expression of the wild-typeaged. Three independent experiments were conducted with

similar results. GPR1 gene from a plasmid complemented the gpr1D/Detection of the Gpr1-GFP hybrids: The Gpr1-GFP fusion gpr1D filamentous growth defect (Figure 1). The gpr1D

protein was localized as described (Xue et al. 1998), using a mutation conferred a pseudohyphal defect on mediatrp1 derivative of strain YJM836 3 YJM837 (kindly providedcontaining a variety of limiting nitrogen sources, includ-by J. McCusker). Cells expressing the Gpr1-GFP fusion proteining ammonium (Figure 1), glutamine, proline, aspar-were grown either at 308 in liquid synthetic medium or at

room temperature on SLAD solid medium and viewed using tate, asparagine, and serine (data not shown).either the fluorescein isothiocyanate (FITC) filter for fluores- The defect in filamentous growth in gpr1 and gpa2cence microscopy or phase contrast optics using a Zeiss Axio- mutant strains was not absolute, as some modest fila-phot microscope.

ments were still observed in the null mutants. gpr1 gpa2double mutant strains exhibited a filamentation defectsimilar to the gpa2 and gpr1 single mutant strains (dataRESULTSnot shown), consistent with the interpretation that

The G protein-coupled receptor Gpr1 regulates fila- Gpr1p and Gpa2p function in a linear signaling cascade.mentous growth: The G protein-coupled receptor ho- Gpr1p and Gpa2p regulate haploid invasive growthmolog Gpr1 was identified in a two-hybrid screen with and FLO11 gene expression: Haploid strains of S. cerevis-the Ga protein Gpa2 (Yun et al. 1997; Xue et al. 1998). iae undergo a dimorphic transition to invasive growthgpr1 or gpa2 mutations confer a synthetic growth defect that shares several features with pseudohyphal differen-in conjunction with mutations in the small G protein tiation of diploid S. cerevisiae strains (Roberts and FinkRas2 (Xue et al. 1998). These findings indicate that 1994). First, the MAP kinase cascade components Ste-Gpr1p and Gpa2p are physically and functionally re- 20p, Ste11p, Ste7p, and Ste12p regulate both haploidlated. Because the Ga protein Gpa2 is required for pseu- invasive growth and diploid pseudohyphal growth. Sec-

ond, haploid invasive cells are elongated, invade thedohyphal differentiation (Kubler et al. 1997; Lorenz


Figure 2.—Gpr1p and Gpa2p regu-late haploid invasive growth and FLO11expression. (A) Isogenic haploid wild-type (MLY40a), gpr1 (MLY232a), gpa2(MLY132a), gpr1 gpa2 (XPY135a), tpk2(XPY5a), and sch9 (MLY265a) mutantstrains were streaked on YPD medium,grown for 96 hr at 308, nonadherant cellswere washed off, and cells that had in-vaded the medium were photographed.(B) Total RNA was isolated from iso-genic haploid strains in A and grown inliquid YPD medium. RNA was fraction-ated by formaldehyde agarose gel elec-trophoresis, transferred to a nylon mem-brane, and hybridized with radioactiveprobes specific for the FLO11 and ACT1genes.

agar, and switch budding pattern from axial to unipolar a model in which the two function in a linear pathway.The defect in FLO11 expression in the gpr1 and gpa2budding. Because the Gpr1 receptor and Gpa2 Ga pro-

tein regulate pseudohyphal differentiation of diploid mutant strains was somewhat less severe than the defectobserved in a mutant strain of the Tpk2 PKA catalyticstrains, we addressed whether these proteins also regu-

late haploid invasive growth. subunit, suggesting that some activation of PKA canoccur in gpr1 and gpa2 mutant strains, possibly via Ras2As shown in Figure 2A, gpr1 and gpa2 haploid mutant

strains exhibited a marked defect in agar invasive activation of adenylyl cyclase (Figure 2B; Pan and Heit-man 1999). In conclusion, the Gpr1 receptor and thegrowth. The agar invasion defect of the gpr1 and gpa2

mutant strains was comparable to that observed with coupled Ga subunit Gpa2 are required for haploid inva-sive growth and regulate expression of the cell surfacemutant strains lacking components of the MAP kinase

cascade (data not shown), to a gpr1 gpa2 double mutant flocculin Flo11.Gpr1p receptor signals via a pathway involvingstrain (Figure 2A), and to a mutant strain lacking the

PKA catalytic subunit Tpk2 (Figure 2A). Thus, Gpr1p Gpa2p, Ras2p, and cAMP: Given the connection be-tween the functions of Gpr1p and Gpa2p and the similarand Gpa2p are required for haploid invasive growth.

The PKA and MAP kinase signaling cascades regulate phenotypes conferred by the gpr1 and gpa2 mutations(Figures 1 and 2), we tested by epistasis analysis whetherhaploid invasive growth and diploid filamentous growth,

in part by regulating expression of the cell surface floc- the Gpr1 receptor functions upstream of the Gpa2 Gaprotein and other components of the cAMP-PKA signal-culin Flo11 (Lo and Dranginis 1998; Pan and Heitman

1999; Rupp et al. 1999). We therefore addressed whether ing cascade. Expression of the dominant active GPA2allele containing a Gly132Val mutation in the GTPasethe Gpr1 receptor and the Ga protein Gpa2 regulate

FLO11 gene expression. RNA was isolated from haploid active site enhances pseudohyphal differentiation(Lorenz and Heitman 1997). Expression of the domi-strains grown in YPD rich medium and analyzed by

Northern blot analysis with probes directed to the nant active GPA2 allele suppressed the filamentationdefect of gpr1D/gpr1D mutant strains (Figure 3A), pro-FLO11 gene, and also to the ACT1 gene as a loading

control. FLO11 expression was readily detected in the viding genetic evidence that the Ga protein Gpa2 func-tions downstream of the Gpr1 receptor.wild-type strain, but little or no FLO11 expression was

observed in isogenic mutant strains lacking Gpr1, Gpa2, In previous studies, we demonstrated that the fila-mentation defect of gpa2 mutant strains could be sup-or both Gpr1 and Gpa2 (Figure 2B). The defect in

FLO11 expression in the gpr1 and gpa2 mutant strains pressed by activation of the cAMP signaling pathway,either by expression of a dominant active RAS2 allelewas comparable to that observed in mutant strains lack-

ing both Gpr1p and Gpa2p (Figure 2B), consistent with (RAS2Gly19Val) or by the addition of cAMP. If the Gpr1


receptor acts upstream of Gpa2p, then either theRAS2Gly19Val dominant active mutant or cAMP should re-store filamentation in a gpr1D/gpr1D mutant strain. Thisis indeed the case, as expression of RAS2Gly19Val (Figure3B) or addition of 1 to 10 mm cAMP restored filamen-tation in gpr1D/gpr1D pde2D/pde2D mutant strains (Fig-ure 3C). These findings provide a further link betweenthe functions of the Gpr1 receptor and Gpa2 Ga proteinand suggest a role in regulating the cAMP signalingpathway.

The Gpa2-cAMP-PKA pathway functions, in part, in-

Figure 4.—The effects of the gpr1D mutation are partiallyindependent of the MAP kinase cascade. Wild-type (MLY61a/a), gpa2D/gpa2D(MLY132a/a), and gpr1D/gpr1D (MLY232a/a) strains expressing either a control vector or the indicatedallele (vector, YEplac195; STE11-4, pSL1509; pGAL-STE12,pNC252) were expressed on SLAD medium for 4 days at 308.SLADG medium was used to induce expression of the pGAL-STE12 construct.

dependently from the MAP kinase pathway in regulatingpseudohyphal differentiation. The filamentation defectof gpa2/gpa2 mutant strains is suppressed by markedoverexpression of STE12, while expression of the domi-nant active STE11-4 allele does not (Lorenz and Heit-man 1997, 1998a,b) (Figure 4). Similar epistasis resultswere observed in gpr1D/gpr1D mutant strains (Figure4). The gpr1 and gpa2 mutations also had no effect onexpression of an FRE-lacZ reporter gene that is knownto be regulated by the MAP kinase cascade, whereas aras2 mutation inhibited FRE-lacZ expression (data notshown). Taken together, these findings suggest thatGpr1p and Gpa2p regulate the cAMP signaling cascadeand not the MAP kinase pathway.

Gpr1 receptor plays a role in sensing fermentablesugars: Pseudohyphal growth occurs on medium con-taining not only limiting concentrations of nitrogen

Figure 3.—gpr1D is suppressed by dominant active GPA2or RAS2 mutations or by cAMP. (A) Isogenic diploid wild-type(MLY61a/a), gpr1D/gpr1D (MLY232a/a), and gpa2D/gpa2D(MLY132a/a) mutant strains containing a plasmid expressingGPA2wt (pML180) or the dominant active GPA2-2 allele Gpa2-Val132 (pML160) were incubated on inducing SLARG me-dium. (B) The same strains as in A containing plasmids ex-pressing RAS2wt (pMW1), or RAS2Val19 (pMW2) were incubatedon SLAD medium. (C) pde2D/pde2D (MLY162a/a), gpr1D/gpr1D pde2D/pde2D (MLY259a/a), and gpa2D/gpa2D pde2D/pde2D (MLY171a/a) mutant strains were incubated on SLADmedia containing 0, 1, or 10 mm cAMP. Each experiment wasincubated on the indicated media for 4 days at 308.


Figure 5.—Gpr1p andGpa2p regulate filamentousgrowth and cAMP produc-tion in response to fer-mentable sugars. (A) Iso-genic wild-type (MLY61a/a),gpr1D/gpr1D (MLY232a/a), gpa2D/gpa2D (MLY132a/a), and tpk2D/tpk2D(XPY5a/a) diploid strainswere grown on SLA mediumcontaining 2% glucose, su-crose, galactose, or maltose,incubated for 96 hr at 308,and representative colonieswere photographed at 325magnification. (B) Isogenicwild-type (MLY41a), gpr1D(MLY232a), and gpa2D(MLY132a) strains weregrown to stationary phase inYPD medium. Cells werewashed and incubated inbuffer in the absence of glu-cose for 2 hr. Subsequently,glucose (Glu), sucrose(Suc), galactose (Gal), ormaltose (Mal) was added tothe cultures at 2%. Cellswere collected at varioustime points prior to (0 min)or after (0.5, 1, 3, and5 min) the addition of thesesugars, and cell extractswere prepared and cAMPwas measured as describedin materials and meth-ods. Samples were assayedin duplicate and averaged.The results shown here arerepresentative of three simi-lar experiments. (r) Wild-type; (j) gpr1; (m) gpa2.

source, such as 50 mm ammonium sulfate, but also an ure 5A and data not shown). In contrast, the monosac-charide galactose supported growth but only poor fila-abundant level of a fermentable carbon source, such as

2% glucose. In contrast, on a medium with low nitrogen mentation was observed (Figure 5A). The disaccharidesucrose, which is rapidly metabolized to fructose andlevels and a nonfermentable carbon source, such as

acetate, sporulation occurs. Thus, yeast cells must sense glucose by invertase, also supported pseudohyphalgrowth in SLA medium (Figure 5A). Interestingly, theboth abundant fermentable carbon source and nitrogen

deprivation to undergo pseudohyphal differentiation. disaccharide maltose dramatically enhances pseudohy-phal growth. This finding may be related to the earlierWhen glucose is readded to glucose-starved yeast cells,

cAMP is rapidly produced and growth ensues. Recent observation that starches, glucose polysaccharides thatare metabolized to maltose by starch-utilizing yeaststudies reveal that this response to glucose readdition

requires Gpr1p, Gpa2p, and Ras2p, suggesting a role strains, stimulate filamentous growth (Lambrechts etal. 1996; Figure 5A).in carbon source sensing (Colombo et al. 1998; Jiang

et al. 1998; Yun et al. 1998; Kraakman et al. 1999). Importantly, both gpr1/gpr1 and gpa2/gpa2 mutantstrains exhibited defects in pseudohyphal differentia-We therefore assessed filamentous growth of the iso-

genic wild-type, gpr1/gpr1, and gpa2/gpa2 mutant strains tion on medium containing the monosaccharides glu-cose, mannose, or fructose, or the disaccharide sucroseon synthetic low ammonium (SLA) medium containing

different carbon sources (2%). In wild-type cells, the (Figure 5A and data not shown). These findings suggesta specific role for the Gpr1 receptor/Gpa2 G proteinmonosaccharides glucose, fructose, and mannose all

supported pseudohyphal growth to similar extents (Fig- complex in sensing the presence of abundant structur-


ally related fermentable monosaccharides in the growth tous growth or FLO11 gene expression (data notshown). These observations provide support for themedium. The defect in gpr1/gpr1 or gpa2/gpa2 mutant

cells on glucose medium could be suppressed by the model that glucose and structurally related sugars areagonists of the Gpr1 receptor.addition of 1% ethanol, which induces hyperfilamenta-

tion in diploid yeast strains, further indicating that these Gpr1p does not localize to hyphal projections: Detec-tion of Gpr1p in vegetative cells using a green florescentmutant cells do not have an intrinsic defect in fila-

mentation (data not shown). The gpr1 and gpa2 muta- protein (GFP)-tagged variant localized the protein tothe plasma membrane (Xue et al. 1998). We postulatedtions had no effect on the robust pseudohyphal fila-

mentation induced by maltose, or on the low level of that the Gpr1p receptor might localize to the tips ofpseudohyphal filament cells. In this model, Gpr1pfilamentation observed with galactose, indicating that

maltose and galactose are not sensed by the Gpr1p- would act in the same manner as the pheromone recep-tors, Ste2p and Ste3p, which are localized to the shmooGpa2p pathway (Figure 5A). Finally, mutant strains

lacking the PKA catalytic subunit Tpk2 exhibited a fila- tip and stimulate morphogenesis in the direction of thehighest pheromone concentration (Jackson et al. 1991;mentation defect on glucose, mannose, sucrose, fruc-

tose, and maltose (Figure 5A and data not shown), indi- Segall 1993; Chenevert 1994). In contrast, we foundthat the uniform plasma membrane staining of thecating that PKA plays a role in the responses to all of

these sugars. These findings suggest that the functions Gpr1-GFP fusion protein was maintained even in elon-gated cells grown under pseudohyphal inducing condi-of Gpr1p, Gpa2p, and PKA are coupled in sensing car-

bon sources. tions (data not shown). The overall level of the Gpr1-GFP fusion protein was, however, lower in filamentousFinally, we sought to establish whether the role of

Gpr1p and Gpa2p in regulating filamentation in re- than in vegetative cells (data not shown). These findingssuggest that Gpr1p and Gpa2p may not function tosponse to different carbon sources is correlated with

the production of cAMP. To this end, isogenic wild- regulate the direction of polarized growth.Gpr1p and Gpa2p signal in parallel with Sch9p duringtype, gpr1, and gpa2 mutant strains were starved for

carbon source, and glucose, sucrose, galactose, or malt- vegetative growth: In previous studies, the Sch9 kinasewas implicated as a downstream signaling componentose was added to the cells. As shown in Figure 5B, wild-

type cells respond to glucose or sucrose addition by of the Gpa2p signal transduction cascade. Namely, ex-pression of a dominant active Gpa2 mutant causes heatproducing cAMP, and this requires both Gpr1p and

Gpa2p. In marked contrast, neither galactose nor malt- shock sensitivity and this effect is blocked by an sch9mutation, suggesting that Sch9p might be a downstreamose stimulated cAMP production (Figure 5B). Because

maltose dramatically enhances filamentous growth in- mediator of Gpa2 signaling (Xue et al. 1998). The Sch9kinase was originally identified via its ability to suppressdependently of Gpr1p and Gpa2p and does not stimu-

late cAMP production, another sensing mechanism lethality of tpk1,2,3 triple mutant strains when overex-pressed, indicating a role in parallel with PKA down-likely operates to detect maltose and regulate fila-

mentation, which may involve the maltose permease (X. stream of cAMP (Toda et al. 1988). In addition, theGpr1 receptor and Gpa2 G protein signal in a pathwayWang and C. Michels, personal communication).

Taken together, our observations indicate that the that functions in parallel with Ras2p, regulates cAMPproduction, and is required for vegetative growth. ForGpr1 receptor/Gpa2 G protein complex regulates the

PKA pathway, FLO11 transcription, and filamentous example, ras2 gpr1 and ras2 gpa2 mutants have a severesynthetic growth defect, and normal growth of thesegrowth based on the availability of glucose and other

structurally related saccharides in the extracellular me- mutant strains is restored by cAMP or a pde2 mutationthat increases intracellular cAMP levels (Kubler et al.dium. Several models can be envisioned to explain these

findings. The first and most parsimonious is that these 1997; Xue et al. 1998).To address a possible role of Sch9p in signaling down-sugars are the direct ligands of Gpr1p. Alternatively,

these saccharides may bind to other cell surface proteins stream of Gpr1p, Gpa2p, and Ras2p, we employed ge-netic epistasis analysis. We considered three possiblethat then interact with Gpr1p. Finally, the Gpr1p ligand

could be a compound that is rapidly produced and models: Sch9p could signal downstream of Gpr1p andGpa2p, downstream of Ras2p, or in a pathway parallelsecreted following glucose readdition. Yeast cells are

known to secrete H1, ATP, and the lipid second messen- to both the Ras2p and Gpr1p-Gpa2p pathways. Ourepistasis analysis supports this third model. First, an sch9gers glycerophosphatidylinositol-4-phosphate (gPI4P)

and -4,5 bisphosphate (gPI4,5P2) in response to glucose mutation is synthetically lethal with a ras2 mutation,indicating that Sch9p does not signal solely downstream(Serrano 1983; Hawkins et al. 1993; Brandao et al.

1994; Boyum and Guidotti 1997). However, we found of Ras2p (Figure 6). Second, an sch9 mutation was alsosynthetically lethal with either a gpr1 or a gpa2 mutationthat altering the pH of SLAD medium with buffer inhib-

ited filamentous growth, whereas ATP and other nucleo- (Figure 6). This last finding does not support a modelin which one pathway consists of a linear Gpr1p-Gpa2p-tides and nucleosides had no effect (data not shown).

Finally, gPI4P and gPI4,5P2 did not stimulate filamen- Sch9p cascade that functions in parallel with Ras2p and


Figure 6.—sch9 mutationis synthetically lethal withgpr1, gpa2, or ras2 mutations.The diploid strains sch9::HygB/SCH9 (XPY163a/a),sch9::HygB/SCH9 gpr1::G418/gpr1::G418 (XPY164a/a), sch9::HygB/SCH9 gpa2::G418/gpa2::G418 (XPY-165a/a), and sch9::HygB/SCH9 ras2::G418/ras2::G418(XPY166a/a) were sporu-lated, tetrads were dissectedby micromanipulation intovertical grids, and sporeswere germinated on YPD me-dium for 3 days and then rep-lica-plated to YPD mediumcontaining G418 or hygro-mycin B to identify segreg-ants that contained the sch9(HygB), gpr1, gpa2, and ras2mutations (G418).

is required for vegetative growth on rich medium; it mutations (see Figure 6). Finally, overexpression of theSch9 kinase from a 2m multicopy plasmid did not en-instead supports an alternative model in which Sch9p

functions in an independent pathway parallel to Gpr1p hance or inhibit pseudohyphal differentiation in a wild-type strain (data not shown).and Gpa2p.

Regulation of filamentous growth by Gpr1p and To investigate the upstream elements that regulateSch9p, we expressed the dominant RAS2 and GPA2 al-Gpa2p does not require Sch9p: We next addressed

whether Sch9p plays a role in the regulation of pseu- leles in the sch9/sch9 mutant strain. Deletion of SCH9had no effect on the ability of the active RAS2Val19 alleledohyphal differentiation by Gpr1p and Gpa2p. As

shown in Figure 7, an sch9/sch9 mutant strain had no to enhance filamentous growth (Figure 8A). Moreover,expression of the dominant active GPA2-2 allele (Gly13-defect in filamentous growth on SLAD medium at early

time points (12 hr), whereas a filamentation defect was 2Val) still stimulated filamentation, though not to wild-type levels, in an sch9/sch9 mutant strain (Figure 8B),readily apparent in gpr1/gpr1 or gpa2/gpa2 mutant mi-

crocolonies. Following 3 days of incubation, a fila- indicating that activation of filamentous growth byGpa2p is not dependent upon Sch9p.mentation defect was apparent in the sch9 mutant strain

but was not as severe as the defect observed with either We also investigated whether Sch9p regulates haploidinvasive growth or FLO11 gene expression. In contrastgpr1 or gpa2 mutant strains (Figure 7). This filamenta-

tion defect of sch9 mutant strains may be in part attribut- to gpr1, gpa2, or tpk2 mutations, the sch9 mutation didnot block invasive growth; in fact, sch9 mutant strainsable to a nonspecific effect of the growth defect that is

conferred by the sch9 mutation but not by gpr1 or gpa2 were hyperinvasive (Figure 2A). Moreover, gpr1, gpa2,

Figure 7.—sch9 mutationconfers a modest defect inpseudohyphal growth. Iso-genic diploid wild-type(MLY61a/a), gpr1/gpr1(MLY232a/a), gpa2/gpa2(MLY132a/a), and sch9/sch9 (MLY265a/a) mutantstrains were grown on SLADmedium at 308 and micro-colonies were photo-graphed at 325 magnifica-tion following incubationfor 12 hr or 3 days.


Figure 9.—A model for the regulation of pseudohyphaldifferentiation in yeast. The Gpr1 receptor is depicted as anutrient sensor that detects glucose as a ligand and transmitsa signal via Gpa2p to regulate cAMP production and the activa-tion of PKA. The Gpr1p-Gpa2p-cAMP signaling cascade func-tions in parallel with the MAP kinase cascade, and these twosignaling pathways converge to regulate expression of the cellsurface flocculin Flo11 that is required for haploid invasivegrowth and diploid filamentous growth. The Sch9 kinase isrequired for normal vegetative growth and heat shock sensitiv-ity in cells expressing activated Gpa2 mutants.

clude that Sch9p functions in an independent signalingcascade, parallel to the Gpr1p-Gpa2p-cAMP-PKA signal-ing pathway, which regulates vegetative growth and re-Figure 8.—sch9 mutation does not block filamentation in

response to GPA2-2 or RAS2Val19. (A) Wild-type (MLY61a/a), sponses to heat shock and inhibits haploid invasivegpa2D/gpa2D (MLY132a/a), and sch9D/sch9D (MLY265a/a) growth and FLO11 gene regulation. Further, Sch9p isstrains, expressing a control vector (YEplac195), RAS2wt

not directly downstream of Ras2p, because sch9 and ras2(pMW1), or RAS2Val19 (pMW2), were incubated on SLAD me-mutations are also synthetically lethal and the RASVal19

dium. (B) The strains from A, expressing a control vectorallele still stimulates hyperfilamentation in an sch9 mu-(YEplac195), GPA2wt (pML180), or GPA2-2 (pML160), were

incubated on inducing SLARG medium. Each experiment was tant strain. Sch9 may regulate diploid filamentousincubated on the indicated media for 4 days at 308. growth, although this interpretation must be qualified

because the sch9 mutation confers a marked growthdefect whereas gpr1, gpa2, tpk2, and flo11 mutationsand tpk2 mutations each blocked FLO11 expression,do not.whereas FLO11 expression was enhanced in an sch9 mu-

tant strain (Figure 2B), consistent with the hyperinvasivephenotype.

DISCUSSIONThe findings that gpa2 and sch9 mutations confer

opposite phenotypes with respect to haploid invasion The Gpr1 G protein-coupled receptor regulates fila-mentous growth: Our studies reveal that the G protein-and FLO11 expression, that the sch9 mutation does not

block hyperfilamentation conferred by the dominant coupled receptor Gpr1 regulates pseudohyphal differ-entiation in S. cerevisiae (Figure 9). The Gpr1 receptoractive GPA2 allele, and that sch9 mutations are syntheti-

cally lethal when combined with gpr1 or gpa2 mutations, also regulates invasive growth of haploid strains and isrequired for expression of the FLO11 gene encoding aall support a model in which Sch9p is not the direct

target of the Gpr1p-Gpa2p signaling cascade. We con- cell surface flocculin. The phenotypes of mutant strains


lacking the Gpr1 receptor are similar to those of mutant pathway serves as a dual sensor of both carbon abun-dance and nitrogen limitation.strains lacking the Ga protein Gpa2, and genetic evi-

dence supports a model in which ligand binding to the Sch9p signals in a pathway distinct from Gpr1p andGpa2p: Previous studies suggested that the PKA-relatedGpr1 receptor activates Gpa2p via a direct interaction.

The downstream elements of this signaling pathway con- protein kinase Sch9 might participate in a signalingpathway directly downstream of the Gpr1 receptor andsist of adenylyl cyclase, which is activated by Gpa2p to

produce cAMP (Nakafuku et al. 1988; Colombo et al. Gpa2 Ga protein. Namely, expression of an activatedGPA2 allele confers a heat shock sensitive phenotype1998), resulting in activation of PKA (Figure 9). Recent

studies have revealed that the Tpk2 catalytic subunit of that can be blocked by an sch9 mutation, whereas theheat shock sensitive phenotype conferred by a dominantPKA regulates pseudohyphal differentiation via the Sfl1

and Flo8 transcription factors that regulate expression active RAS2 mutant allele is not (Xue et al. 1998).Here we have further analyzed the role of the Sch9of the cell surface flocculin Flo11 (see Figure 9; Robert-

son and Fink 1998; Pan and Heitman 1999). kinase in signaling regulated by Gpr1p and Gpa2p. First,we found by genetic epistasis analysis that sch9 mutationsThe two-hybrid screen that identified Gpr1p also

identified portions of three additional proteins that are synthetically lethal with ras2, gpr1, or gpa2 mutations.That sch9 mutations are synthetically lethal with ras2bind to Gpa2p: Ime2p, YAL056, and YGL121 (Xue et

al. 1998). These proteins do not regulate filamentous mutations, as is also the case with gpr1 ras2 and gpa2ras2 double mutants, could have been interpreted togrowth as assayed by deletion analysis (data not shown),

and thus these proteins may regulate additional pheno- suggest that Sch9 kinase functions downstream of Gpr1pand Gpa2p. However, the finding that sch9 gpr1 andtypes of Gpa2p, such as heat shock, sporulation, or glyco-

gen accumulation. sch9 gpa2 double mutant strains are also inviable is notconsistent with a simple linear pathway consisting of aGpr1p as a receptor for fermentable sugars: Pseu-

dohyphal differentiation occurs in response to two nu- Gpr1p-Gpa2p-Sch9p cascade that signals in parallel withRas2p. Second, we find that sch9 mutations confer atritional signals: nitrogen starvation and the presence

of abundant fermentable carbon source. The Gpr1 re- modest defect in pseudohyphal growth that is not assevere as gpr1 or gpa2 mutations, and sch9 mutations failceptor plays a role in sensing both of these extracellular

signals. First, we have presented evidence here that to block hyperfilamentation in response to an activatedRAS2 allele and only partially block filamentous growthGpr1p plays a role in sensing the presence of glucose

or other fermentable sugars, resulting in cAMP produc- in response to a dominant activated GPA2 mutant.These effects may be attributable to the growth defecttion. The simplest model for explaining these findings

is that sugar binding to Gpr1p results in activation of the conferred by the sch9 mutation. Finally, gpr1 and gpa2mutations block haploid invasive growth and FLO11coupled Ga protein Gpa2, and the Gpa2-GTP complex

then binds to and activates cAMP production by adenylyl expression, whereas sch9 mutations enhance invasivegrowth and increase FLO11 gene expression.cyclase (see Figure 9). More complex models in which

glucose addition to cells results in the production and Taken together, these findings suggest that Sch9pdoes not mediate Gpr1p-Gpa2p signaling for vegetative,secretion of an unknown Gpr1 ligand cannot be ex-

cluded, and further studies employing biochemical ap- invasive, or filamentous growth, but may contribute bysignaling in a parallel pathway (outlined in Figure 9).proaches will be required to test the hypothesis that

glucose directly binds to Gpr1p. This model is consistent Sch9p is required for heat shock sensitivity induced byactivation of Gpa2p, which could either be the resultwith previous studies demonstrating that glucose stimu-

lates cAMP and that this requires both Gpr1p and Gpa2p of a direct regulation of Sch9p by the Gpa2p pathway,or reflect an indirect role for the Sch9 kinase in the(Colombo et al. 1998; Yun et al. 1998; Kraakman et

al. 1999). This glucose sensing signaling pathway also action of the Gpa2p-regulated target PKA.Gpr1 receptor homologs in other organisms: Gpr1pappears to be conserved in both S. pombe and Kluyvero-

myces lactis (Hoffman and Winston 1990, 1991; Nocero is the first G protein-coupled receptor to be identifiedin fungi that has a ligand other than mating pheromoneet al. 1994; Savinon-Tejeda et al. 1996).

The Gpr1p-Gpa2p signaling also plays a role in sens- peptides. Given the importance of nutrients, and inparticular carbon and nitrogen sources, a role for Gpr1ping nitrogen starvation. For example, activation of the

Gpr1-coupled Ga protein Gpa2 or the addition of exog- as a novel nutrient sensor could be broadly conserved.For example, a protein that shares identity with Gpr1penous cAMP can, in part, bypass the requirement for

nitrogen starvation for filamentous growth (Lorenz and from S. pombe, Git3, is known to play a role in a cAMP-dependent signal transduction pathway that mediatesHeitman 1997). Second, the GPR1 gene is known to

be transcriptionally induced in response to nitrogen repression of gene expression in response to glucose(Nocero et al. 1994). Further, the C. albicans genomestarvation (Xue et al. 1998). Thus, nitrogen starvation

induces the expression of the Gpr1 receptor, increasing sequencing project has also identified a Gpr1 homolog.Other Gpr1 receptor homologs likely remain to bethe sensitivity of this signaling cascade for the extracellu-

lar ligand. By this means, the Gpr1p-Gpa2p signaling identified in other fungi, because homologs of the Gpr1-


coupled Ga protein Gpa2 have been identified and cerevisiae Gpa2 protein, plays a conserved role in nutrientcorrelated with differentiation in S. pombe, C. neoformans, sensing, regulates virulence factor expression, and isU. hordei, U. maydis, and Podospora anserina (Isshiki et required for virulence (Alspaugh et al. 1997). The re-al. 1992; Alspaugh et al. 1997; Lichter and Mills 1997; ceptor binding portions of the C. neoformans Gpa1 andRegenfelder et al. 1997; Kruger et al. 1998; Loubra- S. cerevisiae Gpa2 proteins share marked identity, anddou et al. 1999). Previous studies have demonstrated thus a Gpr1 homolog that could be targeted for thera-that the C-terminal regions of Ga proteins interact with peutic intervention is likely coupled to Gpa1 in thisG protein-coupled receptors. The C-terminal domains pathogenic fungus. Similar approaches could also beof this family of Ga proteins share up to 80% identity. used to combat plant fungal pathogens.Thus, the Gpr1 receptor likely belongs to a family of We thank Gerry Fink, Steve Garrett, and John McCusker for strainsconserved receptors that regulate differentiation in nu- and plasmids; Danny Lew, Steve Garrett, Charles Hoffmann, Corinnemerous fungal species in response to carbon source and Michels, and Alan Myers for helpful discussions; and Bob Lefkowitz,

Marc Caron, and Pat Casey for advice and encouragement. This proj-via Ga protein-cAMP signaling cascades. Finally, Gpr1ect was supported by a grant-in-aid from the American Heart Associa-receptor homologs could function in mammals as thetion, New York City affiliate (to J.P.H) and by K01 career developmentsweet receptors of the tongue, which are known to be award CA77075 from the National Cancer Institute (to M.E.C.). Joseph

G protein-coupled, or as part of the glucose sensing Heitman is an associate investigator of the Howard Hughes Medicalapparatus that regulates insulin production by pancre- Institute and a Burroughs Wellcome Scholar in Molecular Pathogenic

Mycology.atic b-cells.Nutrient receptors: The Gpr1 receptor joins a grow-

ing family of other novel cell surface proteins that serveas receptors for extracellular nutrients in yeast and LITERATURE CITEDfungi. For example, the ammonium permease Mep2

Alspaugh, J. A., J. R. Perfect and J. Heitman, 1997 Cryptococcusplays a novel role as an ammonium ion sensor requiredneoformans mating and virulence are regulated by the G-protein

for filamentous differentiation in response to ammo- a subunit GPA1 and cAMP. Genes Dev. 11: 3206–3217.Bolker, M., 1998 Sex and crime: heterotrimeric G proteins in mat-nium ion limitation (Lorenz and Heitman 1998a,b).

ing and pathogenesis. Fungal Genet. Biol. 25: 143–156.The glucose permease homologs Rgt2 and Snf3 haveBoyum, R., and G. Guidotti, 1997 Glucose-dependent, cAMP-medi-

diverged from other glucose permeases and no longer ated ATP efflux from Saccharomyces cerevisiae. Microbiology 143:1901–1908.transport sugar, but instead play a novel signal transduc-

Brandao, R. L., N. M. D. Magalhaes-Rocha, R. Alijo, J. Ramostion role, activating a signaling cascade that detects theand J. M. Thevelein, 1994 Possible involvement of a phosphati-

extracellular concentration of glucose and regulates dylinositol-type signaling pathway in glucose-induced activationof plasma membrane H1-ATPase and cellular proton extrusiontranscription of glucose permease genes (Liang andin the yeast Saccharomyces cerevisiae. Biochim. Biophys. Acta 1223:Gaber 1996; Ozcan et al. 1996, 1998; Jiang et al. 1997;117–124.

Schmidt et al. 1999). A related glucose permease homo- Broach, J., 1991 RAS genes in Saccharomyces cerevisiae: signal trans-log, RCO3, appears to play a similar signaling function duction in search of a pathway. Trends Genet. 7: 28–33.

Chenevert, J., 1994 Cell polarization directed by extracellular cuesin regulating glucose transport and conidiation in Neu-in yeast. Mol. Biol. Cell 5: 1169–1175.rospora crassa (Madi et al. 1997). Finally, several recent Colombo, S., P. Ma, L. Cauwenberg, J. Winderickx, M. Crauwels

studies reveal that a novel amino acid permease homo- et al., 1998 Involvement of distinct G-proteins, Gpa2 and Ras,in glucose- and intracellular acidification-induced cAMP signal-log, Ssy1, has diverged from other amino acid permeasesling in the yeast Saccharomyces cerevisiae. EMBO J. 17: 3326–3341.and now plays a novel role as a receptor for extracellular Cook, J. G., L. Bardwell and J. Thorner, 1997 Inhibitory and

amino acids that regulates expression of amino acid activating functions for MAPK Kss1 in the S. cerevisiae filamentous-growth signalling pathway. Nature 390: 85–88.permease genes (Didion et al. 1998; Iraqui et al. 1999;

Didion, T., B. Regenberg, M. U. Jorgensen, M. C. Kielland-BrandtKlasson et al. 1999). Taken together, these studies re-and H. A. Andersen, 1998 The permease homologue Ssy1p

veal a diverse and growing family of novel nutrient re- controls the expression of amino acid and peptide transportergenes in Saccharomyces cerevisiae. Mol. Microbiol. 27: 643–650.ceptors that play diverse roles in regulating gene expres-

Gietz, R. D., and A. Sugino, 1988 New yeast-Escherichia coli shuttlesion and differentiation in microorganisms and thatvectors constructed with in vitro mutagenized yeast genes lacking

could be conserved in multicellular eukaryotes. six-base pair restriction sites. Gene 74: 527–534.Potential role of Gpr1 receptor homologs in virulence Gimeno, C. J., and G. R. Fink, 1994 Induction of pseudohyphal

growth by overexpression of PHD1, a Saccharomyces cerevisiae geneof human fungal pathogens: Given the correlation be-related to transcriptional regulators of fungal development. Mol.tween differentiation and virulence in pathogenic fungi, Cell. Biol. 14: 2100–2112.

the understanding of extracellular signaling events is Gimeno, C. J., P. O. Ljungdahl, C. A. Styles and G. R. Fink, 1992Unipolar cell divisions in the yeast S. cerevisiae lead to filamentousparticularly important. If, for example, hyphal or pseu-growth: regulation by starvation and RAS. Cell 68: 1077–1090.dohyphal development in C. albicans is regulated via

Hawkins, P. T., L. R. Stephens and J. R. Piggott, 1993 Analysisthe Gpr1 receptor homolog, receptor antagonists that of inositol metabolites produced by Saccharomyces cerevisiae in re-

sponse to glucose stimulation. J. Biol. Chem. 268: 3374–3383.inhibit signaling would be potent antifungal agents, asHoffman, C. S., and F. Winston, 1990 Isolation and characteriza-the ability to filament is required for virulence (Lo et

tion of mutants constitutive for expression of the fbp1 gene ofal. 1997). In the pathogenic basidiomycete C. neo- Schizosaccharomyces pombe. Genetics 124: 807–816.

Hoffman, C. S., and F. Winston, 1991 Glucose repression of tran-formans, the Ga protein Gpa1p is homologous to the S.


scription of the Schizosaccharomyces pombe fbp1 gene occurs by a tion of development and vegetative incompatibility. Genetics 152:519–528.cAMP signaling pathway. Genes Dev. 5: 561–571.

Iraqui, I., S. Vissers, F. Bernard, J. O. D. Craene, E. Boles et al., Madhani, H. D., C. A. Styles and G. R. Fink, 1997 MAP kinaseswith distinct inhibitory functions impart signaling specificity dur-1999 Amino acid signaling in Saccharomyces cerevisiae: a perme-

ase-like sensor of external amino acids and F-Box protein Grr1p ing yeast differentiation. Cell 91: 673–684.Madi, L., S. K. McBride, L. A. Bailey and D. J. Ebbole, 1997 rco-3,are required for transcriptional induction of the AGP1 gene,

which encodes a broad-specificity amino acid permease. Mol. a gene involved in glucose transport and conidiation in Neurosporacrassa. Genetics 146: 499–508.Cell. Biol. 19: 989–1001.

Isshiki, T., N. Mochizuki, T. Maeda and M. Yamamoto, 1992 Char- Mosch, H. C., R. L. Roberts and G. R. Fink, 1996 Ras2 signals viathe Cdc42/Ste20/mitogen-activated protein kinase module toacterization of a fission yeast gene, gpa2, that encodes a Ga sub-

unit involved in the monitoring of nutrition. Genes Dev. 6: 2455– induce filamentous growth in Saccharomyces cerevisiae. Proc. Natl.Acad. Sci. USA 93: 5352–5356.2462.

Jackson, C. L., J. B. Konopka and L. H. Hartwell, 1991 S. cerevisiae Mosch, H.-U., E. Kubler, S. Krappmann, G. R. Fink and G. H. Braus,1999 Crosstalk between the Ras2p-controlled mitogen-activatedalpha pheromone receptors activate a novel signal transduction

pathway for mating partner discrimination. Cell 67: 389–402. protein kinase and cAMP pathways during invasive growth ofSaccharomyces cerevisiae. Mol. Biol. Cell 10: 1325–1335.Jiang, H., I. Medintz and C. A. Michels, 1997 Two glucose sensing/

signaling pathways stimulate glucose-induced inactivation of malt- Nakafuku, M., T. Obara, K. Kaibuchi, I. Miyajima et al., 1988 Isola-tion of a second yeast Saccharomyces cerevisiae gene (GPA2) codingose permease in Saccharomyces. Mol. Biol. Cell 8: 1293–1304.

Jiang, Y., C. Davis and J. R. Broach, 1998 Efficient transition to for guanine nucleotide-binding regulatory protein: studies on itsstructure and possible functions. Proc. Natl. Acad. Sci. USA 85:growth on fermentable carbon sources in Saccharomyces cerevisiae

requires signaling through the Ras pathway. EMBO J. 17: 6942– 1374–1378.Neer, E. J., 1995 Heterotrimeric G proteins: organizers of transmem-6951.

King, K., H. G. Dohlman, J. Thorner, M. G. Caron and R. J. Lefkow- brane signals. Cell 80: 249–257.Nocero, M., T. Isshiki, M. Yamamoto and C. S. Hoffman, 1994itz, 1990 Control of yeast mating signal transduction by a mam-

malian b2-adrenergic receptor and Gs a subunit. Science 250: Glucose repression of fbp1 transcription in Schizosaccharomycespombe is partially regulated by adenylate cyclase activation by a G121–123.

Klasson, H., G. R. Fink and P. O. Ljungdahl, 1999 Ssy1p and protein a subunit encoded by gpa2 (git8). Genetics 138: 39–45.Ozcan, S., J. Dover, A. G. Rosenwald, S. Woelfl and M. Johnston,Ptr3p are plasma membrane components of a yeast system that

senses extracellular amino acids. Mol. Cell. Biol. 19: 5404–5416. 1996 Two glucose transporters in Saccharomyces cerevisiae are glu-cose sensors that generate a signal for induction of gene expres-Kraakman, L., K. Lemaire, P. Ma, A. W. R. H. Teunissen, M. C. V.

Donaton et al., 1999 A Saccharomyces cerevisiae G-protein cou- sion. Proc. Natl. Acad. Sci. USA 93: 12428–12432.Ozcan, S., J. Dover and M. Johnston, 1998 Glucose sensing andpled receptor, Gpr1, is specifically required for glucose activation

of the cAMP pathway during the transition to growth on glucose. signaling by two glucose receptors in the yeast Saccharomyces cerevis-iae. EMBO J. 17: 2566–2573.Mol. Microbiol. 32: 1002–1012.

Kron, S. J., 1997 Filamentous growth in budding yeast. Trends Mi- Pan, X., and J. Heitman, 1999 Cyclic AMP-dependent protein kinaseregulates pseudohyphal differentiation in Saccharomyces cerevisiae.crobiol. 5: 450–454.

Kruger, J., G. Loubradou, E. Regenfelder, A. Hartmann and R. Mol. Cell. Biol. 19: 4874–4887.Parent, C. A., and P. N. Devreotes, 1996 Molecular genetics ofKahmann, 1998 Crosstalk between cAMP and pheromone sig-

nalling pathways in Ustilago maydis. Mol. Gen. Genet. 260: 193– signal transduction in Dictyostelium. Annu. Rev. Biochem. 65: 411–440.198.

Kubler, E., H. U. Mosch, S. Rupp and M. P. Lisanti, 1997 Gpa2p, Reed, R. R., 1992 Signaling pathways in odorant detection. Neuron8: 205–209.a G-protein alpha-subunit, regulates growth and pseudohyphal

development in Saccharomyces cerevisiae via a cAMP-dependent Regenfelder, E., T. Spellig, A. Hartmann, S. Lauenstein, M.Bolker et al., 1997 G proteins in Ustilago maydis: transmissionmechanism. J. Biol. Chem. 272: 20321–20323.

Lambrechts, M. G., F. F. Bauer, J. Marmur and I. S. Pretorius, of multiple signals? EMBO J. 16: 1934–1942.Roberts, R. L., and G. R. Fink, 1994 Elements of a single MAP1996 Muc1, a mucin-like protein that is regulated by Mss10, is

critical for pseudohyphal differentiation in yeast. Proc. Natl. kinase cascade in Saccharomyces cerevisiae mediate two develop-mental programs in the same cell type: mating and invasiveAcad. Sci. USA 93: 8419–8424.

Liang, H., and R. F. Gaber, 1996 A novel signal transduction path- growth. Genes Dev. 8: 2974–2985.Robertson, L. S., and G. R. Fink, 1998 The three yeast A kinasesway in Saccharomyces cerevisiae defined by Snf3-regulated expres-

sion of HXT6. Mol. Biol. Cell 7: 1953–1966. have specific signaling functions in pseudohyphal growth. Proc.Natl. Acad. Sci. USA 95: 13783–13787.Lichter, A., and D. Mills, 1997 Fil1, a G-protein a-subunit that

acts upstream of cAMP and is essential for dimorphic switching Rupp, S., E. Summers, H. Lo, H. Madhani and G. Fink, 1999 MAPkinase and cAMP filamentation signaling pathways converge onin haploid cells of Ustilago hordei. Mol. Gen. Genet. 256: 426–435.

Liu, H., C. A. Styles and G. R. Fink, 1993 Elements of the yeast the unusually large promoter of the yeast FLO11 gene. EMBO J.18: 1257–1269.pheromone response pathway required for filamentous growth

of diploids. Science 262: 1741–1744. Sambrook, J., E. F. Fritsch and T. Maniatis, 1989 Molecular Clon-ing: A Laboratory Manual, Ed. 2. Cold Spring Harbor LaboratoryLo, H.-J., J. R. Kohler, B. DiDomenico, D. Loebenberg, A. Caccia-

puoti, et al., 1997 Nonfilamentous C. albicans mutants are aviru- Press, Cold Spring Harbor, NY.Savinon-Tejeda, A. L., L. Ongay-Larios, J. Ramırez and R. Coria,lent. Cell 90: 939–949.

Lo, W.-S., and A. M. Dranginis, 1998 The cell surface flocculin 1996 Isolation of a gene encoding a G protein a subunit in-volved in the regulation of cAMP levels in the yeast KluyveromycesFlo11 is required for pseudohyphae formation and invasion by

Saccharomyces cerevisiae. Mol. Biol. Cell 9: 161–171. lactis. Yeast 12: 1125–1133.Schiestl, R. H., P. Manivasakam, R. A. Woods and R. D. Gietz,Lorenz, M. C., and J. Heitman, 1997 Yeast pseudohyphal growth

is regulated by GPA2, a G protein a homolog. EMBO J. 16: 1993 Introducing DNA into yeast by transformation. Methods5: 79–85.7008–7018.

Lorenz, M. C., and J. Heitman, 1998a The MEP2 ammonium per- Schmidt, M. C., R. R. McCartney, X. Zhang, T. S. Tillman, H.Solimeo et al., 1999 Std1 and mth1 proteins interact with themease regulates pseudohyphal differentiation in Saccharomyces

cerevisiae. EMBO J. 17: 1236–1247. glucose sensors to control glucose-regulated gene expression inSaccharomyces cerevisiae. Mol. Cell. Biol. 19: 4561–4571.Lorenz, M. C., and J. Heitman, 1998b Regulators of pseudohyphal

differentiation in Saccharomyces cerevisiae identified through Segall, J. E., 1993 Polarization of yeast cells in spatial gradients ofa mating factor. Proc. Natl. Acad. Sci. USA 90: 8332–8336.multicopy suppressor analysis in ammonium permease mutant

strains. Genetics 150: 1443–1457. Serrano, R., 1983 In vivo glucose activation of the yeast plasmamembrane ATPase. FEBS Lett. 156: 11–14.Loubradou, G., J. Begueret and B. Turcq, 1999 MOD-D, a Ga

subunit of the fungus Podospora anserina, is involved in both regula- Sherman, F., 1991 Getting started with yeast, pp. 3–21 in Methods


in Enzymology, edited by C. Guthrie and G. R. Fink. Academic Wach, A., A. Brachat, R. Pohlmann and P. Philippsen, 1994 Newheterologous modules for classical or PCR-based gene disruptionsPress, San Diego.in Saccharomyces cerevisiae. Yeast 10: 1793–1808.Spiegel, A. M., 1997 Inborn errors of signal transduction: mutations

Ward, M. P., C. J. Gimeno, G. R. Fink and S. Garrett, 1995 SOK2in G proteins and G protein-coupled receptors as a cause ofmay regulate cyclic AMP-dependent protein kinase-stimulateddisease. J. Inherited Metab. Dis. 20: 113–121.growth and pseudohyphal development by repressing transcrip-Sprague, G. F., Jr., and J. W. Thorner, 1992 Pheromone responsetion. Mol. Cell. Biol. 15: 6854–6863.and signal transduction during the mating process of Saccharo-

Xue, Y., M. Batlle and J. P. Hirsch, 1998 GPR1 encodes a putativemyces cerevisiae, pp. 657–744 in The Molecular and Cellular BiologyG protein-coupled receptor that associates with the Gpa2p Gaof the Yeast Saccharomyces: Gene Expression, edited by E. W. Jones,subunit and functions in a Ras-independent pathway. EMBO J.J. R. Pringle and J. R. Broach. Cold Spring Harbor Laboratory17: 1996–2007.Press, Cold Spring Harbor, NY.

Yun, C., H. Tamaki, R. Nakayama, K. Yamamoto and H. Kumagai,Stevenson, B. J., N. Rhodes, B. Errede and G. F. Sprague, Jr., 1992 1998 Gpr1p, a putative G protein coupled receptor, regulatesConstitutive mutants of the protein kinase STE11 activate the glucose-dependent cellular cAMP level in yeast Saccharomyces cere-pheromone response pathway in the absence of the G protein. visiae. Biochem. Biophys. Res. Commun. 252: 29–33.Genes Dev. 6: 1293–1304. Yun, C.-W., H. Tamaki, R. Nakayama, K. Yamamoto and H. Kumagai,

Toda, T., S. Cameron, P. Sass and M. Wigler, 1988 SCH9, a gene 1997 G-protein coupled receptor from yeast Saccharomyces cere-of Saccharomyces cerevisiae that encodes a protein distinct from, but visiae. Biochem. Biophys. Res. Commun. 240: 287–292.functionally and structurally related to, cAMP-dependent proteinkinase catalytic subunits. Genes Dev. 2: 517–527. Communicating editor: M. D. Rose

the g protein-coupled receptor gpr1 is a nutrient sensor

Documents