Proc. Natl. Acad. Sci. USAVol. 93, pp. 5352-5356, May 1996Genetics

Ras2 signals via the Cdc42/Ste20/mitogen-activated proteinkinase module to induce filamentous growth inSaccharomyces cerevisiaeHANS-ULRICH MOSCH, RADCLYFFE L. ROBERTS, AND GERALD R. FINK*Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142

Contributed by Gerald R. Fink, January 29, 1996

ABSTRACT RAS2Val19, a dominant activated form of Sac-charomyces cerevisiae Ras2, stimulates both filamentousgrowth and expression of a transcriptional reporterFG(TyA)::kacZ but does not induce the mating pathway re-porter FUSI::lacZ. This induction depends upon elements ofthe conserved mitogen-activated protein kinase (MAPK)pathway that is required for both filamentous growth andmating, two distinct morphogenetic events. Full inductionrequires Ste2O (homolog of mammalian p65PAK protein ki-nases), Stell [an MEK kinase (MEKK) or MAPK kinase(MEK) kinase], Ste7 (MEK or MAPK kinase), and thetranscription factor Stel2. Moreover, the Rho family proteinCdc42, a conserved morphogenetic G protein, is also a potentregulator offilamentous growth and FG(TyA)::lacZ expressionin S. cerevisiae. Stimulation of both filamentous growth andFG(TyA)::lacZ by Cdc42 depends upon Ste2O. In addition,dominant negative CDC42AIall8 blocks RAS2vall9 activation,placing Cdc42 downstream of Ras2. Our results suggest thatfilamentous growth in budding yeast is regulated by anevolutionarily conserved signaling pathway that controls cellmorphology.

starvation, loss of carbohydrate reserves, and a block tosporulation.Recent work on S. cerevisiae has revealed that activated

Ras2Vall9 stimulates filamentous growth (12), a morphogeneticpathway that requires the p65PAK kinase homolog Ste2O, theStell (MEKK), and Ste7 (MEK; MAPK kinase) proteinkinases and the transcription factor Stel2-proteins that con-stitute part of the MAPK module required for mating (13).However, the upstream effectors of mating, the pheromonereceptors (Ste2 and Ste3), the heterotrimeric G protein (Gpal,Ste4, and Stel8), and two MAP kinases (Fus3 and Kssl) arenot required for filamentous growth. In addition, recentevidence suggests that the Rho family protein Cdc42 mayregulate the Ste2O/MAPK module for pheromone signaling(14, 15). Here, we show that (i) Ras2 stimulates filamentousgrowth depending upon Ste20, Stell, Ste7, and Stel2; (ii) Ras2regulation via the Ste2O/MAPK module is independent of Akinase; (iii) Cdc42 is a potent regulator of filamentous growth,and is likely to act downstream of Ras2; (iv) an Stel2-dependenttranscriptional reporter, FG(TyA)::lacZ, discriminates betweenactivation of the Ste2O/MAPK cascade during mating and fila-mentous growth.

The small G protein Ras plays a key role in signaling processesthat regulate proliferation and differentiation in eukaryotes. Inmammals, flies, worms, and fission yeast, Ras regulates con-served mitogen-activated protein kinase (MAPK) pathwaysthat convey signals from the plasma membrane to the nucleus(for reviews see refs. 1-3). In metazoans, Ras mediates twodifferent pathways, one via Raf, a serine/threonine kinase (forreviews see refs. 4 and 5) and another, distinct from theRaf/MAPK pathway, which involves Rho family proteins suchas Cdc42h (6, 7). The situation in fission yeast is similar to thatin metazoans: the Ras homolog rasl triggers two distinctsignaling pathways (8), a mating pathway involving a MEKK-like serine/threonine kinase (byr2) and a morphogeneticpathway involving a Rho family protein (Cdc42sp) and ap65PAK kinase homolog, Shkl (9).By contrast, in the budding yeast Saccharomyces cerevisiae,

neither RAS1 nor RAS2 (the two redundant RAS genes) hasbeen linked to any of the MAPK signaling cascades thatcontrol mating, osmotic sensitivity, the protein kinase Cpathway, or sporulation in this organism (for review see ref.10). The sole characterized target of Ras in S. cerevisiae isadenylate cyclase (for review see ref. 11). Activation of Ras inS. cerevisiae results in elevated intracellular cAMP levels thatin turn activate the A kinase, which is composed of aninhibitory subunit Bcyl and a catalytic subunit encoded bythree redundant genes TPKI, TPK2, and TPK3. Activation ofthe A kinase, either by a dominant RAS2Val19 mutation or bcylnull mutation confers sensitivity to heat shock and nutrient

MATERIALS AND METHODSMedia, Growth Conditions, and Yeast Strains. Standard

yeast culture medium was prepared essentially as described(16). Low ammonia medium for scoring pseudohyphal growthor j3-galactosidase assays was prepared as described (12) withcarbon sources at the following concentrations: synthetic lowammonia dextrose (SLAD) (2% glucose), synthetic low am-monia raffinose (SLAR) (3% raffinose), SLAR + 0.1%galactose (3% raffinose and 0.1% galactose), or synthetic lowammonia galactose (SLAG) (3% galactose).

All strains used in this study are congenic to the 11278bgenetic background (13, 17). The derivation of ste2, ste4, stel8,ste2O, steS, stell, ste7, fus3, kssl, and stel2 strains has beendescribed (13, 18). The bcyl mutation was introduced usingthe disruption plasmid pbcyl::LEU2 (19). The FG(TyA)::lacZtranscriptional reporter was transformed into strains usingcentromere-based plasmids pFG(TyA)::lacZ-URA3, pFG(TyA)::lacZ-LEU2, or pFG(TyA)::lacZ-H1S3, and integratedFUS1::lacZ has been described (18). The STE11-4 mutation(20) was expressed using plasmid pSL1509 (G. Sprague, Uni-versity of Oregon, Eugene). For high copy expression of Ste20or Tpkl, plasmids pRS426-STE20 or YEp-TPK1 (21), respec-tively, were used. The dominant RAS2Vall9 mutation allele wasexpressed using plasmids YCp50-RAS2Val19 or YCpLEU2-RAS2vall9. Dominant CDC42 alleles were expressed from agalactose-inducible promoter (GALl-10) using plasmids

Abbreviations: MAPK, mitogen-activated protein kinase; MEK,MAPK kinase; MEKK, MEK kinase.*To whom reprint requests should be addressed at: Whitehead Insti-tute for Biomedical Research, Nine Cambridge Center, Cambridge,MA 02142.

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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pGAL-CDC42Val l2, pGAL-CDC42I U-6l, pGAL-CDC42Ala-118, or pGAL-CDC42Ser-l88 (22). Plasmids pRS315, pRS316,YCp5O, or pRS426 were used as vector controls. Standardprocedures were used for yeast transformation (23, 24) andgenetics (16).

Plasmids. Plasmid pFG(TyA)::lacZ-URA3 is plasmid pIL30(25) and contains a 97-bp fragment derived from transposonTyl harboring an Ste12 binding site in a TDH3::lacZ reportergene expression system. Plasmids pFG(TyA)::lacZ-LEU2 andpFG(TyA)::lacZ-HIS3 were constructed by homologous re-combination in yeast (26) using pFG(TyA)::lacZ-URA3 and alinear ura3::LEU2 or ura3::HIS3 URA3-disruption cassette(both from Yona Kassir, Technion, Haifa, Isreal). PlasmidYCpLEU2-RAS2Vall9 was obtained by homologous recombi-nation in yeast using plasmid YCp50-RAS2Vall9 and a linearura3::LEU2 disruption fragment. Plasmid pRS426-STE20 wasconstructed by cloning a 4.9-kb KpnI-NotI fragment fromplasmid pSTE20-5 (27) into high copy plasmid pRS426. Mo-lecular biological techniques were performed as described(28).

j8-Galactosidase Assays. f-Galactosidase assays were per-formed with extracts from exponentially growing strains(OD600 of 0.5-1.0) (29). For pheromone induction, cells inexponential growth phase were washed into 5 ml of freshmedium with or without 5 ,uM a-factor and incubated at 30°Cfor 2 hr before being harvested. For assays under nitrogenstarvation conditions, cells in exponential growth phase werewashed with 2% glucose or 3% galactose, spread on SLAD orSLAG plates, incubated at 30°C for 3 days (SLAD) or 22 hr(SLAG), and harvested by resuspension from the plates. Cellswere always washed once with water prior to lysis to removeexternal enzyme. Broken cell extracts (29) were assayed for,B-galactosidase activity at 37°C. The activities were normal-ized to the total protein in each extract using a Bio-Rad proteinassay kit. 13-Galactosidase specific activity equals (OD420 X1.7)/(0.0045 x protein concentration x extract volume xtime).

Qualitative and Quantitative Assays of FilamentousGrowth. The qualitative growth assay for filament formationand light microscopy of microcolonies and invasive cells wereperformed as described (12, 30). Quantitative pseudohyphalcell development was performed based on the method forcharacterization of cell shape in Candida albicans (31). Strainswere grown on SLAD medium for 3 days at 30°C, andnoninvasive cells were removed by washing the plates withwater. Invasive cells were scraped off the plate, suspended in50 ,ul of water, and analyzed for cell shape by light microscopy.Cell shape was quantified by determining the length-to-widthratio of typically 200 cells and dividing them into the classes:Yeast form cells with a length-to-width ratio of less than 2;pseudohyphal cells with a length-to-width ratio of greater than2. The data were verified by taking photographic pictures of asmaller number of cells and directly measuring cell dimensions.

RESULTSFG(TyA)::acZ Discriminates Between Activation of the

Ste2O/MAPK Cascade During Mating and FilamentousGrowth. Because both mating and filamentous growth requirethe same transcription factor, Stel2, we sought an Stel2-dependent transcriptional reporter that discriminates betweenthe signals that induce mating and filamentation. Previousstudies had described a lacZ fusion derived from transposonTyl whose transcription depends on STE12 (25). We foundthat expression of this fusion FG(TyA)::lacZ (previously de-scribed as pIL30) correlates extremely well with activity of thefilamentous Ste2O/MAPK cascade (Table 1) and with fila-mentous growth (13, 18). Nitrogen starvation, which stimulatesfilamentous growth (12), also induces FG(TyA)::lacZ. In con-trast, FG(TyA)::lacZ is not induced by alpha pheromone at

Table 1. Expression of FG(TyA)::lacZ in haploid anddiploid strains

Activity

GenotypeSTESTESTESTEste2ste4stel8ste2OsteSstellste7fus3ksslfus3 ksslstel2

Haploids Diploids

(low [N]*)(a factort)(Stell-4O)

1013309732055889133100102.5

13029661.7

1089

38

2.2

0.751.4

140.66

FG(TyA)::lacZ expression was measured in exponentially growingcells in nitrogen-rich medium (synthetic complete medium), andP-galactosidase assays were performed with cellular extracts as de-scribed (18, 29). Activities are expressed in nmol of O-nitrophenyl-,j-D-galactopyranoside hydrolyzed per min times mg of protein.*Exponentially growing cells were transferred onto SLAD and incu-bated for 3 days prior to ,3-galactosidase assays (details in Materialsand Methods).tExponentially growing cells were exposed to 5 ,uM a factor for 2 hrprior to ,3-galactosidase assays.tSTE11-4 is a dominant activated mutant allele (20).

concentrations that induce the mating specific reporterFUS1::lacZ approximately 100-fold. Previous work had shownthat FG(TyA)::lacZ responds to Stell, Ste7, and Stel2 (25).We find that both haploid and diploid strains containingmutations in STE20, STE11, STE7, and STE12 reduce theexpression of FG(TyA)::lacZ, whereas overexpression ofSTE20 or expression of the dominant gain of function alleleSTE11-4 induce FG(TyA)::lacZ expression (Table 1 and Table2). However, strains deleted for Ste2, Ste4, Stel8, Ste5, or forboth mating MAPKs, Fus3 and Kssl, show normal levels ofFG(TyA)::lacZ. Thus, FG(TyA)::lacZ is a sensitive STE12-dependent transcriptional reporter that reflects the activity ofthe filamentous growth kinase cascade and the extent offilamentous growth.Ras2 Stimulation of Filamentous Growth Depends upon

STE20, STEII, STE7, and STE12. To test whether enhance-ment of filament formation by a constitutively activated Ras2protein requires the filamentous Ste2O/MAPK cascade, weexpressed the dominant RAS2Vall9 allele in strains carryingnull mutations in STE20, STE11, STE7, and STE12 andassayed both the FG(TyA)::lacZ reporter and filamentousgrowth under nitrogen starvation conditions. The ste2O mu-tation completely blocks the RAS2Vall9 enhancement of fila-mentous growth and the stell, ste7, and stel2 mutationspartially block this enhancement (Fig. 1). These morphologicalobservations were corroborated by measurement ofFG(TyA)::lacZ expression in these strains. RAS2vall9 inducesFG(TyA)::lacZ (about 8-fold in diploids and 3-fold in haploids)under conditions that induce filamentous growth and thisenhancement is completely dependent upon Ste2O, Ste7, andStel2 (Table 2). In contrast, FUS1::lacZ expression is notinduced by RAS2VaIl9 either in the presence or absence of afactor (data not shown). Taken together, these results suggestthat Ras2 regulates filamentous growth by activating the Stel2signal transduction pathway.Ras2 Activation ofFG(TyA)::kawZ Does Not Occur via the A

Kinase. We tested whether activation of the A kinase wasinvolved in the stimulation of FG(TyA)::lacZ by Ras2. Neitherdeletion of BCY1 nor overexpression of Tpkl significantly

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Table 2. Regulation of FG(TyA)::lacZ expression by Ras2and Cdc42

Genotype Relative activity

SLAD medium*STE/STE 1.0STE/STE STE11-4 8.2STE/STE STE20 (high copy) 3.2STE/STE RAS2VaI19 7.6ste20/ste2O 0.5ste20/ste2O RA52VaI19 0.5ste7/ste7 0.6ste7/ste7 RAS2Va119 0.3stel2/stel2 0.6stel2/stel2 RAS2VaI19 0.2STE/STE TPK1 (high copy) 1.8ste2O/ste2O TPK1 (high copy) 1.9bcyl/bcyl 1.7bcyl/bcyl, 0.6

ste2O/ste2OSLAG mediumt

STE/STE 1.0ste2O/ste2O 0.4STE/STE PGAL::CDC42Val12 5.2ste2O/ste2O PGAL::CDC42Val12 0.8STE/STE PGAL::CDC42Leu6l 9.2ste2O/ste2O PGAL::CDC42Leu6l 1.1STE/STE PGAL::CDC42A1a118 1.1ste2O/ste2O PGAL::CDC42A1all8 0.7STE/STE PGAL::CDC42Serl88 0.9ste2O/ste2O PGAL::CDC42Serl88 0.4STE/STE RAS2Val19 11.8STE/STE RAS2Va119 PGAL::CDC42AIall8 6.1STE/STE STE11-4 5.4STE/STE STEII-4, PGAL::CDC42A1all8 5.3

FG(TyA)::lacZ expression was measured in nitrogen-starved cells,and f3-galactosidase assays were performed with cellular extracts.Numbers represent 3-galactosidase activities normalized to the activ-ity measured in wild-type diploid strains under nitrogen starvationconditions (Table 1), with a value of 1 corresponding to 89 nmol ofO-nitrophenyl-f3-D-galactopyranoside hydrolyzed per min times mgprotein. Under genotype the first designation is the chromosomalarrangement and the second is the plasmid genotype. Where noplasmid is shown a vector was used as control.*Strains were grown on SLAD for 3 days.tStrains were grown for 20 h on SLAG for expression of the variousPGAL::CDC42 mutant alleles.

enhances FG(TyA)::lacZ expression (Table 2) or filamentousgrowth. The results with the reporter are critical here becausethe bcyl mutation has morphological abnormalities that couldconfuse the analysis. Moreover, double mutants betweenRAS2Vall9 or bcyl and ste2O, stell, or ste7 are still sensitive toheat shock. Thus, Ras2 regulation of filamentous growth viathe Ste2O/MAPK cascade is independent of A kinase and Akinase phenotypes appear to be independent of the Ste2O/MAPK cascade.Ras2 Stimulates Filamentous Growth via Cdc42. To deter-

mine whether stimulation of filamentous growth by Ras2depends upon Cdc42, a Rho-type GTP-binding protein, weanalyzed the effects ofvarious dominant alleles ofCDC42 (22).Expression of the CDC42 alleles was controlled by growingstrains with pGAL::CDC42 constructs on raffinose to avoidthe inhibition of growth caused by high levels of these mutantproteins. Under these conditions expression of the dominantnegative allele CDC42Alall8 completely suppressed filamen-tous growth (Fig. 2). In contrast, the dominant active CDC42alleles CDC42Vall2 and CDC42L-u61 induce both filamentousgrowth and the FG(TyA)::lacZ reporter (Fig. 2 and Table 2).This induction is completely dependent upon the presence of

A* :*::~~~~~~~

B.

c

E

VI

UzLIa. WI

~ ~ ~ ~ I

LnM I

Cs~,T

~ ~ ~

4) vI *

FIG. 1. Filamentous growth stimulation by RAS2Val19 depends onSTE20, STE11, STE7, and STE12. Filamentous growth ofSTE/STE (Aand B) and ste20/ste20 mutant (C and D) diploid strains containingcontrol plasmid YCp5O (A and C) or plasmid YCp50-RAS2Vall9 (Band D) after 4 days of growth on SLAD medium. The scale bar in Aapplies to panelsA-D and equals 100 ,uM. (E) Quantitative measure-ment of pseudohyphal cell development upon nitrogen starvation.STE/STE and mutant ste/ste diploids containing YCp5O (solid bars)or YCp50-RAS2Vall9 (open bars) were grown for 4 days on SLADmedium. The percentage of cells that had undergone pseudohyphaldevelopment (% PH cells) was then determined (for details seeMaterials and Methods). Cells were considered as pseudohyphal whentheir length/width ratio was greater than two.

a functional STE20 gene, showing that Cdc42 activates fila-mentous growth via Ste2O.Double mutants containing the activated RAS2 allele and

the dominant negative CDC42 allele (RAS2Vall9, CDC42Alal18)fail to form filaments (Fig. 3) and have lower expression ofFG(TyA)::lacZ than does the RAS2Va119 single mutant (Table2). In contrast, the CDC42Alall8 allele does not block theSTE11-4 enhancement ofFG(TyA)::lacZ expression (Table 2).Taken together these data suggest that Cdc42 is required forRas2 activation of filamentous growth and acts downstream ofRas2.

DISCUSSIONThese experiments resolve some of the disparity between thefunction of the Saccharomyces RAS genes and the RAS genesfrom other organisms. In fission yeast, flies, worms, andhumans, Ras protein activity signals through an evolutionarilyconserved MAPK cascade to activate gene expression. Infission yeast, for example, rasl signals through an MAPKcascade to stimulate mating (8). However, in Saccharomyces,no role for Ras in mating has been uncovered despite theinvolvement of a conserved MAPK cascade consisting of

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A

C

FIG. 2. Stimulation of filamentous growth by CDC42 dependsupon STE20. STE/STE (A, B, C, D, I, and J) and ste20/ste20 mutant(E, F, G, and H) diploids containing control plasmid pRS315 (A, B, E,and F), plasmid pGAL-CDC42Leu-6l (C, D, G, and H) or plasmidpGAL-CDC42AIa-118 (I and J) were streaked on (SLAR) medium(A-H) or SLAR medium containing 0.1% galactose (I and J), andincubated at 30°C. Pictures of representative microcolonies were takenafter 17 hr of growth using a light microscope (A, C, E, G, and I). After4 days, pseudohyphal development of cells at the edges of the colonieswas visualized under the microscope using Nomarski optics (B, D, F,H, and J). The scale bar in A applies to A, C, E, G, and I and equals50 t,M; the scale bar in B applies to B, D, F, H, and J and equals 10,uM.

Ste2O, Stell, Ste7, and two MAPKs (Fus3 and Kssl). Activa-tion of this mating MAPK cascade by mating pheromone isreadily measured by the transcriptional reporter FUSI::lacZ.However, activated alleles of Ras (e.g., Ras2vall9) fail to induceFUS1::lacZ. Previous work had shown that some elements ofthe mating MAPK cascade also function in vegetative cells topromote filamentous growth (13). Although RAS2Vall9 wasknown to stimulate filamentous growth (12), it was not clearwhether this stimulation occurred via the mating MAPKcascade.Our study shows that Saccharomyces Ras2 signals through

the MAPK cascade for filamentous growth. Mutations in theMAPK cascade (ste2O, stell, ste7, and stel2) block the stim-ulation of filamentation by RA52Vall9. Moreover, theFG(TyA)::lacZ reporter, which responds to the activity of theSte2O, Ste 1l, and Ste7 kinase cascade in vegetative cells but notto mating pheromones, is induced by RAS2VaI19. Our doublemutant data place Cdc42 in the pathway downstream fromRas2.

This arrangement of the filamentous growth signaling path-way in Saccharomyces is similar to that described for mamma-lian cells and fission yeast (Fig. 4). In mammalian cells Ha-Ras

FIG. 3. Genetic interactions between RAS2 and CDC42 duringfilamentous growth. Strain CGX179 (ura3/ura3, leu2/leu2) was trans-formed with plasmids YCp50 and pRS315 (A), YCp50-RAS2Vall9 andpRS315 (B), YCp5O and pGAL-CDC42Ala-1l8 (C), or YCp50-RAS2Vall9 and pGAL-CDC42Ala-ll8 (D) and grown on SLAR mediumcontaining 0.1% galactose. After 4 days of growth, pseudohyphaldevelopment of strains was visualized under a microscope and pho-tographed. The scale bar in A applies to A-D and equals 100 t,M.

(33) as well as Racl and Cdc42Hs have recently been shownto activate c-Jun N-terminal kinase/stress-activated proteinkinase (MAP kinases), with Racl proposed as an intermediatebetween Ha-Ras and JNK/SAPK (32, 34). In addition, inmammals Ras is thought to act via the Rho family proteinsRacl and Cdc42Hs to control reorganization of the actincytoskeleton (7). A similar pathway has been described inSchizosaccharomyces pombe, where the rasi protein is thoughtto regulate cell morphogenesis via cdc42sp (8). In addition,Shkl, a homolog of the S. cerevisiae Ste2O and mammalianp65PAK protein kinases, is thought to be a component of theRasl/Cdc42sp signaling module (9).

peptide pheromone

Ste2 or Ste3

Gpal Ste4 Ste18

Cdc42Ste2O

Stel1Ste7

Fus3 or Kssl

Ste12

mating responseFUS1 transcription

growth factors

Ras2

Cdc42Ste2O

Stel 1Ste7

4Ste12

filamentous growthFG(TyA) transcription

budding yeast

TyrK

Ha-Ras

Racl/2; Cdc42HsPAK(s)

MEKKJNKK

JNK; p38

c-Jun

stress responses

mammals

FIG. 4. Signal transduction through members of the Ras and Rhofamily proteins in budding yeast and mammals. Pheromone signalinghas been reviewed (10), and a role for Cdc42 in mating has beendescribed (14, 15). The model for Ha-Ras mediated regulation of c-JunN-terminal kinases (JNK) by growth factors in mammalian cells isadapted from ref. 32.

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Previous work suggested the involvement of the A kinase infilamentous growth because overexpression of the cAMPphosphodiesterase (PDE2) reduced the stimulation of filamen-tous growth by RAS2Val19 (35). However, our results indicatethat Ras2 regulation of filamentous growth via the Cdc42/Ste2O/MAPK pathway is independent of A kinase activity.These findings could be explained if Ras2 has a dual role inactivation of filamentous growth; activation of a Cdc42/Ste2O/MAPK signaling module (cAMP independent) and stimula-tion of A kinase activity (cAMP dependent). Alternatively, thecAMP-dependent effects upon cell morphology could resultfrom some general disruption in cell physiology caused byabnormal A kinase activity rather than a specific effect on thefilamentous growth pathway. A reporter whose activity re-flected the extent of filamentation and the A kinase levelsmight clarify this issue.The region around the Stel2 binding sites in the two

reporters that discriminate between the signals for mating andfilamentation may provide clues to the mechanism by whichthis transcription factor differentially activates the two path-ways. The FUSJ::lacZ construct contains four Stel2 bindingsites (36), whereas the FG(TyA)::lacZ contains both an Stel2binding site and an adjacent sequence which binds a secondfactor (25, 37). This factor might be Tecl, because TECI isrequired for the expression of FG(TyA)::lacZ (25). Moreover,TEC] is also required for filamentous growth but not mating(ref. 38; H.-U.M. and G.R.F., unpublished). Thus, one possi-bility is that Tecl binds in concert with Stel2 to promote thetranscription of genes required for filamentous growth. Alter-natively, the genes upstream of STE12 could differentially alterthe activity of Stel2 because activation of Ste 12 by pheromonerequires a MAP kinase (Fus3/Kssl) and scaffolding protein(Ste5) that are not required for filamentous growth.

Mating and filamentous growth require different G proteinsfor activation, a heterotrimeric G protein and Ras2, respec-tively. How does Ras regulate the Rho Cdc42 protein toactivate Ste20 for filamentous growth? A number of recentstudies indicate the existence of a protein complex includingCdc42 and Ste2O (14, 15, 39) thought to function in budemergence, cell polarity, and pheromone signaling. The Ras-like protein Rsrl is thought to recruit a protein complexcontaining Cdc42 to the site of the emerging bud therebycontrolling cell polarity (40). By analogy with Rsrl, Ras2 couldrecruit a Cdc42/Ste20 complex to the membrane therebyregulating cell morphology during filamentous growth. In-deed, recent work has suggested that Rsrl function overlapswith that of Rasl and Ras2 in cell cycle control (41). Thefactors that control the orderly interchange of G proteinswithin Ste2O complexes should help to explain how a singlemap kinase pathway can activate two different developmentalpathways.

We thank Michael Wigler, Douglas Johnson, Evelyne Dubois,George Sprague, Ekkehard Leberer, and Yona Kassir for kindlyproviding plasmids. We thank Amir Sherman and other members ofthe Fink laboratory for many fruitful discussions and Hiten Madhani,Steffen Rupp, Eric Summers, and Eric Kubler for helpful commentson the manuscript. This work was supported by National Institues ofHealth Research Grant GM40266 to G.R.F. H.-U.M. was supportedby postdoctoral fellowships from the International Human FrontierScience Program Organization (HFSPO) and of the Swiss NationalScience Foundation. G.R.F. is an American Cancer Society Professorof Genetics.

1. McCormick, F. (1994) Curr. Opin. Genet. Dev. 4, 71-76.2. van der Geer, P., Hunter, T. & Lindberg, R. A. (1994) Annu. Rev.

Cell Biol. 10, 251-337.3. Burgering, B. M. & Bos, J. L. (1995) Trends Biochem. Sci. 20,

18-22.

4. Daum, G., Eisenmann-Tappe, I., Fries, H. W., Troppmair, J. &Rapp, U. R. (1994) Trends Biochem. Sci. 19, 474-480.

5. McCormick, F. (1994) Trends Cell Biol. 4, 347-350.6. Prendergast, G. C., Khosravi-Far, R., Solski, P. A., Kurzawa, H.,

Lebowitz, P. F. & Der, C. J. (1995) Oncogene 10, 2289-2296.7. Khosravi-Far, R., Solski, P. A., Clark, G. J., Kinch, M. S. & Der,

C. J. (1995) Mol. Cell. Biol. 15, 6443-6453.8. Chang, E. C., Barr, M., Wang, Y., Jung, V., Xu, H.-P. & Wigler,

M. (1994) Cell 79, 131-141.9. Marcus, S., Polverino, A., Chang, E., Robbins, D., Cobb, M. H.

& Wigler, M. H. (1995) Proc. Natl. Acad. Sci. USA 92,6180-6184.10. Herskowitz, I. (1995) Cell 80, 187-197.11. Broach, J. R. & Deschennes, R. J. (1990) Adv. Cancer Res. 54,

79-139.12. Gimeno, C. J., Ljungdahl, P. O., Styles, C. A. & Fink, G. R.

(1992) Cell 68, 1077-1090.13. Liu, H., Styles, C. A. & Fink, G. R. (1993) Science 262, 1741-

1744.14. Simon, M. N., De Virgilio, C., Souza, B., Pringle, J. R., Abo, A.

& Reed, S. I. (1995) Nature (London) 376, 702-705.15. Zhao, Z., Leung, T., Manser, E. & Lim, L. (1995) Mol. Cell. Biol.

15, 5246-5257.16. Sherman, F., Fink, G. R. & Hicks, J. (1986) Methods in Yeast

Genetics (Cold Spring Harbor Lab. Press, Plainview, NY).17. Grenson, M., Mousset, M., Wiame, J. M. & Bechet, J. (1966)

Biochim. Biophys. Acta 127, 325-338.18. Roberts, R. L. & Fink, G. R. (1994) Genes Dev. 8, 2974-2985.19. Toda, T., Cameron, S., Sass, P., Zoller, M., Scott, J. D., Mc-

Mullen, B., Hurwitz, M., Krebs, E. G. & Wigler, M. (1987) Mol.Cell. Biol. 7, 1371-1377.

20. Stevenson, B. J., Rhodes, N., Errede, B. & Sprague, G., Jr. (1992)Genes Dev. 6, 1293-1304.

21. Toda, T., Cameron, S., Sass, P., Zoller, M. & Wigler, M. (1987)Cell 50, 277-287.

22. Ziman, M., O'Brien, J. M., Ouellette, L. A., Church, W. R. &Johnson, D. I. (1991) Mol. Cell. Biol. 11, 3537-3544.

23. Ito, H., Fukuda, Y., Murata, K. & Kimura, A. (1983) J. Bacteriol.153, 163-168.

24. Gietz, D., St. Jean, J. A., Woods, R. A. & Schiestl, R. H. (1992)Nucleic Acids Res. 20, 1425.

25. Laloux, I., Jacobs, E. & Dubois, E. (1994) Nucleic Acids Res. 22,999-1005.

26. Ma, H., Kunes, S., Schatz, P. J. & Botstein, D. (1987) Gene 58,201-216.

27. Leberer, E., Dignard, D., Harcus, D., Thomas, D. Y. & White-way, M. (1992) EMBO J. 11, 4815-4824.

28. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seid-man, J. G., Smith, J. A. & Struhl, K. (1993) Current Protocols inMolecular Biology (Greene/Wiley-Interscience, New York).

29. Rose, M. & Botstein, D. (1983) Methods Enzymol. 101, 167-180.30. Gimeno, C. J. & Fink, G. R. (1994) Mol. Cell. Biol. 14, 2100-

2112.31. Merson-Davies, L. A. & Odds, F. C. (1989) J. Gen. Microbiol.

135, 3143-3152.32. Minden, A., Lin, A., Claret, F. X., Abo, A. & Karin, M. (1995)

Cell 81, 1147-1157.33. Derijard, B., Hibi, M., Wu, I. H., Barrett, T., Su, B., Deng, T.,

Karin, M. & Davis, R. J. (1994) Cell 76, 1025-1037.34. Coso, 0. A., Chiariello, M., Yu, J. C., Teramoto, H., Crespo, P.,

Xu, N., Miki, T. & Gutkind, J. S. (1995) Cell 81, 1137-1146.35. Ward, M. P., Gimeno, C. J., Fink, G. R. & Garrett, S. (1995) Mol.

Cell. Biol. 15, 6854-6863.36. Hagen, D. C., McCaffrey, G. & Sprague, G., Jr. (1991) Mol. Cell.

Biol. 11, 2952-2961.37. Company, M., Adler, C. & Errede, B. (1988) Mol. Cell. Biol. 8,

2545-2554.38. Gavrias, V., Andrianopoulos, A., Gimeno, C. J. & Timberlake,

W. E. (1996) Mol. Microbiol., in press.39. Leeuw, T., Fourest-Lieuvin, A., Wu, C., Chenevert, J., Clark, K.,

Whiteway, M., Thomas, D. T. & Leberer, E. (1995) Science 270,1210-1213.

40. Zheng, Y., Bender, A. & Cerione, R. A. (1995)J. Biol. Chem. 270,626-630.

41. Morishita, T., Mitsuzawa, H., Nakafuku, M., Nakamura, S.,Hattori, S. & Anraku, Y. (1995) Science 270, 1213-1215

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