the pheromone receptors inhibit the pheromone response ...fusl expression, and suppression of...

Copyright 0 1993 by the Genetics Society of America

The Pheromone Receptors Inhibit the Pheromone Response Pathway in Saccharomyces cerevisiae by a Process That Is Independent of Their

Associated Ga! Protein

Jeanne P. Hirsch*91 and Frederick R. Cross? *Department $Cell Biology and Anatomy, Mount Sinai Medical Center, New York, New York 10029, and ?Rockefeller University,

New York, New York 10021 Manuscript received MaySO, 1993

Accepted for publication August 1 1, 1993

ABSTRACT Dominant mutations at the DAF2 locus confer resistance to the cell-cycle arrest that normally occurs

in M A T a cells exposed to a-factor. One of these alleles, DAF2-2, has also been shown to suppress the constitutive signaling phenotype of null alleles of the gene encoding the a subunit of the G protein involved in pheromone signaling. These observations indicate that DAF2-2 inhibits transmission of the pheromone response signal. The DAF2-2 mutation has two effects on the expression of a pheromone inducible gene, FUSI. In DAF2-2 cells, FUSl RNA is present at an increased basal level but is no longer fully inducible by pheromone. Cloning of DAF2-2 revealed that it is an allele of STE3, the gene encoding the a-factor receptor. S T E 3 is normally an a-specific gene, but is inappropriately expressed in a cells carrying a STE3DAF2-2 allele. The two effects of STE3DAFz-2 alleles on the pheromone response pathway are the result of different functions o f the receptor. The increased basal level of FUSl RNA is probably due to stimulation of the pathway by an autocrine mechanism, because it required at least one of the genes encoding a-factor. Suppression of a null allele of the G, subunit gene, the phenotype associated with the inhibitory function of STE3 , was independent of a-factor. This suppression was also observed when the wild-type S T E 3 gene was expressed in a cells under the control of an inducible promoter. Inappropriate expression of STEP in a cells was able to suppress a point mutation, but not a null allele, of the G, subunit gene. The ability of the pheromone receptors to block the pheromone response signal in the absence of the G, subunit indicates that these receptors interact with another component of the signal transduction pathway.

T HE pheromone response pathway of the budding yeast Saccharomyces cerevisiae is activated when

haploid cells of one cell type are exposed to pheromone from the other cell type [for review, see HIRSCH and CROSS (1992) and MARSH, NEIMAN and HER- SKOWITZ (1991)l. Each of the two haploid cell types express a set of specific genes under the control of the MAT locus. Thus, MATa cells secrete a-factor and have receptors for a-factor, and MATa cells secrete CY-

factor and have receptors for a-factor. The response of haploid yeast cells to the presence of mating factors includes arrest in the G, phase of the cell cycle and induction of specific gene expression.

The pheromone receptor sequences predict proteins that contain seven membrane-spanning domains and, like other members of this receptor class, are thought to be linked to a heterotrimeric G protein. Genes encoding the subunits of this G protein have been identified as GPAI (or S C G I ) for the a subunit, STE4 for the p subunit and STE18 for the y subunit. Deletion of the gene encoding the a subunit of the G protein produces cells that grow extremely slowly and

' To whom correspondence should be addressed.

Genetics 135 943-953 (December, 1993)

display features consistent with constitutive signaling (DIETZEL and KURJAN 1987b; JAHNC, FERCUSON and REED 1988; MIYAJIMA et al. 1987), whereas deletion of either of the genes encoding the P and y subunits produces cells that are viable but sterile (HARTWELL 1980; WHITEWAY et al. 1989). A current model for activation of the response pathway involves interaction of the G protein with occupied receptor resulting in guanine nucleotide exchange on the G, subunit and release of the Pr subunits from the complex. The released Py dimer activates downstream components of the pathway that ultimately produce the physiolog- ical responses required for mating. The a subunit was assigned a negative role in the signaling pathway, that of sequestering the Pr complex in the absence of pheromone, based on the recessive phenotype of constitutive signaling seen in a subunit deletion alleles. A dominant allele of the P subunit gene, STE4Hp', has also been shown to result in constitutive signaling (BLINDER, BOUVIER and JENNESS 1989), consistent with this model.

The DAF2-2 mutation was identified by its ability to confer dominant resistance to the cell-cycle arrest that normally occurs in MATa cells exposed to a-

944 J. P. Hirsch and F. R. Cross

mating factor (CROSS 1990). DAF2-2 was also found to suppress the extremely slow growth that occurs in cells containing G protein mutations in the a-subunit ( A g p a l ) or @-subunit (STE4Hp’) gene that cause constitutive signaling (BLINDER, BOUVIER andJENNEsS 1989; DIETZEL and KURJAN 1987b; MIYAJIMA et al. 1987). When the DAF2-2 allele is present, these mutants grow at the wild-type rate and appear normal, In addition, DAF2-2 confers a modest mating defect on MATa cells

DAF2-2 also has an effect on the expression of a pheromone-inducible gene, the FUSl gene. In wild- type cells, FUSl RNA is present at a low basal level which greatly increases when cells are exposed to pheromone (MCCAFFREY et al. 1987; TRUEHEART, BOEKE and FINK 1987). In DAF2-2 mutants, FUSl RNA is present at a high basal level which increases only slightly when cells are exposed to pheromone (CROSS 1990). The STE4, STE5 and STE18 genes are required for the elevated basal expression of FUSI, but STE2, the a-factor receptor gene, is not required.

The phenotypes of DAF2-2 are all specific to MATa cells and can be thought of as having two apparently opposite components. One aspect of the phenotype is suggestive of an inability to activate the pheromone response pathway as evidenced by resistance to growth inhibition by pheromone, inability to fully induce FUSl expression, and suppression of constitutive signaling mutations. The other aspect of the phenotype appears to result in a moderate constitutive increase in the activity of the signal transduction pathway reflected in the high basal level of FUSl RNA.

Here we show that the DAF2-2 mutation is an allele of the STE3 gene, which encodes the a-factor receptor. DAF2-2 caused inappropriate expression of STE3 in MATa cells. This phenotype resulted in cells that express both a-factor and the a-factor receptor, a situation that could create an autocrine loop for signaling. The two aspects of the DAF2-2 phenotypes are produced by different functions of the a-factor receptor: constitutive activation probably results from autocrine signaling through the G protein, whereas inability to respond may result from an inhibitory process that is independent of the G, subunit.

(CROSS 1990).

MATERIALS AND METHODS

Strains and media: Strains used in this study are listed in Table 1. Strains designated with an “H,” “D” or “JH” are derived from W303. Some STEjDAF2-’ alleles were made using a 2.7-kb HpaI-Sac1 fragment from cloned STE3DA‘2-2 DNA that was integrated at the STE3 locus. The STE3DAFZ-

integrating plasmid (piDAF2 m-5) was linearized with Sal1 and transformed into the following strains: Dl 1 1 to give H24; a MATa GPAlA‘“322 strain (KURJAN, HIRSCH and DIETZEL 1991) to give JH10-1 and JH10-4; a MATa

give JH11-1 and JH11-2; H20 to give H23. Strain H20 was constructed by disruption of the SST2 gene in strain Dl09

GpA l o c h 4 6 8 strain (HIRSCH, DIETZEL and KURJAN 1991) to

(DIETZEL and KURJAN 198713) using a Not1 fragment that contained a transposon carrying LEU2 inserted into the SST2 gene (DIETZEL and KURJAN 1987a). Strain H27 was constructed by disruption of the KSSl gene in strain H24 using a 1.9-kb EcoRI-SphI fragment containing the kssl::URA3 allele (COURCHESNE, KUNISAWA and THORNER 1989). Strain H46 was constructed from a diploid homozygous for the gpalLYs388 mutation, which provided an un- marked null allele of GPAl (KURJAN, HIRSCH and DIETZEL 1991). The g p ~ l ~ y ” ~ ~ strain was transformed with a 4.6-kb BglII-XbaI fragment containing the mfal-Al::LEUZ allele (MICHAELIS and HERSKOWITZ 1988) to disrupt the MFAl gene, a 2.0-kb EcoRI fragment containing the mfa2- Al::URA3 allele (MICHAELIS and HERSKOWITZ 1988) to disrupt the MFA2 gene, and plasmid piDAF2 m-5 linearized with SalI to produce a DAF2-2 allele. The STE3 gene was disrupted using a 4.4-kb Hind111 fragment containing the ste3::LEUZ allele (HAGEN, MCCAFFREY and SPRAGUE 1986). All strain constructions involving transformations were con- firmed by Southern blot.

Strains were grown on YEPD (2% glucose) or YEP-Gal (3% galactose), and strains under selection were grown on synthetic dropout media, as described (SHERMAN, FINK and HICKS 1989).

Genomic library contruction: A yeast genomic library, called 3JDAF2, was made in the vector YCp50, which contains the URA3, CEN4 and ARSl sequences. Genomic DNA from strain 21 1-4C was partially digested with Sau3A and size fractionated by centrifugation for 5 hr at 40,000 rpm through a 10-30% glycerol gradient. The high molecular weight fraction was cloned into the BamHI site in the vector. The library contains 70% inserts with an average size of 10 kb and represents 5.2 X genomes.

Plamid construction and polymerase chain reaction (PCR): pDAF2 m-1, 2 and 3 were plasmids obtained from screening the genomic library 3JDAF2 for clones that conferred a-factor resistance. pDAF2 m-4 was constructed by subcloning the 3.2-kb SphI-Sac1 fragment from pDAF2 m- 3 into the vector pSE358, which contains the TRPl, CEN4 and ARSl sequences. piDAF2 m-5 was constructed by digestion of pDAF2 m-4 with HpaI and BglII, treatment with S1 nuclease, and recircularization, to produce an integrating plasmid. piDAF2 m-7 was constructed using the following oligonucleotides containing STE3 sequences as PCR primers: oSTE3-1, 5’-TTTGTTTGCTAGCTG-3’; oSTE3-

oSTE3-1 contains wild-type STE3 sequence that includes a NheI site; oSTE3-2 inserts a stop codon and a Sal1 site near the carboxyl-terminal region of the STE3 gene that results in a 163-amino acid truncation of the protein. The PCR product obtained using these primers and the plasmid pDAF2 m-4 as a template was cloned into the NheI-Sal1 sites of piDAF2 m-5 to create piDAF2 m-7. pDAF2 m-8 was constructed by digestion of pDAF2 m-1 with SalI and recircularization of the plasmid. pGAL-STE2.2 was constructed using the following oligonucleotides containing STE2 sequences as PCR primers: oSTE2-1, 5”CCGGATCCTT-

TAACCTTATACC-3’. These oligonucleotides result in the insertion of a BamHI site 26 nucleotides upstream of the STEP initiation codon, and a SalI site 40 nucleotides downstream of the STEP stop codon. The PCR product obtained using these primers and the plasmid pSL611 (BENDER and SPRACUE 1986) as a template was cloned into the vector pBM272, to create pGAL-STE2.2, which places STE2 under the control of the GAL1 promoter.

PCRs were performed using Taq DNA polymerase (Pro- mega), 50 p~ concentrations of oligonucleotides and 1 ng

2,5‘-GGGTCGACTATCTTTTCCACATGTC-3‘.

AAGAACCATATCC-3’; oSTE2-2, 5”GGGTCGACTAG-

Pheromone Response Inhibition 945

TABLE 1

Strain genotypes

Strain Genotype Source

211-1A 21 1-4C 2 1 1-5A 311-13 F16-1D K23-1 B K213-1A K215-1B W303

MATa canl SUP4-3 lys.2' his4" barl ura3 leu2 hm1a::LEUZ MATa canl SUP4-3 lys2O trpl" bar1 cyh2 ura3 leu2 hm1a::LEUZ STE3DAFz-z MATa canl SUP4-3 lys2O his4" trpl" barl ura3 leu2 STE3DAF2-z MATa canl SUP4-3 lys.2' his4" trpl" barl ura3 leu2 hm1a::LEUZ MATa canl SUP-4-3 lys2O his4" trpl" barl cyh2 ura3 hm1a::LEUZ STEJDAF2.' MATa canl SUP4-3 lys2O his4" trpl" barl cyh2 ura3 hm1a::LEUZ STE3DAFz-z MATa canl SUP4-3 1ysY his4" trpl" barl cyh2 ura3 hmla::LEU2 STE3DAF2-3 MATa canl SUP4-3 lys2O hisC trpl" barl cyh2 ura3 hm1a::LEUZ STE3DAFz-4 MATaIa leu2-3,112 trpl-1 canl-100 ura3-1 ade2-1 his3-11,15

All of the following strains are isogenic t o W303

H24-1C.8A H24-2C.6D

H24-1A.2D H24-3C,5D

JH10-1,4 JH11-1,2 D l 1 1 H23 H24 H27 H36 H40 H46 H47 H48 H49 H54-4D96A H54-1B,7A H54-1C,4B H54-2A,9D

MATa MATa MATa STE3DAFz-Z MATa STE3DAFz-Z gpa1::LEUZ MATa STE3DAFz-2 GPAlA'a3zz MATa STE3DAFz-z GPAloch468 MATala gpal::LEUZ/GPAl MATaIa gpal::URA3/GPAl STE3DA"-2/STE3 sstZ::TnLEUZ/SSTZ MATala gpal::LEUZ/GPAl STE3DAFz-Z/STE3 MATa/a gpal::LEU2/GPAl STE3DAFz-2/STE3 kssl::URA3/KSS1 MATala gpalLy3u8/gpalLy3u8 MATaIa gpa1::LEUZIGPAl STE3DAFZ-ZT-A163/STE3 MATala gpa1L9s388/gpalLys3u8 mfal-Al::LEUZ/MFAl mfaZ-Al::URA3/MFAP STE3DAF2-z/STE3 MATala gpalLy'388/gpalLy388 STE3DAFZ.Z /STE3 MATa/a gpa1::LEUZIGPAl STE3DAFz-2T/STE3 MATaIa gpal::URA3/GPAl STE3DAFZ-Z/STE3 steZ::LEUZ/STEZ MATa STE3DAF2-2 MATa STE3DAFZ.Z mfal-Al::LEUZ MATa STE3DAFz-2 mfaP-Al::URA3 MATa STE3DAFz-z mfal-A1::LEUP mfaZ-Al::URA?

CROSS (1 990) CROSS ( 1 990) CROSS (1 990) CROSS ( 1 990) CROSS (1 990) CROSS (1 990) CROSS ( 1 990) CROSS ( 1 990) R. ROTHSTEIN

This s tudy This s tudy This s tudy This s tudy This s tudy This s tudy J. KURJAN This s tudy This s tudy This s tudy This s tudy This s tudy This s tudy This s tudy This s tudy This s tudy This s tudy This s tudy This s tudy This s tudy

of template, as described by SCHARF, HORN and ERLICH (1 986).

Yeast methods and Northern blot hybridization: Yeast transformations were performed by the lithium acetate method (ITO et al. 1983) with the following modifications. A 50-ml culture grown to a density of 5-7 X lo' cells/ml was pelleted and washed with water. The washed pellet was suspended in 20 ml of LTE (0.1 M lithium acetate, 10 mM Tris hydrochloride, pH 7.8, 1 mM EDTA) and incubated for 30 min at 30" with shaking. The culture was pelleted and suspended in 2 ml LTE. Plasmid DNA and 50 r g of denatured carrier DNA were added to 0.2-ml aliquots of this suspension, and the cultures were incubated for 30 min at 30". An equal volume of 60% polyethylene glycol 3350 in water was added and the procedure was continued as described by ITO et al. (1 983).

Yeast RNA was isolated as described previously (CROSS and TINKELENBERC 1991). RNA was transferred to a nitrocellulose membrane after formaldehyde-agarose gel electro- phoresis as described by LEHRACH et al. (1977). The membranes were dried and UV cross-linked using a Stratalinker UV box. Prehybridization and hybridization were done at 65" in a buffer containing 0.9 M sodium chloride, 0.09 M sodium citrate, 0.5% sodium dodecyl sulfate, 5 X Denhardt's solution, and 0.1 mg/ml denatured DNA. The probes used were all gel-purified DNA restriction fragments '*P-labeled by random primer labeling using a Prime-It kit (Stratagene). The fragments used were: FUSl , a 1.4-kb EcoRI-Hind111 fragment from plasmid pSL589 (MCCAFFREY et al. 1987);

TCMl (SCHULTZ and FRIESEN 1983), a 0.8-kb HpaI-Sal1 fragment from plasmid pAB309A; STE3, a 0.6-kb ScaI-Sal1 fragment from plasmid pDAF2 m-8.

RESULTS

The dominant DAF2-2 mutation confers resistance to pheromone-induced GI arrest in a MATa cell-specific manner (CROSS 1990). This phenotype as well as its suppression of constitutive signaling due to deletion of the G, subunit gene indicates that DAF2-2 causes a defect in the response to pheromone. Mutations that result in signaling defects, such as ste alleles, often result in a decrease in the basal level of the pheromone response pathway (MARSH, NEIMAN and HERSKOWITZ 199 1). DAF2-2, however, results in a higher basal level of the pathway than in wild type cells (CROSS 1990). The interaction of DAF2-2 with mutations in another gene that result in signaling defects was therefore examined.

DAF2 is not epistatic to sterile alleles of GPAI: DAF2-2 was thought to act at the G protein step of the pheromone response signaling pathway based on results from epistatic analyses (CROSS 1990). It was therefore of interest to determine the effect of the DAF2-2 mutation on basal activity of the response


STE3 + + DAF2 OAF2 DAF2 OAF2

SCGl + + + - Ala322 Och468

l a ~ a l a l a ~ a l a l

0

FUS7 - 0 0 0 0 -

. TCM7 . o m . m . . m o . . m

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2

FIGURE 1 .-FUSl RNA expression in DAF2-2 and G P A l mutant strains. Total RNA from yeast strains with the indicated genotypes was fractionated, transferred to nitrocellulose, and hybridized with ;I 1.4-kb SaP-labeled EcoRI-Hind111 fragment containing the FUSI coding region. The blot was rehybridized with a 0.8-kb s2P-labeled H/JalSalI fragment containing the T C M l coding region to determine the amount of RNA per lane. Two strains of each genotype were analyzed. The strains used were: H24-1C and H24-8A. M A T a DAF2+ G P A P (lanes 1 , 2); H24-2C and H24-6D. MATa DAF2+ GPAI' (lanes 3, 4); H24-3C and H24-5D. M A T a D A F 2 - 2 G P A P (lanes 5,6); H24-1A and H24-2D, MATaDAF2-2gpal : :LEU2 (lanes 7. 8); JH10-1 and JH10-4, MATa DAF2-2 CPA1A'o'22 (lanes 9, IO); JHIl-I andJHl1-2, MATaDAF2-2CPA1°'h"68(lanes 1 1 , 12).

pathway in cells containing sterile mutations in the gene encoding the G, subunit. The two G, mutations used in this study are thought to prevent signaling by different mechanisms based on the effects of analo- gous mutations in mammalian G, proteins that have been studied biochemically (HIRSCH, DIETZEL and KURJAN 1991; KURJAN, HIRSCH and DIETZEL 1991). One mutation, G P A I ~ " ' ~ ~ , results in sterility most likely from an inability to become activated by bound GTP (KURJAN, HIRSCH and DIETZEL 199 1). The other mutation, a carboxyl-terminal truncation of G P A l , encodes a G, subunit that is thought to be uncoupled from the receptors (HIRSCH, DIETZEL and KURJAN

An RNA blot containing samples from strains of different genotypes was hybridized with a labeled fragment from the pheromone inducible gene FUSI. In this genetic background, the basal level of FUSl RNA is higher in M A T a cells than in MATa cells (Figure 1, lanes 1-4). As reported previously (CROSS 1990). MATa cells that carry the DAF2-2 allele had a higher basal level of RNA expression from the pheromone inducible gene FUSl than wild-type cells (Fig- ure 1, lanes 3-6). Likewise, cells carrying both DAF2- 2 and a disruption of the G P A l gene had the increased level of FUSl RNA that is characteristic of DAF2-2 cells (Figure 1, lanes 7, 8). In contrast, cells carrying DAF2-2 in combination with a mutation in G P A l that causes sterility, Ala322 (KURJAN, HIRSCH and DIETZEL 199 l), had a very low level of FUSl RNA (Figure 1,

199 1).

lanes 9, I O ) . The effect of the yeast G, Ala322 mutation is probably similar to its effect in the mammalian Gas subunit, where it results in a protein that is unable to undergo the conformational change associated with GTP binding (MILLER et al. 1988). Cells carrying DAF2-2 and another mutation in GPAl that causes sterility, Och468 (HIRSCH, DIETZEL and KURJAN 1991), also contained a very low level of FUSl RNA (Figure 1, lanes 1 1, 12). The defect in response caused by the Och468 mutation is thought to result from an inability of the protein to interact with occupied receptor, similar to the Gas unc mutation (RALL and HARRIS 1987; SULLIVAN et al. 1987). This result was surprising because the effect of DAF2-2 on the basal level of FUSl RNA does not require the a-factor receptor gene, STE2, and was therefore not expected to require a G, subunit capable of coupling to the receptor

In summary, G P A l was epistatic to DAF2-2 with respect to FUSl RNA expression in double mutant combinations carrying DAF2-2 and GPAlST" alleles. However, DAF2-2 is epistatic to GPAl with respect to the constitutive signaling phenotype of gpal cells (CROSS 1990), and DAF2-2gpa1 cells had the DAF2-2 phenotype of high basal expression of FUSl RNA. These genetic results can be interpreted in two ways. One possibility is that DAF2-2 acts at the level of the G, subunit, so that their functions cannot be ordered. This situation is unlikely because DAF2-2 can block the signal from a constitutive allele of the GB subunit, which acts downstream of the G, subunit (CROSS 1990). The other possibility is that the DAF2-2 mutation has an effect on two processes, one that functions in suppression of gpal and another that regulates FUSl expression.

DAF2-2 is an allele of STE3: The gene encoding the mutant dominant allele DAF2-2 was cloned in the expectation that it would provide information about the phenotypes caused by the mutation. A genomic library was constructed in the CEN vector YCp50 using DNA from a DAF2-2 strain. The library was transformed into a wild-type strain and transformants were replicated to plates containing a-factor. Three overlapping plasmids were obtained which conferred resistance to growth inhibition by a-factor. The region of overlap in the three clones had the same restriction map as the STE3 gene, which encodes the a-factor receptor (Figure 2). The 5"flanking sequence of STE3 in the DAF2-2 clones had a different restriction map from wild-type DNA and contained a repetitive element in the region between approximately 100 and 900 nucleotides upstream of the initiation codon (data not shown).

T o confirm that the cloned gene was STE3, a plasmid containing the 3.2-kb SphI-Sac1 fragment from pDAF2 m-3 (Figure 2) was transformed into a MATa

(CROSS 1990).


N S a p Bg S a B g S a S a FIGURE 2.-Restriction maps of

€ E \ € S B I I 1 I I I - pDAF2m1 dasmids containing the STE3"AFZ'2 al-

Bo Bg Bg N Sa s I I

DAF2+ I 1 2 3 4 DA F2-

STE3

TCMl

FIGURE 3 . 4 T E 3 RNA expression in DAF2 strains. Log phase cultures of strains with the indicated genotypes were split into two fractions, and one set was treated with a-factor at lo" M for 20 min. Total RNA from these samples was fractionated, transferred to nitrocellulose and hybridized with a 0.8-kb '*P-labeled ScaISall fragment containing part of the STE3 coding region. The blot was rehybridized with a fragment containing TCMl as described in Figure 1. The strains used were: 311-13, MATa STE3+; 211-1A, MATa STEP; F16-1D. MATa STE3DAFz"; K23-1B, MATa STE3D"Fz- '; K213-1A, MATa STE3DAFZ"; K215-1B. MATa STE3DAF24.

ste3::LEU2 strain. The DAF2-2 plasmid complemented the ste3::LEU2 strain for the ability to mate, confirm- ing that it contained the STE3 gene.

T o establish that the DAF2-2 mutation resides in the STE3 gene, a DAF2-2 strain was transformed with a fragment containing a ste3::LEU2 disruption allele. Disruption of STE3 in a DAF2-2 strain restored a- factor sensitivity, demonstrating that DAF2-2 is an allele of STE3.

D M 2 alleles of STE3 cause inappropriate expression in MATa cells: The identification of DAF2-2 as an allele of STE3 was unexpected because STE3 is an a-cell specific gene, and the DAF2-2 mutation was isolated in an a cell. The expression of STE3 in DAF2 strains was examined by RNA blot to determine if the mutation affects the cell type specificity of STE3 transcription. Wild type a cells contained STE3 RNA, whereas wild type a cells did not, as expected (Figure 3, DAF2+ lanes). a cells carrying the DAF2-1, 2 and 4 alleles contained a detectable level of STE3 RNA, suggesting that the mutation involves a change in the STE3 promoter (Figure 3, DAF2-1, 2 and 4 lanes). This interpretation is consistent with the finding that the cloned DAF2-2 allele contains a repetitive element inserted in its 5"flanking region. The DAF2-3 allele,

B lele. Stippled box shows the position pDAF2m-2 of the repetitive element that is not

present in the wild-type allele. Filled pDAF2m3 box shows the position of STE3 cod-

ing region. Restriction site abbrevia-

1 kb Pstl; S , Sacl; Bg, BgllI; B, BamHl; H tions: E, EcoRI; 9, Nhel; sa, Sall; P.

Sp, SphI.

pGAL-STE3 scgl::L€U2/SCGl

glucose galactose FIGURE 4.-A gpal::LEU2/GPAl diploid strain (Dl 1 I ) contain-

ing a GALSTE3 plasmid (pSL552) was sporulated, and tetrads were dissected on either glucose or galactose. Four spores from a single ascus were placed in each vertical column. The plates were examined microscopically to insure that all of the spores had germinated.

unlike the other DAF2 alleles, does not cause an increase in the basal level of FUSl RNA (F. R. CROSS, unpublished observation). DAF2-3 a cells contained no detectable STE3 RNA when untreated, but did show RNA accumulation after treatment with a-factor (Figure 3, DAF2-3 lanes). This result suggests that the DAF2-3 allele of STE3 has acquired a pheromone- inducible promoter that causes it to be expressed inappropriately. STE3 was not overexpressed in DAF2 a cells as determined by comparison to wild-type a cells.

Expression of wild-type STE3 in MATa cells sup presses a null allele of GPA1: The novel finding that a receptor gene promoter mutation suppressed activation of signaling caused by the absence of its associated G, protein suggested that wild-type STE3, if expressed in a cells, could also perform this function. To test this idea, a plasmid containing the STE3 gene under the control of the GAL promoter (pSL552) was transformed into a yeast strain heterozygous for a disruption of the GPAl gene. This strain normally segregates 2:2 for the constitutive signaling phenotype, which results in extremely small colonies containing abnormally shaped cells (DIETZEL and KURJAN 1987b; JAHNC, FERCUSON and REED 1988; MIYAJIMA et al. 1987). The gpal::LEU2IGPA1 diploid transformed with GAL-STE3 was sporulated and dissected on plates containing either glucose or galactose. Te- trads dissected on glucose displayed 2:2 segregation for the constitutive signaling phenotype (Figure 4, glucose; gpal::LEU2 spore colonies not visible). Te- trads dissected on galactose to induce expression of


TABLE 2

Expression of STE3 or MATal suppresses the growth defect of an gpal::LEU2 strain

Glucosea Galactose

Spore viability in tetrads Percent Space viability in tetrads Percent plasmid

Plasmid plasmid

4:Ob 3:l 2:2 Total transmissionC 4:O 3: l 2 2 Total transmission

GAL-STE3 (pSL552) 0 0 22 22 64 5 8 12 25 54 GAL-MATal (pSL599) 0 0 22 22 77 5 7 8 20 81 GAL-STE2 (pSL611) 0 0 22 22 55 0 0 22 22 75 GALSTEP (pCAL-STE2.2) 0 0 17 17 50 0 0 20 20 48 Vector (YCp50) 0 0 21 21 40 0 0 23 23 70

A heterozygous diploid gpaZ::LEU2/GPAZ strain containing the plasmids shown was sporulated and dissected on either glucose or

Tetrads were scored as the ratio of large colonies containing normally shaped cells to very small colonies containing abnormally shaped

Plasmid transmission was the ratio of Ura+ spore colonies to the total number of spore colonies.

galactose.

cells.

STE3 showed some cases of 3:l and 4:O segregation (Figure 4, galactose, rows 1, 5 and 7). In all tetrads with more that two wild-type spore colonies, one (in 3: 1 tetrads) or two (in 4:O tetrads) spore colonies were MATa gpal::LEU2 and contained the GAL-STE3 plasmid. When transferred to glucose these segregants displayed the aberrant morphological changes characteristic ofgpal::LEU2 mutants. The results obtained from a larger sample of tetrads are shown in Table 2. Tetrads derived from an gpal::LEU2/GPAI diploid carrying GAL-STE3 (pSL552) always showed 2:2 segregation for the gpal null phenotype when dissected on glucose. About half of the tetrads from the strain carrying GAL-STE3 that were dissected on galactose contained MATa gpaI::LEU2 spore colonies capable of wild-type growth. The expected number of MATa gpal::LEU2 segregants suppressed by expression of STE3 depends on the number of segregants that in- herit the plasmid. In this case, transmission of the plasmid to haploids following sporulation was about 54%, in approximate agreement with the number of tetrads containing suppressed spore colonies. The gpal::LEU2 phenotype segregated 2:2 when a control strain that contained only the vector (YCp50) was dissected on either glucose or galactose. This experiment demonstrated that the wild type STE3 coding region, when expressed inappropriately in a cells, prevented constitutive activation of the pheromone response pathway.

Another way to express the STE3 gene in a cells is to produce the MAT01 protein, an activator of a- specific genes (SPRAGUE, JENSEN and HERSKOWITZ 1983). The gpaI::LEU2/GPAI diploid was transformed with a plasmid containing the MATal gene under the control of the GAL promoter (pSL599), sporulated, and dissected on plates containing either glucose or galactose. Tetrads derived from this diploid that were dissected on galactose showed 3:l and 4:O segregation for large spore colonies (Table 2). Wild-

type growth of the MATa gpal::LEU2 segregants that contained GAL-MATal was dependent on galactose, demonstrating that activation of a-specific genes in a cells also suppressed gpaI::LEU2 (Table 2).

Expression of STE2 in MATa cells suppresses a point mutation in GPA1: T o test whether inappropriate expression of STE2 in a cells could suppress the growth defect of gpal::LEU2 cells, a plasmid containing STE2 under the control of the GAL promoter (pSL611) was transformed into a gpaI::LEU2/GPAI diploid. Sporulation and dissection of this strain on either glucose or galactose produced 2:2 segregation for the gpaI::LEU2 phenotype (Table 2). Because the GAL-STE2 construct present in this plasmid was not expressed very efficiently, as determined by complementation of the mating defect of a ste2 strain, a second GAL-driven STE2 gene was constructed. In this fusion the initiation codon of STE2 was placed closer to the transcription initiation site used by the GAL promoter. Expression of STE2 from this plasmid, pGAL-STE2.2, was also unable to suppress the growth defect of gpal::LEU2 cells (Table 2), although it was able to fully complement a ste2 strain.

The ability of inappropriately expressed STE2 to suppress a different mutation in GPAl was determined using the gpaILYs388 allele, which contains a single amino acid change from asparagine to lysine at position 388 (KURJAN, HIRSCH and DIETZEL 1991). This allele confers growth and morphology phenotypes that are similar to those of the gene disruption, but, unlike the disruption, has the potential to produce a protein product. A diploid homozygous for the un- marked gpaILys388 allele was used in this experiment to allow unambiguous scoring of suppression. This diploid is viable because MATa/a cells do not express the other components of the pheromone signaling pathway, but upon sporulation it produces four invi- able spores. The gpaILYs388 diploid was transformed with pCAL-STE2.2, sporulated, and dissected on

Yeast Pheromone Response Inhibition 949

TABLE 3

Expression of STE2 suppresses the growth defect of a gpalLp388 strain

Glucose" Galactose

Plasmid 2:2b 1:3 0:4 Total 2:2 I :3 0:4 Total

GAL-STE2 (pGAL-STE2.2) 0 0 18 18 5 9 8 22 Vector (pBM272) 0 0 20 20 0 0 22 22

" A homozygous diploid gpalL"'"/gpalL~'*8 strain containing the plasmids shown was sporulated and dissected on either glucose or

Tetrads were scored as the ratio of large colonies containing normally shaped cells to very small colonies containing abnormally shaped galactose.

cells.

plates containing either glucose or galactose. Tetrads derived from this strain that were dissected on glucose did not contain any large spore colonies (Table 3). Out of 22 tetrads dissected on galactose, 14 contained segregants that grew at the wild-type rate and displayed a normal cell morphology. All of these segregants were MATa, contained the GAL-STE2 plasmid, and when transferred to glucose displayed the aberrant morphological changes characteristic ofgpal mutants. Tetrads derived from the gpalLYs388 diploid carrying a vector with the GAL promoter (pBM272) that were dissected on either glucose or galactose did not contain any large spore colonies. Expression of the STEP gene in MATa cells was therefore capable of suppressing an allele of GPAl that confers a phenotype similar to that of a null mutation.

a-Factor is required for the high basal FUSI expression phenotype of STE3DAFz": The identification of DAF2-2 as an allele of STE3 suggested an explanation for the high level of FUSl expression in DAF2-2 strains. Because MATa STE3DAF2-2 strains make both a-factor and the a-factor receptor, the potential exists for stimulation of the pheromone response pathway by an autocrine signaling loop. If this is the case, elimination of a-factor production should decrease the level of FUSl RNA. MATa STE3DAF2-2 strains were constructed that differed at the loci encoding the two a-factor genes, MFAl and MFA2. Expression of FUSl in these strains was examined by RNA blot. STE3DAF2*z strains that contained intact copies of both MFA genes (Figure 5, lanes 1, 2) or either one of the genes (Figure 5 , lanes 3-6) displayed the high basal level of FUSl expression characteristic of the STE3DAF2-2 mutation. Strains containing disrup tions of both MFA genes, however, did not express a detectable level of FUSl RNA (Figure 5 , lanes 7-8). This result demonstrated that a-factor production is required for the high basal level of FUSl RNA seen in STE3DAF2-2 alleles.

a-Factor, the a-factor receptor and the SST2 and KSSZ gene products are not required for the g p d suppression phenotype of STE3DAFz-2: The phenotype of STEPAF2-' cells has aspects of both stimulation of the pheromone response pathway, as evidenced by

MFAl + - + - MFA2

FUS1

1 2 3 4 5 6 7 8

FIGURE 5.-FUSl RNA expression in DAF2-2 and MFAlI.2 mutant strains. A blot containing total RNA from yeast strains with the indicated genotypes was hybridized with a fragment containing FUSl as described in Figure 1; a parallel blot was hybridized with a fragment Containing TCM1. T w o strains of each genotype were analyzed. The strains used were: H54-4D and H54-6A. MATa STE3DAFz-2; H54-1B and H54-7A, MATa STE3DAFz~zmfal-Al::LEU2; H54-1C and H54-4B. MATa mfa2-k:URAA H54-2A and H54-9D. MATa STE3DAF2-2 mfal-Al::LEU2 mfa2-k:URAjr.

high basal expression of FUSl, and inhibition of the pathway, as evidenced by resistance to cy-factor and suppression of mutations in GPAl. One explanation for inhibition of the response pathway is that cells are desensitized to pheromone due to constant production and binding of a-factor by the a-factor receptor. To test the dependence of gpal suppression on a-factor production, a strain was constructed that was homozygous for the gpalLYs388 mutation and heterozygous for STE3DAF2-2, mfal::LEU2 and mfa2::URA3 alleles. Sporulation and dissection of this diploid produced some large spore colonies, all of which had the genotype MATa gpalLYs38RSTE3DAF2-2. These haploids contained MFAl and MFA2 alleles that were approximately evenly distributed among the four possible combinations, including segregants that contained both the mfal::LEU2 and mfa2::URA3 genes (Table 4). A control strain with the genotype gpalLy'388 STE3DAF2-2/STE3 also gave rise to haploids capable of wild type growth, all of which were MATa gpalLYs388STE3DAFz-2. Inhibition of the response pathway by STEPAF2-' was therefore independent of the MFA genes, suggesting that the negative effect of


STE3 expression is not the result of desensitization due to an autocrine signaling loop. This finding was in contrast to the stimulatory effect of STE3DAF2-2 on the response pathway, which required an intact copy of either MFAl or MFA2.

Because MATa STE3DAF2-2 cells express receptors for both a- and a-factor, it was possible that the inhibitory effect of STE3DAF2-2 was due to the presence of both pheromone receptors on the cell surface. A diploid strain heterozygous for the gpal::URA3, STE3DAF2-2 and ste2::LEU2 mutations was constructed to determine if suppression of gpal by STE3DAF2-2 requires the gene encoding the a-factor receptor, STEB. Sporulation and dissection of this strain resulted in the recovery of STE3DAF2-2gpal::URA3 segregants that contained the ste2::LEU2 mutation (Table 4). Suppression of gpal by STE3DAF2-2 therefore does not involve an interaction between the two pheromone receptors.

The effect of genes that may play a role in desensitization was determined with respect to the inhibitory phenotype of STE3DAF2-2. SST2 is thought to be involved in desensitization because mutations in this gene cause a defect in recovery from pheromone- induced arrest (CHAN and OTTE 1982; DIETZEL and KURJAN 1987a); KSSl was isolated as a multicopy suppressor in a strain carrying an sst2 mutation (COUR- CHESNE, KUNISAWA and THORNER 1989). Strains heterozygous for a gpal disruption, STE3DAF2-2, and disruptions of either the SST2 or KSSl genes were con- tructed to test the involvement of these genes in STE3- mediated inhibition of the pheromone response pathway. Sporulation and dissection of these diploids resulted in the recovery of haploids that were suppressed for the gpal null phenotype by STE3DAF2-2 and also contained disruptions alleles of either SST2 or KSSl (Table 4). These results indicated that neither SST2 nor KSSl are required for suppression of gpal by STE3DAF2-2.

Carboxyl terminus of the a-factor receptor is not essential for gpal suppression: Deletion of the carboxyl terminus of the a-factor receptor results in a supersensitive phenotype, suggesting that it may play a negative role in signaling (KONOPKA, JENNESS and HARTWELL 1988; RENEKE et al. 1988). The complete 184-amino acid cytoplasmic tail of STE3 was not thought to be required for the inhibitory effect of the a-factor receptor because one of the original DAF2-2 clones (Figure 2, pDAF2 m-2) encoded a product with a 99-amino acid carboxyl-terminal truncation. T o determine whether the remainder of the STE3 cytoplasmic tail is required for suppression of gpal, con- structs containing STEPAF2-' truncated by either 105 or 163 amino acids were introduced into a diploid heterozygous for a gpal::LEU2 mutation. The 105- and 163-amino acid truncations contain about 79-82

and 21-24 amino acids of the cytoplasmic tail, respec- tively. Truncations that deleted a larger region of the cytoplasmic tail did not produce functional protein as determined by complementation of a ste3::LEU2 allele in MATa cells (data not shown). Analysis of tetrads derived from diploids expressing the receptor truncations showed that the STE3DAF2-2-A105 allele conferred full suppression of gpal, but the STE3DAF2-2- A163 allele conferred only partial suppression (Table 5). The inability of the STE3DAF2-2-A163 construct to completely suppress gpal was inferred from the production of spore colonies that were smaller than their wild-type counterparts yet still viable. The sequences in the a-factor receptor gene that are essential for gpal suppression are therefore not contained within the terminal 163 residues of the protein, although part of this region may be required for full expression of the phenotype. Another explanation for the partial suppression seen with this construct is based on the observation that carboxyl-terminal truncation of the receptor results in supersensitivity. Increased sensitivity of the receptor to pheromone may cause the stimulatory effect of the STE3DAF2-2 trunction to predom- inate over its inhibitory effect.

DISCUSSION

Inappropriate expression of the a-factor receptor gene in MATa cells resulted in both stimulation and inhibition of the pheromone response pathway. These two effects could be distinguished by their require- ments for other gene functions. Stimulation of the pathway resulting in an increased level of FUSl RNA required a functional copy of either the MFAl or MFA2 gene. This effect, therefore, probably occurs by an autocrine signaling mechanism. Inhibition of the pathway occurred in the absence of the GPAl and MFA1/2 genes and thus was not the result of desensitization due to autocrine stimulation. Other mating functions that are independent of the G, subunit include the phenomena of courtship (JACKSON, KON- OPKA and HARTWELL 199 1) and endocytosis of the a- factor (ZANOLARI et al. 1992) and a-factor receptors (N. DAVIS and G. F. SPRAGUE, personal communication). However, both courtship and endocytosis of the a-factor receptor are dependent on the presence of pheromone, unlike inhibition of the response by receptor.

The results presented here invalidate a previous proposal that DAF2-2 acts at the STE4 step of the signaling pathway, which was based on the finding that DAF2-2 acts upstream of a ste4 mutation but downstream of a STE@' mutation (CROSS 1990). It is now apparent that the stimulatory effect of STE3DAF2-

acts upstream of ste4, but the inhibitory effect acts at, or downstream of, STE4Hp'. These two phenotypes are the result of independent functions of STE3DAF2-2

Yeast Pheromone Response Inhibition 95 1

TABLE 4

Suppression of gpal by STE3DApz does not require STE2, SST& KSSl or MFAI and MFA2

Allele configuration of gpal STEjDAf2 Diploid genotypen 4:Ob 3:l 2:2 1.9 0:4 segregants

1L~s388 s~~3D.4Fz-z mfal::LEU2 mfa2:URAjr 0

gpalLJJSB8 STE3 MFAl MFA2 O l o mfa2:mfal mfa2 5:5:7:7 MFAl MFA2:mfaI MFA2:MFAI

gpa 1Lp3.98 STEJDAFZ-Z

gpaILJsS88 STE3 gpal::URA3 STE3D"F2-2 ste2::LEUZ

GPAl STE3 STEP gpal::URA3 STEJDAFZ-' sst2::LEU2

GPAl STE3 SST2 gpal::LEU2 STE3D"Fz-2 kssl::URA3

GPAl STE? KSSl

0 0 4 1 4 0

1 7 1 0 0 0 3:6 STE2:steP

0 14 16 0 0 7:7 SST2:sst2

1 11 10 0 0 10:3 KSS1:kssl

gpal::LEU2 STE3DAF2-2 GPAl STE3

1 11 9 0 0

The STE3DAFZ-Z allele was marked by insertion of the T R P l gene downstream of STE3. Tetrads were scored as the ratio of large colonies containing normally shaped cells to very small colonies containing abnormally shaped

cells.

TABLE 5

Carboxyl terminus of STE3 is not essential for suppression of gpal

Diploid genotypea 4:Ob 3:l 2:2 Total

L- [pSTE3DAf2~2-AI05]c 0 8 15 23 al::LEU2 STE3 GPAl STE3

gpal::LEU2 STE3DAFZ.ZT-A163 GPA 1 STE3

3d l l d 18 32

gpa1::LEUP STE3DAF2-ZT GPA 1 STE3

2 5 6 1 3

Q S T E ~ D A F Z ~ T alleles were made by integration of STE3DAFZ-2 at the T R P l locus.

Tetrads were scored as the ratio of large colonies containing normally shaped cells to very small colonies containing abnormally shaped cells.

YCp5O-based plasmid pDAF2m-8. In these tetrads the gpal::LEU2 STE3DAFZ spore colonies were

somewhat smaller than the wild-type spore colonies.

and do not appear to act at the same step of the signal transduction pathway.

BENDER and SPRACUE (1 989) have previously shown that cells expressing a-factor, the a-factor receptor and the a-factor receptor (equivalent to STE3DAF2-2) have a level of FUSl RNA that is intermediate to the basal and fully induced levels. FUSl RNA is not sig- nificantly induced in these cells by exposure to a- factor, consistent with what is seen in STE3DAF2-2 cells (CROSS 1990). BENDER and SPRACUE (1 989) also found that cells expressing both receptors but not a-factor do not fully arrest after exposure to a-factor. The authors concluded that the presence of unoccupied a- factor receptor interferes with the response to a- factor, but were unable to demonstrate the reciprocal effect of the a-factor receptor. We have extended these findings to show that interference by the a-

factor receptor acts downstream of the G, subunit and does not require the presence of the a-factor receptor. In addition, we have shown that the a-factor receptor is capable of inhibiting the response pathway in a cells, although it is less efficient than the a-factor receptor in a cells. We have also shown that an estab- lished response in a cells can be reversed by the a- factor receptor. This situation occurs in cells carrying DAF2-3, which encodes an allele of STE3 that is not expressed in untreated a cells, but is induced after treatment with a-factor. Newly synthesized a-factor receptor is capable of inhibiting the response, because DAF2-3 cells respond to a-factor by a brief arrest, followed by resumption of growth (F. R. CROSS, unpublished observation).

The cell type specificity of receptor-mediated inhibition of the pheromone response pathway remains one of the most puzzling aspects of this study. Expres- sion of the a-factor receptor suppresses the constitutive activation phenotype of gpal null mutations in MATa cells but not in MATa cells, where it is normally expressed. Likewise, expression of the a-factor receptor suppresses the constitutive activation phenotype of a gpal point mutation only in MATa cells. The fact that suppression of gpa l by STE2 in a cells is limited to an allele of gpa l that is capable of making a protein product indicates that the mechanisms of inhibition by the two receptors are not identical.

The cell type-specific gene responsible for the dif- ferential effect in a and a cells is not STE2 or MFAI/ 2, because null mutations in these genes have no effect on suppression of g p a l . Other cell type-specific genes are mainly involved in pheromone processing and secretion, and do not include obvious candidates for this regulator. Cell type-specific function of STE3


could be due to either an activator of the function that is present in a cells or an inhibitor that is present in a cells. Because MATa1 induces the expression of a-specific genes, a putative a-specific inhibitor would be present in the MATa strain containing GAL-MATal grown on galactose. Suppression of the gpal in this strain therefore suggests that a cell type specific activator of the STE3 inhibitory function is present in a cells.

A negative function for pheromone receptors has been suggested by the observation that receptor mutations that cause an increase in activity are often recessive to wild type. For example, a carboxyl-terminal truncation of the a-factor receptor results in a supersensitive phenotype that is recessive (KONOPKA, JENNESS and HARTWELL 1988; RENEKE et al. 1988), and a point mutation in the a-factor receptor that results in an increase in the basal level of FUSl RNA is also recessive (C. BOONE and G . F. SPRAGUE, personal communication). In the latter case it has been shown that hyperactivity of the receptor is recessive in the complete absence of pheromone. These results and our work are consistent with a model in which unoccupied a-factor receptor interacts directly with both the Pr complex and, in an independent fashion, with the a subunit in order to promote their reasso- ciation. In the absence of a subunit (gpal null mutations), the receptor-/3y interaction would inhibit transmission of the signal to downstream components of the pathway. In wild-type cells, binding of unoccupied receptor to the a subunit and Pr complex would facilitate formation of the heterotrimer, which would promote deactivation of the pathway. This model is supported by, preliminary evidence showing that inhibition of the pheromone response pathway by STE3 occurs upstream of STEl l , a protein kinase that acts downstream of the G protein (J. P. HIRSCH, unpublished observation).

A direct interaction between pheromone receptors and Pr subunits is also supported by recent reports suggesting that mammalian subunits interact with G protein-linked receptors (FAWZI et al. 1991; KLEUSS et al. 1992; PITCHER et al. 1992). For the P-adrenergic receptor, the Pr complex targets a specific kinase to the receptor in a ligand-independent fashion (PITCHER et al. 1992). Although the results presented did not demonstrate direct contact between the receptor and Or, future work may establish that this interaction occurs both in mammals and in yeast. If the pheromone receptors block the signal by sequestering By, the GB subunit encoded by the STE4Hp' allele must be able to bind receptor, because STE3DAF2-2 suppresses the constitutive activation phenotype of this mutation.

The biological function of inhibition of the pheromone signaling pathway by receptors that are not normally expressed in these cell types is unclear,

though several interpretations are possible. One possibility is that this function is important during mating type switching when autocrine signaling could be sig- nificant. A cell undergoing mating type switching may transiently express receptors that are inappropriate to its MAT allele, like the experimental situation described here. Inhibition of the signaling pathway in this situation would eliminate the time it takes for cells to arrest, become desensitized, and recover from arrest. Another possibility is that this function could be important during zygote formation. After the cell membranes of two mating partners fuse, a cell with both receptors on its surface is formed. At this point it may be necessary to inhibit the pheromone response to allow fusion of the nuclei and completion of the mating process. Although further work will be necessary to uncover circumstances under which receptor-mediated inhibition is active, the finding that pheromone receptors are involved in a process that is independent of their associated G, subunit demon- strates that this class of receptors may have multiple functions in cellular signaling pathways.

We thank J. KURJAN, M. JOHNSTON, S. MICHAELIS, J. THORNER, N. DAVIS and G. SPRAGUE for providing plasmids and strains used in this work. We are especially grateful to CHARLIE BOONE, NICK DAVIS and GEORGE SPRAGUE for providing many of the necessary reagents, for valuable discussions and advice, and for communication of results prior to publication. F.R.C. is a Lucille P. Markey Scholar and J.P.H. was supported by a fellowship from the Ameri- can Cancer Society. This work was supported by the Lucille P. Markey Charitable Trust and by the Rockefeller University.

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Communicating editor: D. BOTSTEIN

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