INTRODUCTION

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Cells respond to their external environment by recognizing an extracellular signal, transmitting the signal across the cell membrane, and eliciting a response through activation of the appropriate signal transduction pathway. The binding of a secreted peptide pheromone to its cell surface receptor initiates mating in the budding yeast Saccharomyces cerevisiae. Mating occurs between cells of opposite mating types; haploid yeast cells may be either a or  mating type and produce the secreted peptide pheromone a-factor or -factor, respectively (reviewed in references 20 and 33). These pheromone ligands bind to the appropriate receptor located on the surface of cells of the opposite mating type; the a-factor receptor (encoded by STE3) is present on the surface of MAT cells, and the -factor receptor (encoded by STE2) is present on the surface of MATa cells. The pheromone receptors are members of the G protein-coupled receptor family and are coupled to a heterotrimeric G protein composed of -, -, and -subunits (encoded by GPA1, STE4, and STE18, respectively). In addition to the G protein, the yeast pheromone response pathway utilizes another common eukaryotic signaling module, a mitogen-activated protein (MAP) kinase cascade (13). Transmission of the signal from the G protein complex to the downstream kinase cascade probably occurs through activation of the PAK kinase homologue Ste20p (21). Specificity of the kinases that are sequentially activated during pheromone signaling is thought to be maintained by the scaffold protein Ste5p (38). The ultimate responses to pheromone signaling include arrest in the G₁ phase of the cell cycle, which is mediated by the cyclin-dependent kinase inhibitor Far1p (3, 27, 28). Other responses to pheromone signaling include morphological changes leading to projection formation and transcriptional induction of genes involved in mating.

The differential expression of mating type-specific genes is controlled by regulatory proteins encoded by the mating-type (MAT) locus (reviewed in reference 32). Haploid MATa cells normally express Ste2p, the -factor receptor, and undergo cell cycle arrest and transcriptional induction in response to -factor stimulation. However, MATa cells containing the STE3^DAF mutation inappropriately express the a-factor receptor and exhibit resistance to pheromone-induced cell cycle arrest, a phenomenon termed receptor inhibition (14). The STE3^DAF mutation, originally named DAF2 (for dominant -factor resistance), was isolated in a screen for mutations that resulted in resistance to -factor-induced cell cycle arrest in MATa cells (7). The STE3^DAF allele contains a rearrangement in the 5' flanking region of the STE3 gene which permits expression of wild-type STE3 in all cell types (14). The abundance of STE3 RNA in cells containing STE3^DAF is comparable to the normal level of STE3 RNA in MAT cells, so the STE3^DAF phenotype is not the result of overexpression of the receptor. In addition to conferring resistance to cell cycle arrest, the STE3^DAF allele also causes an increase in the basal expression of a pheromone-inducible gene, FUS1. The increase in FUS1 basal expression is eliminated in STE3^DAF cells that contain deletions of the genes that encode a-factor. This finding demonstrates that the increase in FUS1 basal expression is caused by the presence of both a-factor and the a-factor receptor in the same cell. Thus, expression of a pheromone receptor and its ligand in the same cell causes autocrine stimulation of the pheromone signaling pathway. However, inhibition of cell cycle arrest by the STE3^DAF allele is not affected by deletion of the a-factor genes, indicating that autocrine stimulation does not play a role in this phenotype. STE3^DAF-mediated receptor inhibition can suppress the constitutive cell cycle arrest caused by deletion of GPA1, the G protein -subunit gene, and thus does not require the -subunit for function (7, 14). Expression of STE2, which encodes the -factor receptor, is not required for receptor inhibition; deletion of the STE2 gene does not affect the ability of STE3^DAF to suppress constitutive cell cycle arrest in cells containing null alleles of GPA1 (14). In addition to inhibition of cell cycle arrest, cells containing the STE3^DAF mutation exhibit a block in pheromone-mediated signaling at late time points after pheromone treatment (6). In STE3^DAF cells, initial activation of the pheromone response pathway is similar to that observed in wild-type cells, as measured by Fus3p MAP kinase activity and FUS1 RNA levels. However, at later time points after pheromone induction, STE3^DAF cells display a decrease in signaling when compared to wild-type levels (6). Furthermore, epistasis experiments suggest that STE3^DAF acts at the level of either STE5 or STE4 (6).

In this work, we have isolated mutations in the G protein -subunit gene, called STE4^SD mutations, that suppress receptor inhibition resulting from expression of STE3^DAF in MATa cells. The STE4^SD mutations encode Ste4p proteins that reverse the effects of STE3^DAF on both pheromone-mediated cell cycle arrest and transcriptional activation. The effects of the STE4^SD mutations are specific to cells undergoing receptor inhibition, suggesting that these mutations may define a region of Ste4p that is the target of the signaling block that occurs as a result of receptor inhibition.

	MATERIALS AND METHODS

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Plasmid construction. A centromeric URA3 plasmid containing STE4 was constructed by cloning the 5-kb SphI-BamHI fragment from plasmid M81p12 (5) into YCplac33 (11) to create YCpSTE4. A pUC19 plasmid containing STE4 was constructed by cloning the 5-kb SphI-BamHI fragment from plasmid M81p12 into pUC19 to create pUC-STE4.1. A centromeric LEU2 plasmid containing STE4 was constructed by cloning the 5-kb SphI-BamHI fragment from plasmid M81p12 into YCplac111 (11) to create YCpLSTE4. Centromeric LEU2 plasmids containing other STE4^SD alleles (see Table 2) were constructed by cloning the 5-kb SphI-BamHI fragment from pUC-STE4.1 plasmids that had been subjected to site-directed mutagenesis (Transformer Site-Directed Mutagenesis kit; Clontech) into YCplac111. The FUS1-lacZ reporter plasmid was constructed by cloning the 6-kb PstI fragment from pSB234 (kindly provided by E. Elion) into YCplac111 to create YCpF1-LZ.

Strains and media. The strains used in this study are listed in Table 1. The gpa1::TRP1 null allele was made by transformation of a strain that contains a gpa1::URA3 allele (10) with a 3.8-kb SmaI fragment from marker swap plasmid pUT11 (8). The FAR1 gene was disrupted by transformation with a 3.8-kb XhoI-SacI fragment from pfar1-U1 (6) to create far1::URA3. The ste2::LEU2 allele was made by transformation with a BamHI fragment from pAB506. All strain constructions involving transformations were confirmed by Southern blotting.

PCR mutagenesis and screen for STE4^SD alleles. STE4^SD alleles were isolated by cotransformation of yeast with a pool of PCR-generated mutagenized linear fragments containing STE4 and with a gapped STE4 plasmid, allowing recombination to occur in vivo as described previously (2, 24). Error-prone PCR was performed with YCpSTE4 as a template in 30 cycles of PCR with oligonucleotide primers oMSTE4.1 (5'-AAGAGTACACTAGATCCATTC-3') and oMSTE4.3 (5'-AAAGGAAGCAAATGACAATGC-3'). Error-prone incorporation of nucleotides was achieved by performing PCRs under a nucleotide imbalance (80 µM dATP, 400 µM dCTP, 400 µM dGTP, and 400 µM dTTP or 400 µM dATP, 400 µM dCTP, 80 µM dGTP, and 400 µM dTTP) and by using modified Taq polymerase buffer containing 100 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl₂, and 0.25 mM MnCl₂. Approximately 10% of the mutated fragments produced nonfunctional Ste4p proteins as determined by their ability to complement a ste4 mutation.

Yeast methods. Yeast transformations were performed by the lithium acetate method (16), modified as described previously (14). Yeast RNA was extracted from cells as described previously (9).

Northern (RNA) blots. Cells were treated with either 0.1 µM -factor (Sigma) or 40 ng of a-factor (generously provided by Fred Naider) per ml for various periods of time, and RNA was isolated. RNA was transferred to a nitrocellulose membrane after formaldehyde-agarose gel electrophoresis as described previously (22). The membranes were UV cross-linked by using a Stratalinker UV box. Prehybridization and hybridization were done at 65°C in a buffer containing 0.9 M NaCl, 0.09 M sodium citrate, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin, 33 mM sodium pyrophosphate, and 50 mM sodium phosphate monobasic. The probes used were gel-purified DNA restriction fragments ³²P-labeled by random primer labeling with a Prime-It kit (Stratagene). The fragments used were as follows: for FUS1, a 1.4-kb EcoRI-HindIII fragment from plasmid pSL589 (26); for phosphoglycerate kinase gene PGK1, a 0.5-kb BamHI-XbaI fragment from pPGK1.

	RESULTS

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The pheromone response signal transduction pathway is inhibited in MATa cells that contain the STE3^DAF allele, which causes inappropriate expression of STE3, the a-factor receptor gene (6). Receptor inhibition is specific for the late phase of the response and only occurs at times after 1 h of exposure to pheromone. Under conditions of receptor inhibition, the signaling pathway is blocked at a step upstream of MAP kinase cascade activation and at or downstream of activation of the Ste4p G subunit. These results suggest that Ste4p is a likely target for the inhibitory effect of STE3^DAF. A screen was therefore performed to identify altered versions of Ste4p that can signal normally but are insensitive to receptor inhibition.

Mutations in STE4 suppress receptor inhibition. A screen was performed to obtain mutations in STE4 that have the potential to be specific for its receptor inhibition function. This screen was designed to isolate alleles of STE4 that suppress the signaling defect of MATa cells containing the STE3^DAF allele. MATa STE3^DAF cells exposed to -factor do not arrest and do not sustain pheromone-inducible transcription. Therefore, mutations in STE4 that caused cell cycle arrest and sustained transcription to occur in a MATa STE3^DAF strain were sought. This phenotype requires that the altered versions of Ste4p retain the ability to transmit the pheromone response signal. Thus, this screen has the potential to generate mutations in STE4 that are specific to receptor inhibition.

View larger version (85K): [in this window] [in a new window]   FIG. 1.   Mutations in STE4 suppress receptor inhibition. Halo assays were performed with 5 µl of 1 mM -factor. Top left, a MATa STE3 ste4::HIS3 strain (AC17-7B) containing a wild-type STE4 plasmid (YCpLSTE4); bottom left, a MATa STE3DAF ste4::HIS3 strain (AC17-2B) containing a wild-type STE4 plasmid (YCpLSTE4); right, top to bottom, a MATa STE3DAF ste4::HIS3 strain (AC17-2B) containing plasmids with STE4SD1, STE4SD2, STESD3, and STE4SD13 alleles (YCpSD1, YCpSD2, YCpSD3, and YCpSD13), respectively.

Mutations conferring supersensitivity enhance the STE4^SD phenotype. To determine whether the STE4^SD alleles cause increased signaling in wild-type cells, plasmids containing each of the three alleles were transformed into MATa ste4 cells that did not contain the STE3^DAF mutation. Cells carrying each of the STE4^SD alleles displayed increased sensitivity to -factor, as shown by production of halos larger than those produced by cells containing a wild-type STE4 plasmid (Table 2). Therefore, some component of the suppression of STE3^DAF by the STE4^SD mutations is probably due to an increase in the signaling capacity of the encoded Ste4p proteins.

STE4^SD-encoded mutations map to the N terminus and second WD repeat. The crystal structure of mammalian G protein -subunits has revealed that their seven WD domains fold into a symmetric structure in the form of a seven-bladed -propeller (31, 35). The yeast Ste4p -subunit also has seven WD domains that are expected to form a similar structure. Mutations in STE4 that suppress the STE3^DAF phenotype were positioned on a diagram of a -subunit that is based on the crystal structure (Fig. 2). The Q17L and Q21R mutations map to the extreme N terminus of Ste4p, which is composed of a 30-amino-acid extension that is present only in the yeast protein and thus is not represented in the crystal structure. The R162G mutation maps to the turn between the third and fourth strands of the -sheet of the second blade, at the end of WD repeat 2. This residue is not in either of the two regions of the -subunit that are in direct contact with the -subunit (19, 35). The region of the -subunit that makes the most extensive contacts with the -subunit is the base of the -propeller domain (Fig. 2, surface facing away from viewer). Mutations that alter amino acids on this surface, such as changes at residue 136, disrupt the interaction between the - and -subunits and result in constitutive signaling (36). The other region of contact between the two subunits is along the sides of the first and seventh blades of the -propeller. A mutation that changes the amino acid at position 124, which is in the first blade, also disrupts the interaction between the - and -subunits and results in constitutive signaling (36). The finding that the changes encoded by the STE4^SD13 allele are not in the regions of the -subunit that contact the -subunit suggests that the altered residues could constitute part of a binding site for another protein that interacts with the -subunit.

View larger version (33K): [in this window] [in a new window]   FIG. 2.   Location of mutations on G structure. The diagram is a schematic drawing representing the complete Ste4p sequence of 423 amino acids as a seven-bladed -propeller structure. The product of the STE4SD13 allele contains the Q17L and Q21R mutations encoded by STE4SD2 and the R162G mutation encoded by STE4SD1, all of which are represented by filled diamonds in the diagram. Mutations that disrupt the interaction between the - and -subunits and result in constitutive signaling (36) are represented as grey circles. The amino acid sequence shows amino acids 1 to 162 of Ste4p.

STE4^SD13 promotes sustained transcriptional activation in STE3^DAF cells. Expression of STE3 in MATa cells inhibits signaling during the late phase of the pheromone response (6). It was therefore of interest to determine whether the STE4^SD13 allele specifically affects the late phase of the response or whether it affects signaling at other times. To assess the duration of signaling in cells containing STE4^SD13, a time course of RNA accumulation from the pheromone-inducible FUS1 gene was performed. The strains used in this experiment contain deletions of the MFA1 and MFA2 genes, which encode a-factor, to eliminate any contribution to signaling due to autocrine stimulation of the a-factor receptor in MATa cells.

View larger version (47K): [in this window] [in a new window]   FIG. 3.   Effect of STE4SD13 on pheromone-induced transcription. (A) The following strains were treated with -factor (0.1 µM) for the indicated periods of time: a MATa STE3 ste4::HIS3 strain (AC17-7B) containing a wild-type STE4 plasmid (YCpLSTE4) (lanes 1 to 4) and a MATa STE3DAF ste4::HIS3 strain (AC17-2B) containing either a wild-type STE4 plasmid (YCpLSTE4) (lanes 5 to 8) or a plasmid containing the STE4SD13 allele (YCpLSD13) (lanes 9 to 12). RNA was isolated, transferred to nitrocellulose, and hybridized with a FUS1 probe. The blot was rehybridized with PGK1 to determine the amount of RNA per lane. (B) The data were quantified by PhosphorImager analysis, and the level of FUS1 RNA was normalized to the control PGK1 RNA level. Values from the STE3 STE4 strain are represented by black bars; values from the STE3DAF STE4 strain are represented by open bars; values from the STE3DAF STE4SD13 strain are represented by grey bars. The graph shows the average values from duplicate experiments.

STE4^SD13 has no effect in wild-type cells. If the STE4^SD mutations only affect the receptor inhibition function of STE4, then they should have no effect in MATa cells that do not express the a-factor receptor. This idea was tested by expressing STE4^SD13 as the only copy of STE4 in a wild-type strain and assaying its ability to respond to pheromone. Accumulation of FUS1 RNA in cells treated with -factor was very similar at all time points in MATa cells expressing either STE4^SD13 (Fig. 4A, lanes 5 to 8) or STE4 (Fig. 4A, lanes 1 to 4). Quantification of these results showed that the levels of FUS1 RNA differed by less than 20% in MATa cells expressing STE4^SD13 and STE4, even after 3 h of pheromone treatment (Fig. 4B). These experiments demonstrate that the STE4^SD13 allele is capable of transmitting the pheromone signal in a manner indistinguishable from that of wild-type STE4 in MATa cells that do not express STE3.

View larger version (57K): [in this window] [in a new window]   FIG. 4.   Ability of STE4SD13 to signal in wild-type MATa and MAT cells. (A) A MATa STE3 ste4::HIS3 strain (AC17-7B) containing either a wild-type STE4 plasmid (YCpLSTE4) (lanes 1 to 4) or a plasmid containing the STE4SD13 allele (YCpLSD13) (lanes 5 to 8) was treated with -factor (0.1 µM) for the indicated periods of time, and RNA was isolated. RNA blots were prepared and hybridized as described in the legend to Fig. 3. (B) The data from the experiment shown in panel A were quantified by PhosphorImager analysis, and the level of FUS1 RNA was normalized to the control PGK1 RNA level. Values from the STE4 strain are represented by black bars; values from the STE4SD13 strain are represented by grey bars. (C) A MAT STE3 ste4::HIS3 strain (AC18-9C) containing either a wild-type STE4 plasmid (YCpLSTE4) (lanes 1 to 4) or a plasmid containing the STE4SD13 allele (YCpLSD13) (lanes 5 to 8) was treated with a-factor (40 ng/ml) for the indicated periods of time, and RNA was isolated. RNA blots were prepared and hybridized as described in the legend to Fig. 3. (D) The data from the experiment shown in panel C were quantified by PhosphorImager analysis, and the level of FUS1 RNA was normalized to the control PGK1 RNA level. Values from the STE4 strain are represented by black bars; values from the STE4SD13 strain are represented by grey bars.

MAT

MAT STE4

MAT STE4^SD13

The STE4^SD13 phenotype is independent of GPA1 and is specific to MATa cells. Deletion of GPA1, which encodes the G subunit that functions in the pheromone response pathway, causes constitutive signaling due to free -subunits. Therefore, cells containing a gpa1 mutation undergo cell cycle arrest and induction of FUS1 expression. The constitutive signaling seen in a MATa gpa1 strain is blocked by the STE3^DAF allele, demonstrating that receptor inhibition is independent of GPA1 (7, 14). This result supports the idea that inappropriate STE3 expression inhibits a step that is downstream of -subunit activation. If the STE4^SD13 allele produces a -subunit that is only partially inhibited by expression of STE3, then the STE4^SD13 allele should increase signaling in a MATa gpa1 strain that expresses STE3. To test this idea, the level of FUS1 RNA was determined in the absence of pheromone in a MATa gpa1 STE3^DAF strain expressing either STE4^SD13 or wild-type STE4. A MATa gpa1 STE3^DAF strain does not undergo cell cycle arrest due to inhibition of signaling by the STE3^DAF allele; however, to prevent the possibility of cell cycle arrest under conditions where STE3^DAF is suppressed by STE4^SD13, this strain was constructed to contain a far1 mutation. The FAR1 gene encodes a cyclin-dependent kinase inhibitor that is required for cell cycle arrest in response to pheromone (3, 27, 28).

View larger version (38K): [in this window] [in a new window]   FIG. 5.   Effect of STE4SD13 in cells lacking the G and -factor receptor genes. (A) RNA was isolated from the following strains: a MAT STE3DAF ste4::HIS3 gpa1::TRP1 far1::URA3 strain (K39-23D.f) containing either a wild-type STE4 plasmid (YCpLSTE4) (lane 1) or a plasmid containing the STE4SD13 allele (YCpLSD13) (lane 2), and a MATa STE3DAF ste4::HIS3 gpa1::TRP1 far1::URA3 strain (K39-23B.f) containing either a wild-type STE4 plasmid (YCpLSTE4) (lane 3) or a plasmid containing the STE4SD13 allele (YCpLSD13) (lane 4). (B) RNA was isolated from the following strains: a MATa STE3DAF ste4::HIS3 gpa1::TRP1 strain (K39-23B) containing either a wild-type STE4 plasmid (YCpSTE4) (lane 1) or a plasmid containing the STE4SD13 allele (YCpSD13) (lane 2), and a MATa STEDAF ste4::HIS3 gpa1::TRP1 ste2::LEU2 strain (K39-23B.s2) containing either a wild-type STE4 plasmid (YCpSTE4) (lane 3) or a plasmid containing the STE4SD13 allele (YCpSD13) (lane 4). RNA blots were prepared and hybridized as described in the legend to Fig. 3. The data were quantified by PhosphorImager analysis, and the level of FUS1 RNA was normalized to the control PGK1 RNA level. The relative level of FUS1 RNA is shown below each lane.

MAT gpa1 STE3^DAF

MAT gpa1 STE3^DAF

	DISCUSSION

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The STE4^SD mutations were isolated based on their ability to restore pheromone-induced cell cycle arrest in MATa cells expressing STE3. The following evidence confirms the idea that the STE4^SD13 allele is specific for the receptor inhibition function of STE4. First, expression of STE3^DAF in MATa cells inhibits signaling only at late times during the response, and STE4^SD13 causes an increase in FUS1 RNA levels only at late times after pheromone treatment in MATa STE3^DAF cells. Second, STE4^SD13 does not cause an increase in either the basal or induced level of FUS1 RNA in wild-type MATa or MAT cells. Third, the phenotype conferred by STE4^SD13 is independent of GPA1, the G subunit gene. And, fourth, the effect of STE4^SD13 is cell type specific. These findings rule out the possibility that the Ste4p protein encoded by STE4^SD13 increases signaling by a mechanism that is unrelated to receptor inhibition, such as that it has an increased affinity for a downstream activator of the signaling pathway. Isolation of an allele of STE4 that is specific for suppression of receptor inhibition confirms that this phenomenon is an active cellular process that plays a physiological role in some aspect of the yeast life cycle.

G protein -subunits have been shown to interact with a wide variety of other proteins (4). Structural and functional studies of these interactions demonstrate that the binding surfaces for -subunits and downstream effectors are partially overlapping, suggesting that these interactions are mutually exclusive. The Ste4p -subunit has been shown to interact with the Gpa1 -subunit (36) and with the potential downstream effectors Ste20p (21) and Ste5p (15, 37). The binding surfaces on Ste4p that mediate these interactions are probably not altered by the mutations present in STE4^SD13 because the protein encoded by this allele behaves identically to wild-type Ste4p when it is expressed in wild-type cells. It is therefore likely that the mutations present in STE4^SD13 identify a binding surface for a novel -subunit binding partner. Two of the mutations in STE4^SD13 change residues in the N-terminal extension of Ste4p that is not present in the mammalian -subunits that have been crystalized; the other mutation changes an amino acid that faces outward at the turn between the third and fourth strands of the second blade of the propeller. Because the structure of the Ste4p N terminus is unknown, the two regions of Ste4p identified by the STE4^SD13 mutations could be quite close together, thus forming a unique binding surface for a protein that has not yet been identified.

STE4 alleles that confer prolonged signaling resulting in a defect in adaptation to pheromone have been isolated by Li et al. (23). These alleles contain mutations in the first, second, and seventh WD repeats of Ste4p. The mutations present in the STE4 Adp alleles have been proposed to identify a target site for a negative regulator other than the Gpa1p -subunit. The STE4 Adp alleles, like the STE4^SD alleles described here, are thought to confer an increase in signaling at late times during the pheromone response. However, it appears that the STE4 Adp and STE4^SD alleles do not affect the same process, for the following reasons. First, whereas the effects of STE4 Adp alleles are observed in wild-type MATa cells, the effects of the STE4^SD13 allele are only observed in MATa cells containing a STE3^DAF mutation. Second, whereas all of the STE4 Adp alleles confer an increase in the basal and induced levels of signaling, resulting in supersensitivity to pheromone, the STE4^SD13 allele confers normal basal and induced levels of signaling in wild-type cells. And finally, the effects of STE4 Adp alleles are dependent on a process initiated by Gpa1p, but the effects of the STE4^SD13 allele are independent of Gpa1p function.

Some evidence suggests that -subunits have the potential to bind directly to their associated receptors in the absence of an -subunit. For example, fluorescence energy transfer experiments have demonstrated potential interactions between -subunits and the -adrenergic receptor (12) and between -subunits and rhodopsin (29). In addition, one study has demonstrated direct photoaffinity labeling of a -subunit by a peptide derived from the third cytoplasmic loop of the -adrenergic receptor (34). Direct binding of the Ste4p -subunit to the a-factor receptor may play a role in receptor inhibition; however, a model in which inhibition of signaling is caused by binding of Ste4p to Ste3p does not account for the observation that Ste3p only inhibits signaling in MATa cells. One explanation for the cell type specificity of receptor inhibition is that a component required for this process is expressed only in MATa cells, as shown in the model presented in Fig. 6 and described below.

View larger version (18K): [in this window] [in a new window]   FIG. 6.   Model for receptor inhibition. See text for details.

The results presented here are consistent with a model in which the activity of the -subunit complex is inhibited by a MATa cell type-specific regulatory protein (factor R) that binds to or is activated by Ste3p, the a-factor receptor (Fig. 6). The observation that STE3^DAF only affects signaling at late times after pheromone treatment could be due to the fact that factor R is induced by pheromone. Thus, factor R would not be present before pheromone treatment but would gradually increase in abundance during the time course of the response. In a wild-type MATa cell, the presence of factor R would have no effect because Ste3p is not expressed (Fig. 6, a cell). In a MATa STE3^DAF cell, detection of Ste3p by factor R would produce a change in factor R that would cause it (or another factor activated by it) to block signaling by the -subunits (Fig. 6, a STE3^DAF). In the normal life cycle, factor R and Ste3p would be present in the same cell immediately after the fusion of a and  haploid cells that are in the process of mating (Fig. 6, early zygote) because factor R is an a-specific gene product and Ste3p is an -specific gene product. Detection of Ste3p by factor R in early zygotes would result in inhibition of pheromone signaling by the same mechanism as that seen in MATa STE3^DAF cells. Factor R could function by directly binding to the complex and preventing the activation of downstream effectors or it could affect complex activity by an indirect mechanism, such as altering its association with the plasma membrane. In this model, the effect of the STE4^SD mutations would be to reduce the affinity of Ste4p for factor R, causing less Ste4p to interact with factor R. Thus, more Ste4p would be available to activate the pheromone response pathway, resulting in a higher signal. Preliminary studies on a newly identified a-specific gene suggest that its product is a good candidate for factor R (18).

The putative regulatory factor is expected to be specific to MATa cells because inhibition of signaling by STE3 occurs in a MATa gpa1 strain but does not occur in a MAT gpa1 strain. However, factor R cannot be the -factor receptor, which is specific to MATa cells, because a null allele of STE2 does not affect inhibition of signaling by STE3. Previous studies by Bender and Sprague support the concept of a novel inhibitor of mating that is active in MATa cells that express STE3 (1). These studies showed that expression of a given combination of receptor and pheromone had different effects depending on the MAT allele of the cell in which they were expressed. The experiment was performed by expressing STE3 and MF1 from mating type-independent promoters in either mat1 cells or MATa ste2 ste6 cells. Both of these strains are designed to express only -factor and the a-factor receptor (in the MATa strain, the ste2 mutation eliminates expression of the -factor receptor and the ste6 mutation prevents secretion of a-factor). However, the mat1 strain mated with 10-fold greater efficiency than the MATa strain did. The only difference between the two strains is that a-specific genes are not expressed in mat1 cells due to the presence of the Mat2p inhibitor. Therefore, this result supports the existence of an a-specific component that inhibits mating when STE3 is expressed. The a-specific component cannot be Ste2p and therefore is likely to be our proposed factor R.

MATa cells do not express STE3 at any time during their normal life cycle, so the function of this putative regulatory factor is probably not relevant to vegetative haploid growth. However, a potential physiological function of this regulatory factor is to inhibit signaling in mating cells that have recently undergone cell fusion. Fusion of cells would allow factor R to come into contact with Ste3p, which would block the signaling function of Ste4p. This process may function to promote recovery from mating and allow cell cycle progression to resume. Our attempts to demonstrate an effect of the STE4^SD13 allele on recovery from mating have been hampered by an inability to obtain cultures that undergo synchronous mating. It is difficult to observe short-term effects in unsynchronized mating mixtures because recovery from mating is aided by the long-term process of transcriptional inhibition of haploid-specific genes by the Mata1p/Mat2p complex. Further experiments in which cell fusion and recovery can be precisely controlled will allow definitive testing of the model of receptor inhibition.