INTRODUCTION

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The pheromone response of the yeast Saccharomyces cerevisiae is initiated by the binding of a peptide pheromone to its specific receptor on a responding cell. Haploid yeast cells containing the MATa allele secrete a-factor and express receptors for -factor; cells containing the MAT allele secrete -factor and express receptors for a-factor (19, 33, 36). The a- and -factor receptors are G protein-coupled receptors, and they activate a heterotrimeric G protein that is common to both cell types. Activation of the G protein by occupied receptors results in guanine nucleotide exchange on the G protein  subunit, which causes the  and  subunits to be released from the  subunit. The free complex interacts with downstream components of the pathway, resulting in activation of a mitogen-activated protein (MAP) kinase cascade. Signaling through this pathway produces a number of changes in cellular physiology, including arrest in the G₁ phase of the cell cycle, induction of gene expression, redistribution of cell surface proteins, and formation of cellular projections.

In addition to the classical G protein activation process described above, the pheromone response pathway is subject to a process that inhibits G protein signaling. This process, termed receptor inhibition, was uncovered by a mutation that causes the a-factor receptor to be inappropriately expressed in MATa cells. The STE3^DAF mutation was isolated in a screen for dominant mutations that conferred resistance to pheromone-induced cell cycle arrest (4). STE3^DAF, which is an allele of the a-factor receptor gene STE3, contains a rearranged 5' regulatory region that causes it to be expressed in MATa cells (13). Expression of STE3 in MATa cells confers resistance to pheromone-induced cell cycle arrest by blocking signaling through the pheromone response pathway. Although MATa haploid cells do not normally express the -specific gene STE3, a- and -specific gene products do come into contact with each other immediately after the fusion of a MATa cell and a MAT cell during the process of mating. Therefore, a potential physiological function of receptor inhibition is to inhibit signaling in mating cells that have recently undergone cell fusion. This process may function to promote recovery from mating and allow cell cycle progression to resume.

Cells that contain a STE3^DAF allele display a characteristic pattern of signaling. In STE3^DAF cells treated with pheromone, activation of the MAP kinase is normal early in the response but is gradually inhibited at later time points (3). This pattern suggests that prior activation of the signaling pathway must occur before the inhibitory process can function. Another characteristic of receptor inhibition is that it is independent of the G protein  subunit (4, 13). In the pheromone response pathway, the  subunit plays a negative role by sequestering the complex and keeping it inactive. Deletion of GPA1, the G protein  subunit gene, causes constitutive signaling due to release of the complex. The constitutive signaling phenotype of cells with a GPA1 deletion is suppressed by the STE3^DAF mutation, indicating that receptor inhibition of signaling can occur in the absence of Gpa1p. This result demonstrates that the target of receptor inhibition is a signaling component that is downstream of the G protein  subunit.

Two types of evidence suggest that the G protein  subunit, Ste4p, is the signaling component that is targeted by receptor inhibition. Initial results supporting this idea involve the phenotypes of double mutants that carry both STE3^DAF and a constitutive signaling mutation. The STE3^DAF allele blocks signaling in cells that contain constitutive or overexpression alleles of STE4 (3, 13). However, STE3^DAF does not block signaling in cells that overexpress STE20 or that contain a constitutive allele of STE5 (3). STE20 encodes a kinase that activates the MAP kinase cascade, and STE5 encodes a scaffolding protein for the MAP kinase cascade, so both of these signaling components act downstream of Ste4p. These studies are therefore consistent with the idea that receptor inhibition acts at the level of Ste4p, the G protein  subunit. This conclusion was further supported by the isolation of altered versions of Ste4p that can signal normally but that are insensitive to receptor inhibition (17). The altered residues in Ste4p are not in the regions that contact the  subunit, suggesting that they constitute part of a binding site for another protein that interacts with Ste4p. Such a protein could be a negative regulator that prevents Ste4p from interacting with its downstream targets. One potential explanation for the phenotype of the STE3^DAF allele is that a negative regulator of Ste4p is expressed only in MATa cells.

Receptor inhibition appears to require an a-specific component based on the following observation. Deletion of GPA1 causes constitutive signaling in both MATa and MAT cells due to release of the complex. Expression of STE3 inhibits the constitutive signaling conferred by a GPA1 deletion in MATa cells but has no effect in MAT cells (17). One important difference between MATa and MAT cells is that Ste2p, the -factor receptor, is expressed only in MATa cells. However, receptor inhibition does not require the presence of Ste2p because the ability of STE3^DAF to block signaling in MATa gpa1 cells is unaffected by a ste2 mutation (17). These findings indicate that an a-specific component other than Ste2p is required for inhibition of -subunit activity by the a-factor receptor.

Here we investigate further the process by which Ste4p signaling is blocked by receptor inhibition. Examination of the subcellular location of Ste4p indicates that it undergoes altered localization during receptor inhibition. In addition, we show that altered localization of Ste4p and inhibition of signaling require an a-specific gene called ASG7.

	MATERIALS AND METHODS

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Plasmid construction. Plasmid YCp36 was constructed by replacing the 392-bp HpaI-XhoI fragment in plasmid YCpSTE4 (17) with a 284-bp HpaI-XhoI fragment from a plasmid containing the STE4 gene in which the 108 bp encoding amino acids 310 to 346 of Ste4p had been deleted by site-directed mutagenesis.

Strains and media. Strains used in this study are listed in Table 1. The asg7::URA3 null allele was made by transformation of strains with a 1.8-kb fragment generated by two-step PCR, as described above. The gpa1::TRP1 allele was created by transformation of a strain containing the gpa1::URA3 allele (7) with a 3.2-kb EcoRI-XhoI fragment from plasmid pTU10 (5). The far1::LEU2 allele was created by transformation of a strain containing the far1::URA3 allele (3) with a 4.6-kb SmaI fragment from plasmid pUL9 (5). The asg7::HIS3 allele was created by transformation of a strain containing the asg7::URA3 allele with a 3.6-kb XbaI fragment from plasmid pUH7 (5).

Yeast methods. Yeast transformations were performed by the lithium acetate method (15) modified as described previously (13). Yeast RNA was extracted from cells as described previously (6).

Northern blots. Cells were treated with 0.1 µM -factor (Sigma), 60 nM a-factor, or 300 nM a-factor (generously provided by Fred Naider) for various periods of time, and RNA was isolated. RNA was transferred to a nitrocellulose membrane after formaldehyde-agarose gel electrophoresis as described previously (31). The membranes were UV cross-linked 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 using a Prime-It kit (Stratagene). The fragments used were FUS1, a 1.4-kb EcoRI-HindIII fragment from plasmid pSL589 (25), phosphoglycerate kinase gene PGK1, a 0.5-kb BamHI-XbaI fragment from pPGK1, and ASG7, a 0.75-kb HpaI-SacI fragment from pASG7-351.2.

Immunoblots. For immunoblots, cells were treated with 0.1 µM -factor (Sigma) for various periods of time, and 10-ml aliquots of cells were pelleted and washed once with 10 mM Tris-HCl (pH 7.8)-1 mM EDTA. The washed cells were resuspended in 350 µl of lysis buffer (50 mM Tris-HCl [pH 8.0], 1% sodium dodecyl sulfate [SDS], 1 mM phenylmethylsulfonyl fluoride, and 1 µg each of leupeptin, aprotinin, chymostatin, and pepstatin/ml). The cell suspension was lysed by adding approximately 0.25 ml of acid-washed glass beads (0.5 mm; Biospec Products) and vortexing at high speed for 10 min at 4°C. The lysate was cleared by centrifuging for 2 min at 4°C. The protein concentrations of the samples were determined using a bicinchoninic protein assay kit (Pierce), and equal amounts of protein were loaded onto SDS-polyacrylamide gels (10% acrylamide). Separated proteins were transferred to nitrocellulose, and the blot was probed with anti-Ste4p rabbit antiserum (14) at a dilution of 1:1,000. Donkey anti-rabbit immunoglobulin conjugated to horseradish peroxidase (Amersham) was used at a dilution of 1:10,000, and immune complexes were detected with an enhanced chemiluminescence kit (Amersham).

Microscopy. For fluorescence microscopy, cells containing GFP-Ste4p or Asg7p-GFP fusion proteins were observed on a Zeiss Axioskop microscope with a ×100 (1.3-numerical-aperture) objective and a fluorescein isothiocyanate (FITC) filter set (Chroma Technology). Digital images were captured with a Photometrics SenSys 1400-C1.cCCD camera using IPLab Spectrum image acquisition software (Scanalytics).

	RESULTS

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Expression of the a-factor receptor in MATa cells inhibits signal transduction through the pheromone response pathway by a process called receptor inhibition. In cells undergoing receptor inhibition, the signaling pathway is blocked at the level of Ste4p, the G protein  subunit (3, 17). To investigate the mechanism responsible for the signaling block, the effect of receptor inhibition on properties of Ste4p that are required for signaling was examined.

Expression of the a-factor receptor in a cells affects Ste4p localization. Previous studies have shown that association of the complex with the plasma membrane is required for activation of the signaling pathway (29). Therefore, one mechanism to account for signaling inhibition in the presence of the a-factor receptor is that the Ste4p  subunit is not properly localized to the plasma membrane. To investigate this possibility, the localization pattern of a GFP-Ste4p fusion protein was observed in wild-type MATa cells and STE3^DAF MATa cells that were treated with pheromone for various amounts of time. STE3 encodes the a-factor receptor, and the STE3^DAF allele causes expression of STE3 in MATa cells due to an insertion in its promoter region (13). To eliminate any effects of autocrine signaling through the a-factor receptor, both the wild-type and STE3^DAF strains used in these experiments have deletions of the genes encoding a-factor (13).

View larger version (59K): [in this window] [in a new window]   FIG. 1.   Localization of GFP-Ste4p in wild-type and STE3DAF cells. The following strains were treated with -factor (0.1 µM) for the indicated periods of time: AC17-7B, a MATa STE3 ste4::HIS3 strain (STE3), and AC17-2B, a MATa STE3DAF ste4::HIS3 strain (STE3DAF). Both strains contained the low-copy-number GFP-STE4 plasmid BTL49. The live cells were viewed by fluorescence microscopy using an FITC filter set.

Expression of the a-factor receptor in a cells does not affect Ste4p abundance. In cells undergoing receptor inhibition, the localization pattern of Ste4p resembled that of an endocytic compartment (Fig. 1, STE3^DAF, 2 h). If receptor inhibition causes Ste4p to undergo endocytosis, it is possible that the level of Ste4p would decrease under these conditions. To determine the effect of receptor inhibition on Ste4p abundance, the levels of Ste4p in wild-type MATa cells and STE3^DAF MATa cells were investigated.

310-346

310-346

310-346

310-346

View larger version (41K): [in this window] [in a new window]   FIG. 2.   Abundances of Ste4p and Ste4p310-346 in wild-type and STE3DAF cells. (A) The following strains were treated with -factor (0.1 µM) for the indicated periods of time: H67-9D.Ba, a MATa STE3 strain (lanes 1 to 4), H67-6C.Ba, a MATa STE3DAF strain (lanes 5 to 8), and AC17-7B, a MATa STE3 ste4::HIS3 strain (lane 9). Cell extracts were prepared, and immunoblots containing these extracts were probed with anti-Ste4p polyclonal antibody. (B) The following strains were treated with -factor (0.1 µM) for the indicated periods of time: AC17-7B, a MATa STE3 ste4::HIS3 strain carrying plasmid YCp36, which contains the STE4310-346 allele (lanes 1 to 4), and AC17-2B, a MATa STE3DAF ste4::HIS3 strain carrying plasmid YCp36 (lanes 5 to 8). Cell extracts were prepared, and immunoblots containing these extracts were probed with an anti-Ste4p polyclonal antibody.

An altered version of Ste4p localizes normally in cells expressing the a-factor receptor. STE4^SD alleles are mutated versions of the STE4 gene that produce Ste4p variants that are largely insensitive to inhibition by the a-factor receptor (17). In wild-type MATa cells, the Ste4p^SD10 variant signals in a manner indistinguishable from that of wild-type Ste4p. In STE3^DAF MATa cells, Ste4p^SD10 produces a much greater signal than wild-type Ste4p at late time points after -factor treatment. To test whether signaling correlates with localization of Ste4p at the plasma membrane, the localization pattern of Ste4p^SD10 was investigated in MATa cells expressing the a-factor receptor. In STE3^DAF cells treated with -factor for 2 or 3 h, GFP-Ste4p was localized in an internal particulate pattern (Fig. 3, Ste4p). In contrast, a large proportion of the GFP-Ste4p^SD10 signal was localized at the membrane at regions of mating projections (Fig. 3, Ste4p^SD). Therefore, the increased signaling conferred by the Ste4p^SD10 variant correlates with increased membrane localization. These results suggest that inhibition of signaling by the a-factor receptor is effected by localization of Ste4p away from the plasma membrane.

View larger version (69K): [in this window] [in a new window]   FIG. 3.   Localization of GFP-Ste4p and GFP-Ste4pSD10 in STE3DAF cells. Strain AC17-2B (MATa STE3DAF ste4::HIS3) containing a GFP-STE4 plasmid (pBTL49) or a GFP-STE4SD10 plasmid (YCpGFP-SD10) was treated with -factor (0.1 µM) for the indicated periods of time. The live cells were viewed by fluorescence microscopy using an FITC filter set.

ASG7 is required for inhibition of signaling by the a-factor receptor. Previous results indicate that receptor inhibition requires a component that is only present in MATa cells. However, it has been shown that inhibition of signaling does not require any of the known a-specific genes that might be expected to be involved in this process, including STE2 (a-factor receptor gene), MFA1 and MFA2 (a-factor genes), SST1 (-factor protease gene), and STE6 (a-factor transporter gene) (13; A. Couve and J. P. Hirsch, unpublished data). A comprehensive approach to identify a-specific genes was recently performed by screening the Saccharomyces Genome Database for Mat2p-Mcm1p binding sites in the 5' flanking regions of open reading frames (38). The Mat2p-Mcm1p complex functions to repress transcription in MAT cells, and thus these binding sites identify genes that are only expressed in MATa cells. A previously uncharacterized a-specific gene, ASG7, was identified by the screen. The requirement for ASG7 in receptor inhibition was tested by deleting the ASG7 gene in MATa cells containing a STE3^DAF allele.

View larger version (64K): [in this window] [in a new window]   FIG. 4.   Effect of ASG7 on signaling in STE3DAF cells. (A) Halo assays were performed with 5 µl of 1 mM -factor using the following strains (from left to right): H67-9D.Ba, a MATa STE3 ASG7 strain, H67.9D.a7, a MATa STE3 asg7::URA3 strain, H67-6C.Ba, a MATa STE3DAF ASG7 strain, and H67-6C.a7, a MATa STE3DAF asg7::URA3 strain. (B) The following strains were treated with -factor (0.1 µM) for the indicated periods of time: H67-9D.Ba, a MATa STE3 ASG7 strain (lanes 1 to 4), H67-9D.a7, a MATa STE3 asg7::URA3 strain (lanes 5 to 8), H67-6C.Ba, a MATa STE3DAF ASG7 strain (lanes 9 to 12), and H67-6C.a7, a MATa STE3DAF asg7::URA3 strain (lanes 13 to 16). RNA was isolated, transferred to nitrocellulose, hybridized with a FUS1 probe, and rehybridized with a PGK1 probe. (C) MATa STE3DAF gpa1::TRP1 ASG7 (H125-7D) and MATa STE3DAF gpa1::TRP1 asg7::HIS3 (H125-7D.aH) strains containing a plasmid with GPA1 under the control of the GAL promoter were streaked onto galactose- or glucose-containing plates and grown at 30°C for 2 to 3 days.

Expression of ASG7 is a specific and pheromone inducible. Previous results have shown that expression of ASG7 RNA is a specific (38). To investigate further the regulation of ASG7, a time course of RNA expression was performed in both MATa cells treated with -factor and MAT cells treated with a-factor. In these experiments, ASG7 RNA was not detectable in untreated MATa or MAT cells (Fig. 5, lanes 1 and 5). Treatment of MATa cells with -factor for 1 h caused a large increase in the abundance of ASG7 RNA, and this increase was maintained for 3 h (Fig. 5, lanes 2 to 4). Treatment of MAT cells with a-factor had no effect on the expression of ASG7 RNA, although FUS1 RNA was induced normally (Fig. 5, lanes 6 to 8). These results demonstrate that ASG7 RNA expression is completely a specific and that it is pheromone inducible to a large degree.

View larger version (70K): [in this window] [in a new window]   FIG. 5.   Abundance of ASG7 RNA in MATa and MAT cells treated with pheromone. Strain W3031A.Ba was treated with -factor (0.1 µM) for the indicated periods of time (lanes 1 to 4); strain W3031B was treated with a-factor (60 nM) for the indicated periods of time (lanes 5 to 8). RNA was isolated, transferred to nitrocellulose, hybridized with an ASG7 or FUS1 probe, and rehybridized with a PGK1 probe.

The a-factor receptor inhibits signaling in its liganded state. In the experiments described above, the a-factor receptor is not occupied by a ligand because the strains used have deletions of the genes encoding a-factor. Therefore, it was not known whether the a-factor receptor could function in receptor inhibition in its liganded state. To test this idea, the effect of the ASG7 gene on signaling that originates from the liganded a-factor receptor was investigated. Thus, this experiment tests whether the a-factor receptor can carry out its two independent functions, signal transduction and receptor inhibition, in the same cell.

View larger version (63K): [in this window] [in a new window]   FIG. 6.   Effect of ASG7 on signaling in STE3DAF cells treated with a-factor. The following strains were treated with a-factor (300 nM) for the indicated periods of time: H67-6C.Ba, a MATa STE3DAF ASG7 strain (lanes 1 to 4), and H67-6C.a7, a MATa STE3DAF asg7::URA3 strain (lanes 5 to 8). RNA was isolated, transferred to nitrocellulose, hybridized with a FUS1 probe, and rehybridized with a PGK1 probe.

ASG7 affects Ste4p localization. The results described above show that receptor inhibition results in altered localization of the Ste4p  subunit. In addition, they show that ASG7 is required for inhibition of signaling by the a-factor receptor. It was therefore of interest to determine whether deletion of the ASG7 gene would have an effect on the subcellular location of Ste4p. To investigate this possibility, the localization pattern of a GFP-Ste4p fusion protein in MATa STE3^DAF cells that contained the wild-type ASG7 gene or an asg7 mutation was observed. The cells were treated with -factor for 2 h to maximize the difference between signaling and nonsignaling cells (Fig. 2). GFP-Ste4p was localized in an internal particulate pattern in STE3^DAF cells that contained the wild-type ASG7 gene (Fig. 7). In contrast, GFP-Ste4p was localized predominantly at the cell membrane in STE3^DAF cells that contained the asg7 mutation. These results demonstrate that, in addition to its effect on signaling, ASG7 has an effect on Ste4p localization in cells that express the a-factor receptor. Moreover, localization of Ste4p to the cell membrane correlated with activation of the signaling pathway in all experiments, as would be expected for a protein that transmits a signal to membrane-associated target proteins.

View larger version (58K): [in this window] [in a new window]   FIG. 7.   Effect of ASG7 on localization of GFP-Ste4p in STE3DAF cells. The following strains were treated with -factor (0.1 µM) for 2 h: AC17-2B, a MATa STE3DAF ste4::HIS3 ASG7 strain (ASG7), and AC17-2B.aL, a MATa STE3DAF ste4::HIS3 asg7::LEU2 strain (asg7). Both strains contained the low-copy-number STE4-GFP plasmid pBTL49. The live cells were viewed by fluorescence microscopy using an FITC filter set.

Asg7p localization is consistent with a direct effect of Asg7p on Ste4p. The effect of Asg7p on Ste4p localization could occur through an indirect mechanism, such as transcriptional activation of other genes, or through a more direct mechanism, such as binding of Ste4p to a complex containing Asg7p. To determine whether the location of Asg7p in the cell is consistent with a direct effect on Ste4p, a fully functional ASG7-GFP fusion construct was expressed in STE3^DAF cells containing an asg7 mutation. In cells treated with -factor for 2 or 3 h, Asg7p-GFP was localized in an internal particulate pattern (Fig. 8). Asg7p-GFP was observed in this pattern as soon as the signal became visible, at about 1 h after -factor treatment, and the pattern was the same in both STE3^DAF and wild-type cells (E. Bortz and J. P. Hirsch, unpublished data). The subcellular location of Asg7p is similar to that of Ste4p during receptor inhibition. Therefore, these results are consistent with Asg7p having a direct effect on Ste4p localization.

View larger version (51K): [in this window] [in a new window]   FIG. 8.   Localization of Asg7p in STE3DAF cells. Strain H67-6C.a7 (MATa STE3DAF asg7::URA3) containing an ASG7-GFP plasmid (pASG7-111.GFP) was treated with -factor (0.1 µM) for the indicated periods of time. The live cells were viewed by fluorescence microscopy using an FITC filter set.

	DISCUSSION

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Localization and signaling function of Ste4p. Expression of the a-factor receptor in MATa cells inhibits signaling through the pheromone response pathway at the level of Ste4p, the G protein  subunit (3, 17). To investigate the way in which signaling is inhibited, the normal localization pattern of Ste4p during pheromone signaling was examined. Biochemical fractionation of cell membranes had indicated that the fraction of Ste4p that is associated with the plasma membrane does not change after pheromone stimulation (14). We used microscopic examination of live cells to show that treatment of cells with pheromone causes a redistribution of Ste4p to regions of mating projection formation. These studies were therefore able to detect a change in the location of Ste4p that had not been seen previously. Other signaling components of the pheromone response pathway, such as the pheromone receptors, the kinase Ste20p, and the scaffolding protein Ste5p, have also been shown to localize to the membranes of mating projections (16, 20, 23, 26, 29). These results are consistent with a model in which Ste5p is recruited to the plasma membrane by the released Ste4p  subunit upon activation of the G protein (29). Recruitment of Ste5p to the membrane would bring Ste11p, which is bound to Ste5p, into close proximity with the membrane-associated kinase Ste20p. Phosphorylation of the MAP kinase kinase kinase Ste11p by Ste20p would then activate the MAP kinase cascade. This model is also consistent with our observation that localization of Ste4p away from the cell membrane to an internal compartment correlates with inhibition of signaling. In this case, Ste4p would not be able to recruit Ste5p to the plasma membrane and phosphorylation of Ste11p would not occur. Therefore, sequestration of the G protein  subunit away from the plasma membrane provides an efficient posttranslational mechanism for inhibition of signal transduction.

G subunit binding partners and regulation of localization. ASG7 was identified as an a-specific gene that is required for the inhibitory effect of the a-factor receptor on pheromone signaling. In addition, it was shown that the subcellular location of Asg7p and Ste4p is consistent with Asg7p having a direct effect on localization of the Ste4p  subunit. Several proteins that bind directly to subunits and inhibit their signaling activity have been identified. One such protein is phosducin, which undergoes regulated phosphorylation and which binds to in its unphosphorylated form. The crystal structure of the phosducin- complex reveals that would not be able to bind to a G protein  subunit when it is bound to phosducin (9). The binding of phosducin to is also predicted to disrupt the orientation of relative to the membrane (9). This idea is in agreement with experimental evidence demonstrating that the binding of phosducin to causes it to shift from a membrane subcellular fraction to a soluble fraction (21, 37). The ability of phosducin to cause the translocation of to a different subcellular compartment and to inhibit its signaling activity appears analogous to the action of Asg7p on the yeast complex during receptor inhibition. However, unlike the phosducin- complex, the yeast complex remains in the pellet fraction after it has translocated away from the plasma membrane (J. Kim and J. P. Hirsch, unpublished data). Therefore, the process of receptor inhibition does not cause complete solubilization of the complex.

Models for Asg7p function. The a-specific gene ASG7 was shown to be required for receptor inhibition by demonstrating that deletion of ASG7 eliminated the inhibitory effects of STE3 expression on signal transduction. Moreover, MATa cells expressing STE3 but lacking ASG7 displayed normal localization of Ste4p at the cell membrane. These results are consistent with several different models for the way in which Asg7p functions with the a-factor receptor to inhibit signaling by Ste4p. One potential model for receptor inhibition involves a direct interaction between Asg7p and the a-factor receptor that takes place within an intracellular membrane (Fig. 9A). The Asg7p protein is predicted to contain two hydrophobic regions that could function as transmembrane domains. In this model, the alpha-helical bundle of the receptor transmembrane domains would interact with the transmembrane domains of Asg7p. This interaction is not expected to take place in the plasma membrane because Asg7p is not observed on the cell surface. However, the pheromone receptors are associated with several different intracellular membranes during their life cycle. This model assumes that the cytoplasmic domains of the a-factor receptor directly associate with the subunits. The interaction of the receptor with Asg7p would produce a high-affinity binding site for , which would result in the association of with a specific internal compartment. This model is consistent with several studies of mammalian systems, which have indicated that subunits may bind directly to their associated receptors in the absence of an  subunit (12, 27, 34). Moreover, Roth and colleagues have also identified the role of ASG7 in receptor inhibition using a genomics approach and have shown that Asg7p inhibits delivery of the Ste3p receptor to the cell surface by a process that is independent of (30). This finding provides support for the idea that Asg7p and the a-factor receptor directly interact with each other.

View larger version (24K): [in this window] [in a new window]   FIG. 9.   Models for ASG7 function. See text for details.

Physiological role of receptor inhibition. The results presented here document inhibition of signaling by the complex when Asg7p and the a-factor receptor are present in the same cell. Although this situation was generated by a mutant allele in these experiments, it occurs naturally during several transient phases of the yeast life cycle. For example, homothallic strains of yeast undergo mating type switching during haploid growth. During the transition from one mating type to another, a single cell could produce both a particular pheromone receptor and the pheromone that binds to that receptor. This situation could activate the pheromone response pathway and induce the expression of ASG7, resulting in the presence of Asg7p and the a-factor receptor in the same cell. Activation of the process of receptor inhibition would then turn off the pheromone response pathway and allow the cell to resume cycling. Another example of such a situation occurs immediately after fusion of two haploid cells during the mating process. The long-term mechanism for eliminating pheromone signaling in diploids is the establishment of transcriptional inhibition of genes encoding components of the pheromone response pathway. However, it is also possible that a short-term mechanism acts to inhibit signaling in zygotes. The fusion of two mating partners could bring Asg7p from the MATa mating partner into contact with a-factor receptor from the MAT mating partner. In the newly fused zygote, both pheromone receptors are expected to be occupied. The finding that the occupied a-factor receptor can participate in receptor inhibition indicates that signaling could be blocked under these conditions. The function of receptor inhibition in this case would be to prevent multiple rounds of mating or to allow the zygote to recover rapidly from cell cycle arrest. In support of this idea, Roth and colleagues have demonstrated that there is a delay in the emergence of the first mitotic bud from zygotes in which the MATa mating partner has a deletion of ASG7 (30). Identification of ASG7 as an a-specific component required for receptor inhibition provides an opportunity to test multiple physiological conditions for their ability to be affected by this unique process.