Afr1p Regulates the Saccharomyces cerevisiae -Factor Receptor by a Mechanism That Is Distinct From Receptor Phosphorylation and Endocytosis

Corresponding author: James B. Konopka, Department of Microbiology, SUNY, Stony Brook, NY 11794-5222, konopka{at}asterix.bio.sunysb.edu (E-mail).

Communicating editor: M. JOHNSTON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The -factor pheromone receptor activates a G protein signaling pathway that induces the conjugation of the yeast Saccharomyces cerevisiae. Our previous studies identified AFR1 as a gene that regulates this signaling pathway because overexpression of AFR1 promoted resistance to -factor. AFR1 also showed an interesting genetic relationship with the -factor receptor gene, STE2, suggesting that the receptor is regulated by Afr1p. To investigate the mechanism of this regulation, we tested AFR1 for a role in the two processes that are known to regulate receptor signaling: phosphorylation and down-regulation of ligand-bound receptors by endocytosis. AFR1 overexpression diminished signaling in a strain that lacks the C-terminal phosphorylation sites of the receptor, indicating that AFR1 acts independently of phosphorylation. The effects of AFR1 overexpression were weaker in strains that were defective in receptor endocytosis. However, AFR1 overexpression did not detectably influence receptor endocytosis or the stability of the receptor protein. Instead, gene dosage studies showed that the effects of AFR1 overexpression on signaling were inversely proportional to the number of receptors. These results indicate that AFR1 acts independently of endocytosis, and that the weaker effects of AFR1 in strains that are defective in receptor endocytosis were probably an indirect consequence of their increased receptor number caused by the failure of receptors to undergo ligand-stimulated endocytosis. Analysis of the ligand binding properties of the receptor showed that AFR1 overexpression did not alter the number of cell-surface receptors or the affinity for -factor. Thus, Afr1p prevents -factor receptors from activating G protein signaling by a mechanism that is distinct from other known pathways.

CONJUGATION of the yeast Saccharomyces cerevisiae is under investigation because genetic approaches can be used to examine the mechanisms of hormone signal transduction. Conjugation is initiated when haploid cells of mating type MATa and MAT signal each other with peptide pheromones (BARDWELL et al. 1994 ; FERGUSON et al. 1994 ; KURJAN 1993 ; LEBERER et al. 1997 ). The mating pheromone signal is transduced through the action of cell-surface receptors that belong to the large family of G protein-coupled receptors (GPCRs) that includes rhodopsin and the ß-adrenergic receptor (DOHLMAN et al. 1991 ). GPCRs are distinctive because they contain seven transmembrane segments and transduce their signal by stimulating the subunit of a heterotrimeric G protein to bind GTP. The GTP-bound G subunit then dissociates from the other subunits, allowing the free G_ß complex to activate the subsequent steps in the pheromone pathway. The downstream elements in this pathway include protein kinases with similarity to mitogen activated protein (MAP) kinases and a pheromone-responsive transcription factor, Ste12p, that induces the expression of the genes that function in mating. Pheromone signaling also stimulates cell division arrest through inactivation of the Cdc28p cyclin-dependent kinase. Thus, the mating pheromone signal pathway shows remarkable similarity to the signal transduction pathways in mammalian cells. In fact, some mammalian homologs can substitute for the yeast counterparts to activate the yeast pheromone pathway (KING et al. 1990 ; PRICE et al. 1995 ).

Studies on a wide range of organisms including S. cerevisiae (JACKSON and HARTWELL 1990 ), Aspergillus nidulans (YU et al. 1996 ), and Caenorhabditis elegans (KOELLE and HORVITZ 1996 ) indicate that regulation of G protein signaling is also important for the proper activation of cellular responses. In the case of pheromone signaling, regulatory mechanisms have been identified that act on different stages of the pathway including the receptors (CHEN and KONOPKA 1996 ; CHVATCHKO et al. 1986 ; HICKE and RIEZMAN 1996 ; JENNESS and SPATRICK 1986 ), and G protein (CHAN and OTTE 1982 ; COLE and REED 1991 ; DIETZEL and KURJAN 1987A ; DOHLMAN and THORNER 1997 ; STRATTON et al. 1996 ) and the MAP kinases (DOI et al. 1994 ). The SST2 gene, which encodes a member of the RGS family of G protein regulators (DOHLMAN and THORNER 1997 ), has probably the single largest effect on signaling since cells lacking this gene are about 100-fold more sensitive to pheromone (CHAN and OTTE 1982 ; DIETZEL and KURJAN 1987A ). Interestingly, the expression of SST2 and other genes that regulate signaling is induced by pheromone treatment indicating that signaling and adaptation are finely tuned processes in yeast (DIETZEL and KURJAN 1987A ; DOI et al. 1994 ; KRONSTAD et al. 1987 ). The genes that regulate signaling during mating also function to allow cells that fail to mate to adapt to the pheromone signal and resume the normal division cycle.

To help determine the mechanisms used to regulate GPCR signaling, we previously carried out a screen to identify genes that negatively regulate the pheromone signal pathway (KONOPKA 1993 ). One new gene, AFR1, was identified because its overexpression inhibited pheromone signaling. The expression of AFR1 was induced by pheromone as are SST2 and other adaptation genes. AFR1 appears to act on an early step in the pathway because overexpression of this gene did not diminish the signaling caused by mutation of the G gene. In contrast to the effects of AFR1 overexpression, deletion of AFR1 had only weak effects on the overall intensity of pheromone signaling (KONOPKA 1993 ). However, immunofluorescence studies showed that the AFR1 protein (Afr1p) was restricted to the base of mating projections (KONOPKA et al. 1995 ) and only partially overlapped with the expected distribution of -factor receptors (JACKSON et al. 1991 ). Afr1p is apparently localized to the base of mating projections by interaction with the Cdc12p septin (GIOT and KONOPKA 1997 ). This restricted localization of Afr1p indicates that Afr1p may only be responsible for regulating a subset of receptors. Consequently, it may be expected that deletion of AFR1 would only have a weak effect on the overall sensitivity to -factor. Interestingly, cells lacking AFR1 showed a defect in forming normal mating projections that was similar to the defect observed for cells that lack the C-terminal regulatory domain of the -factor receptors (KONOPKA et al. 1988 ). Thus, regulation of receptor signaling appears to be important for the proper formation of the mating projections that become the site of cell fusion during conjugation.

In this study, experiments were carried out to examine the mechanism of AFR1 action. Afr1p does not show strong sequence similarity to any previously characterized proteins in other organisms to suggest its function. However, AFR1 showed an interesting genetic relationship with the receptor C terminus because the effects of AFR1 overexpression were weaker in a cell that produces C-terminally truncated receptors (KONOPKA 1993 ). The cytoplasmic C terminus of the -factor receptor acts as a regulatory domain because yeast cells lacking this domain of the receptor are at least 10-fold more sensitive to -factor (KONOPKA et al. 1988 ; RENEKE et al. 1988 ). One aspect of the regulation is that phosphorylation of the C-terminal sequences promotes adaptation to -factor, similar to what has been observed for many other members of the GPCR family (CHEN and KONOPKA 1996 ). The C terminus is also involved in the down regulation of -factor receptors by endocytosis (CHVATCHKO et al. 1986 ; JENNESS and SPATRICK 1986 ). Ligand-bound receptors are ubiquitinated on C-terminal sequences that promotes their removal from the cell surface and degradation in the vacuole (HICKE and RIEZMAN 1996 ). Therefore, experiments were carried out to determine how AFR1 overexpression promotes resistance to -factor. The results indicate that AFR1 regulates signaling by a mechanism that is independent of the previously characterized effects of receptor phosphorylation and receptor endocytosis that are mediated by the receptor C terminus.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains and media:
Yeast strains are described in Table 1. Cells were grown in media described by SHERMAN 1991 . Plasmid-containing cells were grown in synthetic medium containing adenine and amino acid additives, but lacking leucine or uracil to select for plasmid maintenance. Tetraploid stains were constructed by three cycles of mating a MATa cry1 strain to a MATSTE2 CRY1 strain or a MAT ste2::LEU2 CRY1 strain and then selecting for cells that underwent mitotic recombination to become homozygous MATa cells. A YIp351-STE2 plasmid was integrated into tetraploid strain JK7434-2 to construct a strain with five copies of STE2. Plasmids were transformed into yeast strains using lithium acetate (SCHIESTL and GIETZ 1989 ).

Plasmids:
The YEp24-AFR1 plasmid and the YEp24-GPA1 plasmids were isolated as described previously because they made MATa cells resistant to -factor (KONOPKA 1993 ). The YEp24-AFR1 plasmid contains the AFR1 gene on an 8.5-kb BamHI fragment of S. cerevisiae genomic DNA. A 3.8-kb Sal I-BamHI fragment that contains the AFR1 gene and upstream regulatory sequences was subcloned into the polylinker site in the vectors YEplac195 and YEplac181 (GIETZ and SUGINO 1988 ). For brevity, these vector names will be shortened to YEp181 and YEp195 in this paper. The construction of the YEp24-ste2-I169 and YEp24-ste2-I229 plasmids carrying linker insertion mutants of STE2 was described previously (KONOPKA and JENNESS 1991 ). The YIp351-STE2 plasmid (pJBK-070) was constructed by subcloning a 4.3-kb BamHI fragment containing STE2 into the integrating vector YIp351 (HILL et al. 1986 ).

Pheromone response assays:
Halo assays for -factor-induced cell division arrest were performed by spreading 1.5 x 10⁵ cells from an overnight culture on the surface of a solid medium agar plate. -Factor was added to sterile filter disks (Difco, Detroit) which were then applied to the surface of the agar plate and incubated at 30° for 3 days. Halo assays performed with the end4::LEU2 cells were incubated at 23° for 5 days since mutation of END4 causes temperature sensitive growth (RATHS et al. 1993 ). The plates were then photographed to record the zone of growth inhibition (halo) surrounding the disk. Similar results were observed in at least three independent assays. The stability of -factor in the medium was assayed by adjusting a log phase culture to 4 x 10⁶ cells/ml and then -factor was added to a final concentration of 10^-7 M. Cells were incubated at 30°, and then samples of the culture supernatant were withdrawn at various times and assayed for ability to promote cell division arrest of the supersensitive strain 6360-17-2a in a halo assay.

Ligand binding assays:
-factor binding assays were carried out essentially as described previously (JENNESS et al. 1983 ; SCHANDEL and JENNESS 1994 ). Binding of -factor to tetraploid strains was carried out with cells that were grown overnight to logarithmic phase. Cells were washed twice with ice-cold inhibitor medium (YEPD medium containing 10 mM KF and 10 mM NaN₃) and then resuspended at a density of 5 x 10⁸ cells/ml. Cells (50 µl) and 50 µl of 20 nM ³⁵S--factor were incubated for 30 min, the cells were collected on a Whatman GF/C filter, washed to remove unbound -factor, and then counted in a scintillation counter. Nonspecific binding assays were performed in the presence of a 100-fold excess of non-radiolabeled synthetic -factor (Bachem, King of Prussia, PA). The effects of AFR1 overexpression on -factor binding were examined with cells that were grown overnight to log phase, and then incubated in the presence of 10^-7 M -factor for the indicated time. The cells were then washed to remove the bound -factor essentially as described (JENNESS and SPATRICK 1986 ; ROHRER et al. 1993 ). Basically, cells were collected on a nitrocellulose filter, washed with ice-cold inhibitor medium, and resuspended in inhibitor medium for 2 hr to allow the -factor to dissociate. The cells were then pelleted by centrifugation and resuspended in inhibitor medium three times to remove the -factor, and resuspended at a density of 10⁹ cells/ml. Ligand binding assays were carried out by incubating 50 µl cells and 50 µl ³⁵S--factor for 30 min, and then cells were collected on a Whatman GF/C filter, the bound -factor was quantitated as described above. Cells that were exposed to -factor for various times were assayed for binding using 20 nM ³⁵S--factor. Scatchard plots were carried out on cells that were exposed to -factor for 3 hr prior to the binding assays. The Scatchard plots represent the average values from three independent assays that were each performed in duplicate. ³⁵S-labeled -factor was purified by chromatography on a Bio-Rex 70 column from the culture supernatant of MAT cells labeled with ³⁵S-SO₄ as described previously (SCHANDEL and JENNESS 1994 ).

Western blot analysis:
The production of Ste2p and Afr1p was analyzed in cells that were grown to logarithmic phase and then incubated in the presence or absence of 10^-7 M -factor for the indicated time. Approximately 2.5 x 10⁸ cells were lysed by agitation with glass beads in 250 µl gel loading buffer (2% SDS, 50 mM Tris pH 6.8, 8 M urea). The protein concentration of the extracts was determined using a BCA Protein Assay kit (Pierce, Rockford, IL). Equal amounts of protein (50 µg) were loaded in each lane of a 9% SDS-polyacrylamide gel and resolved by electrophoresis. The proteins were electrophoretically transferred to nitrocellulose, and probed with rabbit anti-Afr1p antibodies (KONOPKA et al. 1995 ) or with anti-N-terminal anti-Ste2p antibodies (KONOPKA et al. 1988 ). The blots were incubated with horse radish peroxidase-conjugated goat anti-rabbit antibodies and the immunoreactive proteins were detected by chemiluminescence using an Amersham ECL kit. The relative levels of Afr1p production were estimated by comparing different dilutions of sample loaded on the same blot. Similar results were always observed in at least two independent Western blots.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Multicopy plasmid overproduction of Afr1p:
Multicopy plasmid overexpession of AFR1 makes MATa cells resistant to -factor so the extent to which these cells overproduce the AFR1 protein (Afr1p) was analyzed. Cells were stimulated with -factor for 2 hr to induce the expression of the AFR1 gene and then the levels of Afr1p were compared by Western blot analysis. As shown in Figure 1A, cells carrying the multicopy YEp181-AFR1 plasmid produced more Afr1p than cells carrying a YEp181 vector plasmid. Comparison of dilutions of these extracts indicates that Afr1p was overproduced about 10-fold (not shown). In addition, a higher basal level of Afr1p was also detected in the absence of -factor for cells carrying the YEp181-AFR1 plasmid. A time course experiment (Figure 1B) showed that the amount of Afr1p increased rapidly for the first 2 hr and then stayed at a high level but decreased slowly over time as cells adapted and became resistant to -factor. Similar levels of Afr1p overproduction were obtained with cells carrying AFR1 on the LEU2-selectable multicopy vector YEp181 or the URA3-selectable multicopy vector YEp195 (Figure 1C and data not shown) that were used in this study.

View larger version (47K): In this window In a new window Download PPT slide   Figure 1. Western blot analysis of Afr1p overproduction. (A) Analysis of Afr1p levels in cells that lack AFR1 (afr1), cells with a single copy of AFR1 (AFR1), and cells carrying AFR1 on a multicopy plasmid (YEp-AFR1) that were incubated in the absence (-) or presence (+) of -factor for 2 hr as indicated above each lane. The afr1 strain (JKY26) carried the vector YEp181 and wild-type strain DJ211-5-3 carried either the vector YEp181 or YEp181-AFR1. (B) Analysis of Afr1p levels after various periods of -factor induction. Strain DJ211-5-3 carrying YEp181-AFR1 was induced with -factor for the number of minutes indicated above each lane. (C) Analysis of Afr1p levels in cells carrying different AFR1 plasmids. Strain DJ211-5-3 carrying YEp24, YEp24-AFR1, and YEp195-AFR1 incubated in the absence (-) or presence (+) of -factor for 3 hr. All experiments were carried out at 30° and cells were induced with 10-7 M -factor. Cells were extracted and protein samples prepared for Western analysis as described in the MATERIALS AND METHODS.

To confirm that Afr1p overproduction makes cells resistant to -factor by negatively regulating signaling, and not by causing the destruction of -factor, we examined the stability of -factor in the medium. This assay was carried out in a bar1-1 mutant strain that lacks the secreted protease that degrades -factor in the medium. As shown in Figure 2A, -factor added to a culture of bar1-1 cells carrying YEp181 or YEp181-AFR1 was stable for at least 6 hr. In spite of the continued presence of -factor, about 29% of cells carrying YEp181-AFR1 had resumed budding whereas less than 2% of cells carrying the YEp181 vector showed evidence of budding. As expected, BAR1 cells did not contain detectable -factor after 1 hr under these conditions indicating that the -factor was degraded. The resistance to -factor promoted by Afr1p overproduction was also independent of cell density when assayed by plating dilutions of cells on plates containing -factor (Figure 2B). Altogether, these results demonstrate that Afr1p overproduction acts to negatively regulate signal transduction, and does not simply lead to the destruction of -factor.

View larger version (52K): In this window In a new window Download PPT slide   Figure 2. Afr1p overproduction inhibits signaling without causing the destruction of -factor. The stability of -factor was measured for strains carrying either a plasmid vector (YEp181) or an AFR1 overexpression plasmid (YEp181-AFR1). The bar1-1 strain was DJ211-5-3 and the BAR1 strain was YLG104-8-3. (A) Cultures were adjusted to 4 x 106 cells/ml, -factor was added to 10-7 M, and then aliquots of the supernatant were tested for ability to form a halo of cell division arrest on a lawn of strain 6360-17-2a. The bar1-1 mutant cells (B) and the BAR1 cells (C) were also adjusted to 2 x 106 cells/ml and then 10-fold serial dilutions were plated on medium in the presence or absence of -factor (10-7 M) for 3 days at 30°.

Inhibition of receptor endocytosis decreases the effects of Afr1p overproduction on signaling:
Our previous studies showed that AFR1 overexpression caused wild-type STE2 cells to become resistant to -factor, but the effects of AFR1 overexpression were not readily detectable in ste2-T326 cells that lack most of the cytoplasmic C-terminal domain of the -factor receptor (KONOPKA 1993 ). The receptor C terminus is known to be a target for two adaptation mechanisms: phosphorylation and receptor endocytosis. To determine whether Afr1p acts in one of these pathways we examined the effects of AFR1 overexpression in various receptor mutant strains. These cells were tested for the ability to promote resistance to -factor-induced cell division arrest in a halo assay. As shown in Figure 3A, cell division arrest was assayed by placing a filter disk containing -factor on a lawn of cells spread on the surface of a solid medium petri plate. Diffusion of -factor into the medium arrested the division of wild-type cells carrying the vector YEp24 resulting in a clear zone (halo) surrounding the filter disk. In contrast, wild-type cells carrying either the YEp24-AFR1 or YEp195-AFR1 multicopy plasmids failed to form a clear zone of cell division arrest indicating that they are resistant to -factor. As we observed previously, ste2-T326 cells carrying YEp24-AFR1 did not display a significant change in sensitivity to -factor. However, ste2-T326 cells carrying YEp195-AFR1 (Figure 3A) or YEp181-AFR1 (not shown) produced a smaller halo indicating they were more resistant to -factor. The effects of AFR1 overexpression were probably more noticeable in the ste2-T326 cells carrying the YEp195-AFR1 or YEp181-AFR1 plasmids because they contain a different genomic fragment of AFR1 than the YEp24-AFR1 plasmid (see MATERIALS AND METHODS) and result in about twofold greater Afr1p overproduction (Figure 1C and data not shown). The YEp181-AFR1 and YEp195-AFR1 plasmids contain the same AFR1 fragment and had equivalent effects on pheromone signaling (Figure 2 and data not shown). In spite of the stronger effects of these new AFR1 plasmids, AFR1 overexpression still appears to act early in the signaling pathway because YEp181-AFR1 did not suppress the cell division arrest caused by deletion of GPA1 that encodes the G subunit (Figure 3B).

View larger version (39K): In this window In a new window Download PPT slide   Figure 3. Effects of Afr1p overproduction on -factor induced cell division arrest. (A) Halo assays demonstrating the effects of Afr1p overproduction on the -factor sensitivity of STE2 strain DJ211-5-3, ste2-T326 strain 7441-4-2, ste2-D4ala strain QCY03, and end4::LEU2 strain JKY99-1 as indicated on the left. The cells carried either a plasmid vector (YEp24) or AFR1 on a multicopy plasmid (YEp24-AFR1 or YEp195-AFR1) as indicated above. Filter discs containing 10 µl of 10-5 and 3 x 10-6 M -factor were applied to a lawn of the indicated cell type on an agar petri plate, which was then incubated to observe the zone of growth inhibition caused by -factor (B) The upper panel shows halo assays for strain DJ211-5-3 carrying the vector YEp181 or YEp181-AFR1. The lower panel shows growth assays for ste5-3ts gpal::URA3 strain 7646-3-4 carrying one of the indicated plasmids. Dilutions of the cells were spotted onto agar plates and then examined for growth after 5 days at the permissive (23°) or restrictive (34°) temperature for ste5-3ts.

The observation that AFR1 overexpression promotes resistance in ste2-T326 cells indicates that Afr1p acts independently of the sequences that promote ligand-induced endocytosis of the -factor receptors since these sequences are deleted in ste2-T326 receptors (ROHRER et al. 1993 ). The ste2-T326 receptors still contain several potential phosphorylation sites so we examined the ability of Afr1p overproduction to promote resistance in a ste2-D4ala strain to determine if Afr1p-mediated resistance to -factor required phosphorylation of the receptor C terminus. The ste2-D4ala receptors are not phosphorylated in vivo because the majority of the potential phosphorylation sites were removed by deletion of residues 297-391, and then the remaining four serine and threonine residues were mutated to alanine (CHEN and KONOPKA 1996 ). AFR1 overexpression made this strain resistant to pheromone indicating that phosphorylation of the receptor C terminus is not required for Afr1p to function (Figure 3). These results suggested that decreased function of Afr1p in the ste2-T326 strain might be related to the increased receptor number caused by the removal of the endocytosis domain from the receptor C terminus. We therefore examined the ability of AFR1 overexpression to promote resistance in an end4::LEU2 yeast strain that is defective in ligand-induced receptor endocytosis (RATHS et al. 1993 ). Overexpression of AFR1 in an end4::LEU2 strain did not cause a significant increase in resistance to -factor even though these cells produce wild-type receptors with a full-length C terminus. Thus, the effects of Afr1p overproduction were weaker in strains that are defective in ligand-induced receptor endocytosis.

Afr1p does not alter stability of -factor receptor protein:
To investigate the mechanism by which Afr1p alters receptor signaling, we examined the level of the receptor protein (Ste2p). This analysis was carried out with cells that were induced with -factor for 3 hr, a time at which Afr1p production is high and cells appear resistant to -factor. As expected, Western blot analysis (Figure 4) showed that the gel mobility of Ste2p is heterogeneous due to glycosylation (BLUMER et al. 1988 ) and becomes even more heterogeneous after -factor treatment due to ubiquination (HICKE and RIEZMAN 1996 ). The Western blot also showed that Afr1p overproduction did not significantly alter the level of Ste2p or the post-translational modification of Ste2p as compared to cells carrying a control vector (Figure 4).

View larger version (62K): In this window In a new window Download PPT slide   Figure 4. Western blot analysis of Ste2p. Strain DJ211-5-3 carrying YEp181 or YEp181-AFR1 was incubated in the absence (-) or presence (+) of 10-7 M -factor for 3 hr at 30° as indicated above each lane. Cells were extracted and protein samples prepared for Western analysis as described in the MATERIALS AND METHODS.

We next examined the possibility that Afr1p overproduction affected signal transduction by promoting the removal of receptors from the cell surface by endocytosis. Normally, the binding of -factor to its receptor causes the receptors to be targeted for endocytosis and degradation in the vacuole (HICKE and RIEZMAN 1996 ; SCHANDEL and JENNES 1994). To determine whether Afr1p overproduction altered the number of cell-surface receptors, we assayed the ability of cells to bind radio-labeled -factor. Cells were treated with cold -factor for different times to induce AFR1 expression and to promote receptor endocytosis. The cells were then washed to allow the bound -factor to dissociate, and assayed for the ability to bind ³⁵S-labeled -factor. The results showed that cells carrying YEp181-AFR1 or the control vector bound similar amounts of -factor at all time points (Figure 5). If anything, it appeared that cells overexpressing AFR1 displayed slightly greater ability to bind -factor. Both of these cell types maintain a high level of receptors in the presence of -factor because the endocytosed receptors are replaced by newly synthesized receptors. These results indicate that AFR1 overexpression does not diminish signaling by decreasing the number of cell-surface receptors.

View larger version (22K): In this window In a new window Download PPT slide   Figure 5. Binding of -factor to pheromone-induced MATa cells. Strain DJ211-5-3 carrying YEp181 or YE181-AFR1 was incubated with -factor (10-7 M) for the indicated time at 30°. The cells were then washed to remove the bound -factor and then the binding of 35S-labeled -factor was quantified as described in the MATERIALS AND METHODS. The results were normalized to the time zero value.

Afr1p overproduction does not alter the receptor affinity for -factor:
The possibility that Afr1p promotes resistance to -factor by changing the affinity of the receptor for -factor was examined in equilibrium binding assays. Cells were treated with -factor for 3 hr to induce Afr1p, washed to allow the cold -factor to dissociate, and then assayed for ability to bind ³⁵S-labeled -factor. The results were analyzed on a Scatchard plot (Figure 6). The K_d for the binding of -factor to its receptor, as reflected by the slope of the line in the Scatchard plot, was about 5.4 nM for cells carrying a control vector and 4.6 nM for cells carrying YEp181-AFR1 plasmid. This indicates that there is no significant difference in binding affinity as a result of Afr1p overproduction. The Scatchard plots also indicated that Afr1p overproduction did not have significant effects on the number of receptors per cell, in agreement with Figure 5. As derived from the X-intercept values, both YEp181 and YEp181-AFR1 cells contained about 4000 surface binding sites per cell.

View larger version (20K): In this window In a new window Download PPT slide   Figure 6. Scatchard plot analysis of -factor binding to MATa cells. Scatchard plot analysis of the binding of 35S-labeled -factor to strain DJ21-5-3 carrying (A) YEp181 or (B) YEp181-AFR1. The cells were induced with -factor (10-7 M) for 3 hr and then washed to remove the bound -factor. 35S-labeled -factor binding was then quantitated and analyzed by the Scatchard method as described in the MATERIALS AND METHODS.

Gene-dosage relationship between AFR1 and STE2:
Since Afr1p overproduction did not increase receptor endocytosis, the observations that the ste2-T326 and end4::LEU2 mutations that inhibit receptor endocytosis also impaired the ability of Afr1p to promote resistance to -factor suggested that Afr1p function is related to the number of receptors, and not directly to the process of endocytosis. To investigate this relationship further, we examined the effects of Afr1p overproduction in a series of yeast strains that vary in receptor production. MATa tetraploid yeast strains were constructed that contain five, four, three, two, or one copy of the receptor gene, STE2. As a control, we showed that the level of cell-surface receptors varied with STE2 gene dosage by assaying the ability of these strains to bind ³⁵S-labeled -factor (Figure 7). The variations in receptor number did not affect the overall sensitivity of these strains since all of the tetraploid strains produced similar sized halos when carrying a control plasmid vector (Figure 8). This result is in agreement with previous studies showing that the number of receptors is not limiting for pheromone signal transduction (KONOPKA et al. 1988 ; SHAH and MARSH 1996 ). The tetraploid strain carrying only a single copy of STE2 consistently formed colonies within the zone of clearing, which may be caused by gene conversion between the three copies of ste2::LEU2 and the one copy of the STE2 gene in these cells.

View larger version (21K): In this window In a new window Download PPT slide   Figure 7. Binding of -factor to tetraploid yeast strains that vary in STE2 gene dosage. Tetraploid MATa yeast strains carrying the indicated number of copies of the -factor receptor gene STE2 were assayed for ability to bind 35S--factor as described in the MATERIALS AND METHODS. The tetraploid yeast strains are described in Table 1.

View larger version (105K): In this window In a new window Download PPT slide   Figure 8. Halo assays for cell division arrest in tetraploid MATa strains. Tetraploid yeast strains carried the number of copies of the STE2 gene indicated on the left. The cells also carried one of the following plasmids YEp24, YEp195-AFR1, or YEp24-GPA1 as indicated above. Cells were plated as a lawn on an agar plate and then filter disks containing 10 µl of either 10-5 or 3 x 10-6 M -factor were placed on the plate. The zone of growth arrest was photographed after incubation at 30° for 3 days.

In contrast to the vector control, tetraploid cells carrying multicopy AFR1 plasmids showed different sized halos depending on the number of copies of STE2. The effects of AFR1 overexpression also appeared to be different in the tetraploid cells, as compared to the haploid cells, because a tetraploid cell carrying four copies of STE2 showed only a weak increase in resistance to -factor. However, the degree of -factor resistance promoted by AFR1 overexpression was inversely proportional to the number of STE2 genes as tetraploid cells carrying fewer copies of STE2 showed proportionally increased resistance to -factor (Figure 8). In addition, tetraploid cells carrying YEp195-AFR1 showed greater resistance to -factor than did cells carrying the YEp24-AFR1 plasmid (not shown); consistent with the observation described above that cells carrying YEp195-AFR1 produced about twofold more Afr1p. As a control, we also examined the effects of overexpressing the GPA1 gene that encodes the subunit of the pheromone-responsive G protein. Overproduction of the G subunit is thought to promote resistance to -factor by sequestering the G_ß moiety (DIETZEL and KURJAN 1987B ; WHITEWAY et al. 1989 ). All of the tetraploid strains carrying a YEp24-GPA1 plasmid showed approximately the same degree of increased resistance to -factor (Figure 8). These results demonstrate that the ability of Afr1p to promote resistance to -factor is specifically related to the number of receptors in the cell.

The gene dosage relationship between AFR1 and STE2 was also examined in haploid cells the overexpress STE2. To overexpress STE2, the cells that carry one copy of STE2 in the genome were transformed with a multicopy YEp24-STE2 plasmid. This plasmid causes about a fivefold increase in the basal number of cell-surface receptors (KONOPKA et al. 1988 ) which is comparable to the increase in receptors found in ste2-T326 cells. However, individual cells within the population can vary in receptor number because the copy number of the plasmid vector is not regulated. Analysis of these strains by halo assay showed that the effects of AFR1 overexpression were weaker in cells that also carry a multicopy YEp24-STE2 plasmid (Figure 9). This effect was specific for cells that overexpress STE2. AFR1 overexpression promoted resistance to -factor in cells carrying the YEp24 vector and in cells that overexpress a defective receptor gene. Cells carrying the linker insertion mutant alleles ste2-I169 or ste2-I229 on a YEp24 plasmid vector overproduce nonfunctional receptor proteins (KONOPKA and JENNESS 1991 ) that did not interfere with the ability of AFR1 to promote resistance to -factor (Figure 9). These results raise the possibility that Afr1p may preferentially act on activated receptors. However, differences in the structure or subcellular localization of the mutant receptor proteins may also account for these results.

View larger version (108K): In this window In a new window Download PPT slide   Figure 9. Effects of overproducing both Afr1p and Ste2p on -factor sensitivity. Halo assays were performed with cells (DJ211-5-3) that carried either YEp24, or YEp24 carrying the -factor receptor gene indicated on the left (wild-type STE2, ste2-I169, or ste2-I229). The cells also carried either YEp181 or Yep181-AFR1 as indicated above. Cells were plated as a lawn on an agar plate and then filter disks containing 10 µl of either 10-5 or 3 x 10-6 M -factor were placed on the plate. The zone of growth arrest was photographed after incubation at 30° for 3 days.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Mechanism of Afr1p action:
Our previous studies indicated that Afr1p acts on an early step in the pheromone signal pathway and suggested that AFR1 may act in a pathway with the receptor C terminus (KONOPKA 1993 ). In this study we investigated the mechanism of Afr1p action by exploring the relationship between Afr1p and the mechanisms that are known to regulate receptor signaling. AFR1 acts independently of receptor phosphorylation since AFR1 regulated signaling in a ste2-D4ala strain that produces a mutant receptor protein that is not phosphorylated in vivo because it lacks the C-terminal phosphorylation sites of the receptor. The receptor C terminus is also involved in ligand-induced receptor endocytosis (HICKE and RIEZMAN 1996 ; ROHRER et al. 1993 ). However, AFR1 did not require the C terminus to act, nor did it detectably influence receptor endocytosis or the stability of the receptor protein. The weaker effects of AFR1 overexpression in ste2-T326 and end4::LEU2 strains that are defective in receptor endocytosis were probably an indirect effect of the increased receptor number of these strains. This was confirmed by gene dosage studies that showed that the ability of AFR1 to regulate signaling was inversely proportional to the number of receptors. Thus, the relative levels of Afr1p and the -factor receptor can determine the sensitivity of the cells to -factor.

AFR1 appears to be distinct from the other regulatory mechanisms that act on the -factor receptor in that it does not require the cytoplasmic C terminus to influence the activity of the central region of the receptor containing the seven transmembrane segments. Instead, Afr1p overproduction was found to inhibit signaling emanating from the central region of the -factor receptor that is responsible for binding ligand and G protein activation. Analysis of the effects of Afr1p overproduction on the ligand binding properties of the receptors showed that Afr1p did not significantly alter the number of cell-surface receptors or the affinity for -factor. These results suggest that Afr1p acts to prevent G protein activation.

An interesting possibility suggested by the data is that Afr1p may act by forming a complex with the receptors. The intracellular loops that connect the transmembrane segments of the receptor play a key role in G protein activation. Afr1p should be able to interact directly with these intracellular loops since both Afr1p and the receptors are present at the plasma membrane ( JACKSON et al. 1991 ; KONOPKA et al. 1995 ; SCHANDEL and JENNESS 1994 ). In addition, the gene dosage relationship between AFR1 and STE2 suggests that Afr1p may form a stoichiometric complex with the receptor. Although these data are consistent with Afr1p forming a complex with receptors, Afr1p may also modify the receptors or act indirectly through other proteins to inhibit receptor signal transduction. For example, it has been suggested that the Sst2p adaptation protein may form a complex between a G protein and an -factor receptor (SHAH and MARSH 1996 ), and the effects of AFR1 overexpression are weaker in sst2^- strains (KONOPKA 1993 ), so it could be inferred that Afr1p may act on this complex. However, the weaker effects of AFR1 in sst2^- strains may simply be due to their extreme 100-fold increased sensitivity to -factor. In addition, sst2^- mutant strains are defective in adapting to even a short pulse of -factor (CHAN and OTTE 1982 ; KONOPKA et al. 1988 ) so they can accumulate a stable post-receptor signaling intermediate by the time that the maximum levels of Afr1p are induced. Thus, the extreme adaptation defect of sst2 cells is also expected to make them appear to be relatively insensitive to Afr1p overproduction. The effects of SST2 and AFR1 also appear to be different in that, unlike AFR1, we did not see a difference in the efficiency with which overexpression of SST2 confers resistance to -factor receptor truncation strains and strains with increased STE2 dosage [(KONOPKA 1993 ) and unpublished data]. Unfortunately, Afr1p is insoluble under relatively nondenaturing conditions and this prevented us from directly examining the ability of Afr1p to interact with the receptors (J. B. KONOPKA, unpublished data).

The regulation of signaling by Afr1p appears to be different from previously identified mechanisms for the regulation of GPCR signaling in other organisms. Regulation of receptor signaling has been studied in detail for rhodopsin and the adrenergic receptors (DOHLMAN et al. 1991 ; LEFKOWITZ 1993 ). Stimulation of these receptors results in their phosphorylation by a receptor-specific protein kinase. The phosphorylated receptors are then bound by a protein termed arrestin that prevents further G protein activation. Afr1p may act in a manner analogous to arrestin. However, Afr1p function is independent of receptor phosphorylation and Afr1p shows at most limited sequence similarity to arrestin. We also failed to detect an interaction between Afr1p and the receptor C terminus in the two-hybrid assay (unpublished data). In the case of the ß-adrenergic receptor, arrestin acts as an adapter that causes this receptor to associate with clathrin and become endocytosed (GOODMAN et al. 1996 ). This contrasts with the yeast mating pheromone receptors for which the ubiquination of the C terminus apparently acts as the signal to promote receptor endocytosis (HICKE and RIEZMAN 1996 ). Although it is possible that Afr1p carries out a function that is unique to yeast for the regulation of receptor signaling, it seems likely that homologs will be found in other systems that have not yet been characterized in detail given the strong evolutionary conservation of G protein signaling components. For example, Afr1p homologs that exist in other organisms may have escaped detection by biochemical methods since Afr1p is present in the insoluble fraction of a cell extract.

AFR1 function during yeast mating:
Studies on mating pheromone-induced morphogenesis indicate that regulation of receptor signaling is important for cells to form typical mating projections that become the site of cell fusion during conjugation. Wild-type cells stimulated with -factor arrest their division cycle in G1 and then undergo polarized morphogenesis to form a mating projection (LEBERER et al. 1997 ; ROEMER et al. 1996 ). In contrast, cells carrying mutations in the C-terminal regulatory domain of the -factor receptor have a defect in forming typical mating projections that is proportional to their defect in adaptation (CHEN and KONOPKA 1996 ). Cells lacking the AFR1 gene are also defective in forming typical projections (KONOPKA 1993 ). Analysis of AFR1 mutants indicates that the same domain of Afr1p functions to regulate signaling and to promote the formation of mating projections (GIOT and KONOPKA 1997 ). These observations suggest that the regulation of signaling may be important to coordinate cellular morphogenesis. Interestingly, immunolocalization studies show that -factor receptors are detected throughout the projection ( JACKSON et al. 1991 ; MARSH and HERSKOWITZ 1988 ), but Afr1p is only detected at the base of the projection (KONOPKA et al. 1995 ). This restricted localization for Afr1p is mediated by interaction with Cdc12p (GIOT and KONOPKA 1997 ), a member of the septin family of filament-forming proteins (LONGTINE et al. 1996 ). One possibility suggested by the spatial distribution of Afr1p is that Afr1p may act as a boundary to restrict the zone of receptor signaling. According to this model, overproduction of Afr1p could promote resistance to -factor by causing mislocalization of Afr1p and allowing it to inhibit signaling in other parts of the cell. However, studies on Afr1p mutants indicate that mislocalization of Afr1p is not sufficient to observe increased resistance to -factor (GIOT and KONOPKA 1997 ). Overproduction was still required to observe the effects of the mutant Afr1p proteins as expected if the sensitivity of the cells is regulated by the relative levels of Afr1p and the receptor. Since the production of Afr1p is induced by pheromone signaling, these results indicate that Afr1p acts as part of a feedback loop to regulate receptor signaling during mating.

	ACKNOWLEDGMENTS

We thank A. NEIMAN, C. ROY, and the members of our lab for their helpful comments on the manuscript. We also thank L. HICKE and H. RIEZMAN for plasmids. This work was supported by grants from the American Cancer Society (VM-40) and the American Heart Association.

Manuscript received September 29, 1997; Accepted for publication October 27, 1997.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

BARDWELL, L., J. G. COOK, C. J. INOUYE, and J. THORNER, 1994  Signal propagation and regulation in the mating pheromone response pathway of the yeast Saccharomyces cerevisiae. Dev. Biol. 166:363-379[Medline].

BLUMER, K. J., J. E. RENEKE, and J. THORNER, 1988  The STE2 gene product is the ligand-binding component of the -factor receptor of Saccharomyces cerevisiae. J. Biol. Chem. 263:10836-10842[Abstract/Free Full Text].

CHAN, R. K. and C. A. OTTE, 1982  Isolation and genetic analysis of Saccharomyces cerevisiae mutants super-sensitive to G1 arrest by a-factor and -factor pheromones. Mol. Cell. Biol. 2:11-20[Abstract/Free Full Text].

CHEN, Q. and J. B. KONOPKA, 1996  Regulation of the G protein-coupled -factor pheromone receptor by phosphorylation. Mol. Cell. Biol. 16:247-257[Abstract].

CHVATCHKO, Y., I. HOWALD, and H. RIEZMAN, 1986  Two yeast mutants defective in endocytosis are defective in pheromone response. Cell 46:355-364[Medline].

COLE, G. M. and S. I. REED, 1991  Pheromone-induced phosphorylation of a G protein ß subunit in S. cerevisiae is associated with an adaptive response to mating pheromone. Cell 64:703-716[Medline].

DIETZEL, C. and J. KURJAN, 1987a  Pheromonal regulation and sequence of the Saccharomyces cerevisiae SST2 gene: a model for desensitization to pheromone. Mol. Cell. Biol. 7:4169-4177[Abstract/Free Full Text].

DIETZEL, C. and J. KURJAN, 1987b  The yeast SCG1 gene: a G-like protein implicated in the a- and -factor response pathway. Cell 50:1001-1010[Medline].

DOHLMAN, H. G. and J. THORNER, 1997  RGS proteins and signaling by heterotrimeric G proteins. J. Biol. Chem. 272:3871-3874[Free Full Text].

DOHLMAN, H. G., J. THORNER, M. G. CARON, and R. J. LEFKOWITZ, 1991  Model systems for the study of 7-transmembrane-segment receptors. Annu. Rev. Biochem. 60:653-688[Medline].

DOI, K., A. GARTNER, G. AMMERER, B. ERREDE, and H. SHINKAWA et al., 1994  MSG5, a novel protein phosphatase promotes adaptation to pheromone response in S. cerevisiae. EMBO J. 13:61-70[Medline].

FERGUSON, B., J. HORECKA, J. PRINTEN, J. SCHULTZ, and B. J. STEVENSON et al., 1994  The yeast pheromone response pathway: new insights into signal transmission. Cell. Mol. Biol. Res. 40:223-228[Medline].

GIETZ, R. D. and A. SUGINO, 1988  New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74:527-534[Medline].

GIOT, L. and J. B. KONOPKA, 1997  Functional analysis of the interaction between Afr1p and the Cdc12p septin, two proteins involved in pheromone-induced morphogenesis. Mol. Biol. Cell 8:987-998[Abstract].

GOODMAN, O. B., JR., J. G. KRUPNICK, F. SANTINI, V. V. GUREVICH, and R. B. PENN et al., 1996  ß-arrestin acts as a clathrin adaptor in endocytosis of the ß₂-adrenergic receptor. Nature 383:447-450[Medline].

HICKE, L. and H. RIEZMAN, 1996  Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis. Cell 84:277-287[Medline].

HILL, J. E., A. M. MYERS, T. J. KOERNER, and A. TZAGOLOFF, 1986  Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast 2:163-167[Medline].

JACKSON, C. L. and L. H. HARTWELL, 1990  Courtship in Saccharomyces cerevisiae: both cell types choose mating partners by responding to the strongest pheromone signal. Cell 63:1039-1051[Medline].

JACKSON, C. L., J. B. KONOPKA, and L. H. HARTWELL, 1991  S. cerevisiae -pheromone receptors activate a novel signal transduction pathway for mating partner discrimination. Cell 67:389-402[Medline].

JENNESS, D. D., A. C. BURKHOLDER, and L. H. HARTWELL, 1983  Binding of -factor pheromone to yeast a cells: chemical and genetic evidence for an -factor receptor. Cell 35:521-529[Medline].

JENNESS, D. D. and P. SPATRICK, 1986  Down regulation of the -factor pheromone receptor in Saccharomyces cerevisiae. Cell 46:345-353[Medline].

KING, K., H. G. DOHLMAN, J. THORNER, M. G. CARON, and R. J. LEFKOWITZ, 1990  Cotnrol of yeast mating signal transduction by a mammalian ß₂-adrenergic receptor and the G_s -subunit. Science 250:121-123[Abstract/Free Full Text].

KOELLE, M. R. and H. R. HORVITZ, 1996  EGL-10 regulates G protein signaling in the C. elegans nervous system and shares a conserved domain with many mammalian proteins. Cell 84:115-125[Medline].

KONOPKA, J. B., 1993  AFR1 acts in conjunction with the -factor receptor to promote morphogenesis and adaptation. Mol Cell. Biol. 13:6876-6888[Abstract/Free Full Text].

KONOPKA, J. B., C. DEMATTEI, and C. DAVIS, 1995  AFR1 promotes polarized apical morphogenesis in Saccharomyces cerevisiae. Mol. Cell. Biol. 15:723-730[Abstract].

KONOPKA, J. B. and D. D. JENNESS, 1991  Genetic fine-structural analysis of the Saccharomyces cerevisiae -pheromone receptor. Cell Regul. 2:439-452[Medline].

KONOPKA, J. B., D. D. JENNESS, and L. H. HARTWELL, 1988  The C terminus of the Saccharomyces cerevisiae -pheromone receptor mediates an adaptive response to pheromone. Cell 54:609-620[Medline].

KRONSTAD, J. W., J. A. HOLLY, and V. L. MACKAY, 1987  A yeast operator overlaps an upstream activation site. Cell 50:369-377[Medline].

KURJAN, J., 1993  The pheromone response pathway in Saccharomyces cerevisiae. Annu. Rev. Genet. 27:147-179[Medline].

LEBERER, E., D. Y. THOMAS, and M. WHITEWAY, 1997  Pheromone signalling and polarized morphogenesis in yeast. Curr. Opin. Genet. Dev. 7:59-66[Medline].

LEFKOWITZ, R. J., 1993  G protein-coupled receptor kinases. Cell 74:409-412[Medline].

LONGTINE, M. S., D. J. DEMARINI, M. L. VALENCIK, O. S. AL-AWAR, and H. FARES et al., 1996  The septins: roles in cytokinesis and other processes. Curr. Opin. Cell Biol. 8:106-119[Medline].

MARSH, L. and I. HERSKOWITZ, 1988  From membrane to nucleus: the pathway of signal transduction in yeast and its genetic control. Cold Spring Harbor Symp. Quant. Biol. 53:557-565.

PRICE, L. A., E. M. KAJKOWSKI, J. R. HADCOCK, B. A. OZENBERGER, and M. H. PAUSCH, 1995  Functional coupling of a mammalian somatostatin receptor to the yeast pheromone response pathway. Mol. Cell. Biol. 15:6188-6195[Abstract].

RATHS, S., J. ROHRER, F. CRAUSAZ, and H. RIEZMAN, 1993  end3 and end4: two mutants defective in receptor-mediated and fluid-phase endocytosis in Saccharomyces cerevisiae. J. Cell Biol. 120:55-65[Abstract/Free Full Text].

RENEKE, J. E., K. J. BLUMER, W. E. COURCHESNE, and J. THORNER, 1988  The carboxyl terminal domain -factor receptor is a regulatory domain. Cell 55:221-234[Medline].

ROEMER, T., L. G. VALLIER, and M. SNYDER, 1996  Selection of polarized growth sites in yeast. Trends Cell Biol. 6:434-441.

ROHRER, J., H. BENEDETTI, B. ZANOLARI, and H. RIEZMAN, 1993  Identification of a novel sequence mediating regulated endocytosis of the G protein-coupled -pheromone receptor in yeast. Mol. Biol. Cell 4:511-521[Abstract].

SCHANDEL, K. A. and D. D. JENNESS, 1994  Direct evidence for ligand-induced internalization of the yeast -factor pheromone receptor. Mol. Cell Biol. 14:7245-7255[Abstract/Free Full Text].

SCHIESTL, R. H. and R. D. GIETZ, 1989  High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr. Genet. 16:339-346[Medline].

SHAH, A. and L. MARSH, 1996  Role of SST2 in modulating G protein-coupled receptor signaling. Biochem. Biophys. Res. Comm. 226:242-246[Medline].

SHERMAN, F., 1991  Getting started with yeast. Methods Enzymol. 194:3-21[Medline].

STRATTON, H. F., J. ZHOU, S. I. REED, and D. E. STONE, 1996  The mating-specific G protein of Saccharomyces cerevisiae down regulates the mating signal by a mechanism that is dependent on pheromone and independent of G_ß sequestration. Mol. Cell. Biol. 16:6325-6337[Abstract].

WHITEWAY, M., L. HOUGAN, D. DIGNARD, D. Y. THOMAS, and L. BELL et al., 1989  The STE4 and STE18 genes of yeast encode potential ß and subunits of the mating factor receptor-coupled G protien. Cell 56:467-477[Medline].

YU, J. H., J. WIESER, and T. H. ADAMS, 1996  The Aspergillus FlbA RGS domain protein antagonizes G protein signaling to block proliferation and allow development. EMBO J. 15:5184-5190[Medline].

This article has been cited by other articles:

				J. C. Lin, K. Duell, and J. B. Konopka A Microdomain Formed by the Extracellular Ends of the Transmembrane Domains Promotes Activation of the G Protein-Coupled {alpha}-Factor Receptor Mol. Cell. Biol., March 1, 2004; 24(5): 2041 - 2051. [Abstract] [Full Text] [PDF]

C. R. DeMattei, C. P. Davis, and J. B. Konopka Point Mutations Identify a Conserved Region of the Saccharomyces cerevisiae AFR1 Gene That Is Essential for Both the Pheromone Signaling and Morphogenesis Functions Genetics, May 1, 2000; 155(1): 43 - 55. [Abstract] [Full Text]