Ceil, Vol. 67, 389-402, October 18, 1991,Copyright© 1991 by Cell Press

S. cerevisiae Pheromone Receptors Activate a Novel Signal Transduction Pathway for Mating Partner Discrimination Catherine L. Jackson, James B. Konopka, and Leland H. Hartwell Department of Genetics, SK-50 University of Washington Seattle, Washington 98195

Wild-type S. cerevisiae cells of both mating types prefer partners producing high levels of pheromone and mate very infrequently to cells producing no pheromone. However, some mutants that are supersensitive to pheromone lack this ability to discriminate. In this study, we provide evidence for a novel role of a pheromone receptors in mating partner discrimination that is independent of the known G protein-mediated signal transduction pathway. Furthermore, in response to pheromone, receptors become localized to the emerging region of morphogenesis that is positioned adjacent to the nucleus, suggesting that receptor localization may be involved in mating partner discrimination. Actin, myosin 2, and clathrin heavy chain are involved in mating partner discrimination, since strains carrying mutations in the genes encoding these proteins result in a small but significant defect in mating partner discrimination.

process of mating partner discrimination. The pheromone receptor in the MATa cell is encoded by the STE2 gene (Jenness et al., 1986; Blumer et al., 1988) and in the MATa cell by the STE3 gene ( Hagen et al., 1986; Nakayama et al., 1985). Both pheromone receptors are thought to act by inducing dissociation of a tripartite G protein (Miyajima et al., 1987; Dietzel and Kurjan, 1987a); dissociation of Ga (SCGIlGPA1 gene product) from the G~ (STE4)-G~( (STE18) complex (Whiteway et al., 1989) permits GI~:G~'to activate the signaling pathway (Figure 1) composed of the STE5, STE7, STE11, and FUS3 gene products; the latter probably constitute a phosphorylation cascade, since three of the four are thought to be protein kinases (see Fields, 1990 for a review). Ultimately, transcription is activated by a DNA-binding protein (Dolan et al., 1989), the STE12 product, whose activity is controlled by phosphorylation (Song et al., 1991). However, although transcription induction is necessary for mating, it is not sufficient to account for the spatial constraints of the mating reaction. Additional processes must be invoked to explain the localization of cell wall growth toward the chosen partner and the reorganization of the cytoskeleton along this axis. The purpose of the work reported in this article is to investigate whether the receptor plays a role in mating partner discrimination that is distinct from its role in signal transduction and to study the cellular location of receptor molecules during response to pheromone.

Introduction

Results

Conjugation in Saccharomyces cerevisiae is the process by which haploid MATs and MATs cells fuse to form a diploid zygote (reviewed in Nasmyth and Shore, 1987; Cross et al., 1988; Herskowitz, 1989). MATa and MATs cells each make a pheromone that is recognized by a complementary receptor on the other cell type; these pheromones and receptors are the primary determinants of MATs or MATs cell mating identity (Bender and Sprague, 1989; Michaelis and Herskowitz, 1988; Nakayama et al., 1987; Bender and Sprague, 1986; Kurjan, 1985). Cells respond to the pheromone signal of the opposite cell type by inducing the transcription of genes that encode functions needed for mating (such as the cell type-specific agglutinins (Watzele et al., 1988; Lipke et al., 1989), proteins needed for karyogamy (Rose et al., 1986), and the FUS1 protein necessary for cell fusion (Trueheart et al., 1987; McCaffrey et al., 1987). Cell wall fusion occurs at the site where the pair of mating cells are apposed; a pore is then formed in the fused walls, the cell membranes fuse, and the nuclei migrate together and fuse at their spindle pole bodies (Byers and Goetsch, 1975; Byers, 1981). Both MATs and MATs cells are capable of choosing a mating partner from a group of neighboring cells and when g~ven a choice of partners that make different levels of pheromone, prefer the partner making the most pheromone (Jackson and Hartwell, 1990a and 1990b). This result strongly implicates the pheromone receptors in the

Role of a Pheromone Receptors in Mating Partner Discrimination a pheromone receptors (encoded by the STE2 gene) are necessary in MATa cells to activate the mating signal transduction pathway, since strains lacking receptors are unable to respond to a pheromone (Burkholder and Hartwell, 1985; Jenness et al., 1983, 1986). For this reason, at least, the STE2 receptor is required for mating by MATs cells. To determine whether the a pheromone receptor plays a role in addition to its role in signal transduction, G-protein signaling was placed under temperature control and made independent of pheromone-receptor binding (Figure 1) by using an scgl steS-3(ts) strain (Blinder et al., 1989). The SCG1 gene, encoding a putative Ga subunit, is a negative regulator of the signal transduction pathway that acts downstream of the STE2-encoded receptor (Dietzel and Kurjan, 1987a; Miyajima et al., 1987; Nakayama et al., 1988; Blinder et al., 1989). In a haploid scgl strain, the mating response is expressed constitutively, and hence such a strain is inviable because of failure to recover from G1 arrest (Jahng et al., 1988; Dietzel and Kurjan, 1987a; Miyajima et ai., 1987). Inactivation of the STE5 gene product inhibits the signal transduction pathway at a point downstream of SCG1 (Blinder et al., 1989; Nakayama et al., 1988). Hence, an scgl ste5-3(ts) strain (in which the steS-3 allele is functional at 23°C but not at 34°C) is viable and sterile at 34°C, but Gl-arrested and

Summary

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Figure 1. The Mating Signal Transduction Pathway In the wild-typeMATacell, a pheromonebinds to STE2receptor, which in turn stimulatesdissociationof the trimericG protein composedof Ga (SCG1),G~ (STE4), and G7 (STE18)subunits. The GI3-G~"complex stimulates STE5, which in turn activates downstream functions (not shown), leading ultimately to transcriptional inductionof genes neededfor mating. In the scgl ste5ts strain,the pathwayis constitutively activated,and signal is controlledby the effect of temperatureon the ste5gene product.

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fertile at 23°C. Because the signal transduction pathway can be activated independently of pheromone-receptor binding in an scgl steS-3 strain, we were able to examine a possible role for receptors in this strain independent of the normal requirement for receptors in activation of the signal transduction pathway. To test the role of pheromone receptor in mating partner discrimination, we employed the following experimental design (Figure 2). The Mata cell is mixed with an excess of Mata cells so that each Mata cell is surrounded by potential Mata mating partners. These partners are of two types in equal number, those that produce a pheromone and those that do not. The two Mata strains carry different auxotrophic markers so that matings with each Mata can be independently selected. In this mating discrimination test, a wild-type Mata strain will mate almost exclusively with the Mata partner that produces pheromone, but some mutant Mata strains will mate randomly, an equal number of dipIoids being formed with each Mata strain (Jackson and Hartwell, 1990b). To determine how efficiently the mating response is activated in an scgl steS-3 strain after a shift in temperature from 34°C to 23°C, we tested the mating efficiency of an scgl::LEU2 ste5-3 strain, an SCGlsteS-3 strain, and a wild-type SCG1 STE5 strain at various times after shifting the temperature from 34°C to 23°C (Figure 3). The scgl:: LEU2 ste5-3 strain mated very poorly (approximately 0.001%) when challenged to mate for 3.5 hr at 23°C immediately after transfer from 34°C. The mating efficiency increased to approximately 0.1% after the scgl::LEU2 ste5-3 strain was held for 3 to 5 hr at 23°C prior to mating. This level of mating was the maximum observed by Nakayama et al. (1988) after shifting an scg1::HIS3 GAL-SCGI ste2 MATa strain to glucose medium to deplete cells of the Scgl protein. This low level of mating was not simply due to poor activity of the Ste5-3 protein at 23°C, since at all time points tested after the shift to 23°C, the SCGI steS-3 strain mated at a level similar to that of the wild-type strain (30%-60% of the wild-type level). Both scgl::LEU2 and scgl::HIS3 alleles contain insertions into the SCG1 gene and hence could potentially express fragments of this gene; we considered the possibility that the low mating efficiency of these strains is due to the

presence of fragments of the Scgl protein interfering in the mating pathway in some way. Therefore, we also tested strains containing the scgl::URA3 gene, which has a large deletion encompassing residues 116-401 of the SCG1 gene (Miyajima et al., 1988). The scgl::URA3 ste5-3 strain also mated very poorly immediately after being shifted to 23°C, and the mating efficiency increased with time of preincubation at 23°C (Figure 3). It is puzzling that the scgl mutant strains mate so poorly. We suggest two possible explanations. First, SCG1 plays a role in adaptation to pheromone signal (Jahng et al., 1988; Miyajima et al., 1989), and the proper kinetics of adaptation may be important in mating. Second, the STE2 receptor coupled to G protein has a higher affinity for ligand than when uncoupled (Blumer and Thorner, 1990), and hence the uncoupled receptor may be less responsive to pheromone.

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Figure 2. The Mating DiscriminationTest The MATa cell is mixed with an excess of MATa cells, so that each MATa cell is surrounded by potential MATa mating partners. These partnersare of two types in equalnumber,thosethat producea pheromone and those that do not. The two MATa strains carry different auxotrophicmarkers so that matingswith each MATacan be independently selected.

Mating Partner Discrimination 391

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Figure 3. Mating Efficiency and Partner Discrimination in Constitutively Signaling Strains with or without a Pheromone Receptors Strains 7667-7 Mata and 7668-1 Mata were grown overnight at 34°C to midlogarithmic phase, shifted to 23°C for the amount of time indicated, then challenged to mate with a 1:1 mixture of pheromoneless and wild-type Mata cells (strains mfalmfa2 and 7609-7-3, respectively) for 4 hr at 23°C. "% Diploids" is the percentage of Mata cells that formed diploids with either the wild-type or pheromoneless Mata cells. "Randomness Index", interpreted to be the fraction of Mata cells choosing a mating partner at random among the wild-type and pheromoneless Mata cells, was calculated as described in Experimental Procedures. Circles, strain 7667-7 scgl ::URA3ste5-3ste2-10:: LEU2; squares, strain 7668-1 scgl::URA3 steS-3STE2;closed symbols, percent total dipIoids; open symbols, randomness index.

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Despite the low mating efficiency of the s c g l ste5-3 cells, we used these strains to test the effect of receptors on the ability to discriminate mating partners. As a control, we first tested scgl::LEU2 ste5-3 ste2-10::LEU2 cells containing a complete deletion of the a pheromone receptor gene (Burkholder and Hartwell, 1985; Konopka et al., 1988) in the discrimination assay. As expected for a strain producing signal independently of receptor (Jahng et al., 1988), essentially all of the scgl::LEU2 ste5-3 ste2-10:: LEU2 cells chose a mating partner randomly among pheromone-producing and pheromone-nonproducing cells (Table 1); it is important to note that 5 0 % mating with the"pheromoneless" strain is equivalent to 100% random matings in these experiments. We then tested the effect of a functional receptor gene in the scgI::LEU2 steS-3 strain. When the scg I::LEU2 ste5-3 STE2 strain was tested in the discrimination assay, only 14% of the cells chose a mating partner randomly (Table 1). Thus, although this

strain mated with the pheromoneless strain to a significant extent, > 8 0 % of the scgl::LEU2 ste5-3 STE2 cells were able to discriminate pheromone-producing and nonproducing cells as mating partners. We tested the scgl:: URA3 ste5-3 STE2 and scgl::URA3 steS-3 ste2-10::LEU2 strains in the discrimination assay at different times after a shift in temperature from 3 4 ° C to 23°C. At each time point, 7 5 % to 100% of the cells containing no receptors chose mating partners randomly, whereas only 15% to 2 9 % of the cells containing receptors chose a mating partner randomly (Figure 3). These results suggest that receptors can function independently of their activation of the known G protein-signaling pathway to bias mating partner discrimination toward the pheromone-producing cells. Mating P a r t n e r Higher

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Table 1. STE2 Enhances the Ability of scgl Cells to Discriminate Mating Partners Responder a Straina

Percent Total Diploidsb

Pheromoneless a Cells Total ~zcellsc

Percent Matings with Pheromoneless Straind

Randomness Indexe

ste5-3 scgl::LEU2 ste2-10::LEU2

0.36 0.25

0.52 0.49

66.0 62.0

1.28 1.30

steS-3 scgl::LEU2 STE2

0.46 0.72

0.48 0.52

6.5 7.1

0.137 0.136

ste5-3 SCG1 STE2

43.6 43.0

0.47 0.47