The Alpha-Factor Receptor C-terminus Is Important for Mating Projection Formation and Orientation in Saccharomyces cerevisiae

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1 The Alpha-Factor Receptor C-terminus Is Important for Mating Projection Formation and Orientation in Saccharomyces cerevisiae Laura G. Vallier, 1 Jeffrey E. Segall, 2 and Michael Snyder 1 * Cell Motility and the Cytoskeleton 53: (2002) 1 Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 2 Department of Anatomy and Cell Biology, Albert Einstein College of Medicine, New York, New York Successful mating of MATa Saccharomyces cerevisiae cells is dependent on Ste2p, the -factor receptor. Besides receiving the pheromone signal and transducing it through the G-protein coupled MAP kinase pathway, Ste2p is active in the establishmentandorientationofthematingprojection.weinvestigatedtheroleofthecarboxyl terminusofthereceptorinmatingprojectionformationandorientationusingaspatial gradientassay.cellscarryingtheste2-t326mutation,truncating105ofthe135amino acids in the receptor tail including amotif necessary for its ligand-mediated internalization,displayslowonsetofprojectionformation,abnormalshmoomorphology,and reduced ability to orient the mating projection toward apheromone source. This reduction was due to the increased loss of mating projection orientation in apheromonegradient.cellswithamutatedendocytosismotifweredefectiveinreorientation in apheromone gradient. ste2-296 cells, which carry acomplete truncation of the Ste2p tail, exhibit asevere defect in projection formation, and those projections that doformareunabletoorientinapheromonegradient.theseresultssuggestacomplex role for the Ste2p carboxy-terminal tail in the formation, orientation, and directional adjustmentofthematingprojection,andthatendocytosisofthereceptorisimportant forthisprocess.inaddition,mutationsinrsr1/bud1andspa2,genesnecessaryfor budding polarity, exhibited little or no defect in formation or orientation of mating projections. We conclude that mating projection orientation depends upon the carboxyl terminus of the pheromone receptor and not the directional machinery used in budding. Cell Motil. Cytoskeleton 53: , Wiley-Liss, Inc. Key words: polarized cell growth; endocytosis; mating; pheromone receptor; pheromone gradient; yeast INTRODUCTION The ability of cells to respond to external cues and generate dynamic changes in cell architecture, cell morphology, and cellular function has been well documented. Examples include axon formation and guidance in neurons, slime mold aggregation, and the response to matingpheromoneinyeastmatingpartners.howsitesof polarized cell growth are selected and how subsequent polarized cell growth is directed and maintained in response to external cues is not well understood. Yeast undergoes polarized cell growth during three distinct developmental stages of its life cycle [Roemer et al., 1996]. Vegetative cells form new buds in aprecise pattern: haploid yeast cells bud adjacent to the previous site of cytokinesis, whereas diploids bud from either pole. When starved for nitrogen, yeast cells form pseudohyphae in which cells elongate and invade their 2002 Wiley-Liss, Inc. environment to forage for sources of nutrients [Gimeno et al., 1992]. Laura G. Vallier s present address is Department of Biochemistry, Columbia University, 701 W. 168th St. HHSC720, New York City, NY Grant sponsor: NIH National Institute of General Medical Sciences National Research Service Award; Grant number: GM ; Grant sponsor: National Science Foundation; Grant number: MCB ; Grant sponsor: National Institute of Health; Grant number: GM *Correspondence to: Michael Snyder, Department of Molecular, Cellular, and Developmental Biology, PO Box , Yale University, New Haven, CT michael.snyder@yale.edu Received 4February 2002; Accepted 15 May 2002 Published online 11 October 2002 in Wiley InterScience (www. interscience.wiley.com). DOI: /cm.10073

2 252 Vallier et al. During mating in yeast, cells differentiate and form projections to become specialized cells called shmoos [Levi, 1956]. Mating is initiated when partners respond to cell-type specific mating pheromones produced by cells of the opposite mating type; MAT cells produce -factor and MATa cells produce a-factor. The pheromone binds a seven-transmembrane receptor specific for the cell type (Ste2p, the -factor receptor, is produced by MATa cells and Ste3p, the a-factor receptor, by MAT cells), which triggers a common G-protein coupled pheromone-responsive MAP kinase cascade that effects many cellular responses. Cells exposed to mating pheromone arrest in G1; the actin cytoskeleton is transiently depolarized and repolarized towards the mating partner resulting in the formation of a mating projection [Gehrung and Snyder 1990; Hasek et al., 1987; Madden et al., 1992; Read et al., 1992]. Projections are directed toward mating partners by means of a pheromone gradient emanating from each partner. Several lines of evidence indicate that mating yeast cells track pheromone gradients to direct projection growth. First, wild-type cells pick partners secreting the highest level of pheromone [Jackson and Hartwell 1990]. Second, addition of exogenous pheromone to a mating mixture drastically reduces mating efficiently [Dorer et al., 1995]. Third, cells orient their projections in a spatial gradient of pheromone [Segall, 1993]. The mechanisms of how cells establish and maintain polarized growth toward their partner once they have initiated projection formation have not been explored. Thus far, a limited number of genes important for shmoo formation have been identified in yeast. Certain mutations in FAR1 lead to projection formation at axial sites and the failure to orient toward mating partners [Chang and Herskowitz 1990; Valtz and Peter 1997; Valtz et al., 1995]. Mutants that have a disrupted actin cytoskeleton are defective in mating projection formation. These include mutations in the gene encoding actin, ACT1, the Rho1p GTPase and its exchange factor Rom2p and the GTPase activating protein, Bem2p [Read et al., 1992; Drgonova et al., 1999; Manning et al., 1997; Wang and Bretscher 1995]. Other mutants that affect cell polarity such as the Cdc42p GTPase [Johnson 1999], its exchange factor Cdc24p, [Simon et al., 1995; Toenjes et al., 1999; Zhao et al., 1995], spa2 [Gehrung and Snyder, 1990], bem1 [Chenevert et al., 1992], ste2-t326 [Konopka et al., 1988], slk1 [Costigan et al., 1992], afr1 [Konopka 1993; Konopka et al., 1995], pea2 [Chenevert et al., 1994; Sheu et al., 1998], and the fig mutants [Erdman et al., 1998] also exhibit mating projection defects. Many of these proteins localize to the shmoo tip where they might be expected to participate in mating projection orientation. These include Spa2p [Snyder 1989; Snyder et al., 1991] Sph1p [Roemer et al., 1998], Cdc24p [Toenjes et al., 1999], Bud6p and Bni1p [Evangelista et al., 1997]. Due to the limitations of mating and morphological assays, it has been impossible to evaluate the roles of the majority of these mutants in both projection formation and the orientation of the projection toward their mating partners under physiological conditions. Standard shmoo assays evaluate the ability of yeast cells to form projections in a vast excess of pheromone. We used a visual technique, the spatial gradient assay, to directly investigate steps and gene products necessary to form the mating projection and to orient the mating projection during the initiation and process of polarized growth. We find that the carboxyl terminus of the pheromone receptor is required for proper mating projection formation and orientation in yeast. We also provide evidence that endocytosis of the receptor is critical for cells to maintain and improve growth toward pheromone sources. MATERIALS AND METHODS Yeast Strains Yeast strains are listed in Table I. Strains constructed for this study were created using standard methods [Sambrook et al., 1989; Sherman et al., 1986]. Strains Y1200 and Y971 were constructed by integrating plasmid sequences linearized with StuI carrying either the STE2 allele (plv17) or the ste2 296 allele (plv15) at the ura3-52 locus in a strain lacking the STE2 gene (KBY16) (gift of K. Blumer). The presence of the allele was monitored by stable uracil prototrophy. Plasmid plv17 was constructed by ligating a EagI -EcoRI STE2 fragment from plv16 into YIp5; plv16 is the STE2 genomic sequences from pyje111 (gift of J. Thorner) on a BamHI fragment ligated into Bluescript SK at the BamHI site; plv15 carries the ste2 296 allele and was constructed from a two kilobase EcoRI - SalI fragment of pyje (gift of J. Thorner) inserted into YIp5 linearized with EcoRI and SalI. The resulting strains were maintained on SC-Ura medium [Sherman et al., 1986] prior to being assayed in the spatial gradient assay [Segall, 1993]. Spatial Gradient Assay The spatial gradient assay (SGA) was performed essentially as outlined in Segall [1993]. Briefly, MATa cells were grown to early log phase and cells were collected and resuspended in 0.5 ml YPD broth [Sherman et al., 1986]. A monolayer of cells was immobilized in low melt agarose on a YPD plate (1% yeast extract, 2% peptone, 2% dextose, 1% agar), then overlaid with YPD broth. A needle discharging -factor (Sigma

3 TABLE I. Strains Used in This Study Ste2p C-Terminus Role in Mating Polarity 253 Strain Genotype Source ABY355 MATa rsr1- ::URA3 ura3 leu2 ade3 trp1 [Bender and Pringle, 1989] KBY16 MATa mf 1::LYS2 mf 2::LEU2 ste2- ::HIS3 sst1-5 ura3-52 trp1-903 ade2-101 [Blumer et al., 1988; Reneke et al., 1988] his3-200 leu2 3, 112 lys2-801 RH1722 MATa leu2 his4 bar1-1 ste2 ::LEU2 ura3::ste2-ura3 [Rohrer et al., 1993] RH1860 MATa leu2 his4 bar1-1 ste2 ::LEU2 ura3::ste2 345Stop-URA3 [Rohrer et al., 1993] RH1906 MATa leu2 his4 bar1-1 ste2 ::LEU2 ura3::ste2 337A,345Stop-URA3 [Rohrer et al., 1993] Y602 MATa ura3-52 lys2-801 ade2-101 trp1 spa2 1::URA3 his3-200 trp1 Lab strain Y604 MATa ura3-52 lys2-802 ade2-101 trp1 his3-200 Lab strain Y971 MATa ura3-52::plv15-ste2-296-ura3 trp1 903 ade2-101 his3 200 This study (see Materials and Methods) leu2 3,112 lys2 801 mf 1::LYS2 mf 2::LEU2 ste2 ::HIS3 sst1 5 Y1200 MATa ura3-52::plv17-ste2-ura3 mf 1::LYS2mf 2::LEU2 This study (see Materials and trp1-903 ade2-101 his3-200 leu2 3, 112 lys2-801 ste2- ::HIS3 sst1-5 (Methods) MATa cry1 SUP4-3 ts ade2-1 o his4-580 a lys2 o trp1 a leu2-3, 112 ura3-52 ste2-t326 [Konopka et al., 1988] MATa cry1 SUP4-3 ts ade2-1 o his4-580 a lys2 o trp1 a leu2-3, 112 ura3-52 [Konopka et al., 1988] T6901) at the indicated concentration was placed 10 above the surface, the dish was overlaid with light mineral oil (Sigma M5904) to prevent evaporation, and the responses of the cells to the resulting diffusion gradient were captured using time lapse video-microscopy. Responses of the cells in the field were analyzed using NIH Image and Microsoft Excel software. Average values are expressed as the standard error of the mean unless otherwise stated. Ideal or optimal concentration of pheromone is defined as the minimal amount of pheromone necessary for cells to respond: wild-type cells require nm pheromone in the pipet for response to occur whereas hypersensitive strains such as bar1 and certain STE2 alleles require less pheromone to elicit response [Segall, 1993]. Strains carrying both bar1 and a hypersensitive STE2 allele show increased sensitivity over those carrying either single mutation [Segall, 1993]. Strains RH1860, RH1906, Y971, and Y1200 were grown to early log in SC-Ura, harvested, and then resuspended in YPD, processed, and assayed as above. Strains Y971, Y1200, RH1860, RH1722, and RH1906 were analyzed at 25 C; all others were analyzed at 30 C. In the reorientation assay, the subset of cells that initiated mating projection formation on the cell surface at an angle greater than 45 away from the pheromone source were analyzed for their ability to redirect their projections toward the pheromone source. Isotropic Pheromone Assay Pheromone assays were performed essentially as in Roemer et al. [1998]. Briefly, logarithmically growing MATa cells were harvested and washed once in SC Ura. Washed cells were incubated at room temperature in 5 g/ml -factor; after 1hanadditional 5 g/ml -factor was added to the cells and incubated for a second hour. Cells were then washed, fixed, and photographed. RESULTS To examine the ability of different yeast strains to orient mating projection growth, we used a spatial gradient assay (SGA) in which a pipet filled with -factor mating pheromone is positioned near a group of MATa cells. In this assay, cells arrest growth and initiate mating projection formation toward the pheromone source [Segall, 1993; Valtz et al., 1995; Nern and Arkowitz, 1998]. We first examined mating projection orientation more extensively using wild-type strains. A pipet filled with -factor was positioned near a group of MATa cells and cell cycle arrest, cell morphology and mating projection orientation were monitored as a function of time. To quantify the ability to orient the mating projection, the angle that occurs between a line from the tip of the projection to the pipet tip and the line along the actual direction of projection growth was measured and converted to a cosine value for each cell. Measurements were taken at the onset of projection formation and 4 h later. Thus, for optimal orientation (angle equals 0 ), the cosine equals one and for random orientation (angle equals 90 ), the cosine equals zero with a large standard deviation. We found that at optimal pheromone concentrations (67 nm), wild-type cells grow toward the mating source with an orientation cosine at4hof (average cosine standard error of the mean) consistent with that reported previously (see Table IIA) [Segall, 1993]. This high degree of orientation might be due to the fact that the projections initiate in a favorable direction relative to the pheromone source or that they continually track the pheromone gradient and improve their orientation. Consistent with the latter possibility, we note that projection growth at the start of the experiment is less well oriented than the same cells 4 h later (0.54 vs. 0.84). To examine this possibility more directly, we analyzed

4 254 Vallier et al. TABLE II. Components Required for Vegetative Polarity Are Not Required for Mating Polarized Cell Growth* Relevant genotype Time required to initiate projection (min) A. Spatial gradient assay Direction of projection formation at0h Direction of projection formation at4h rsr1 ::URA spa2 ::TRP WT Relevant genotype Direction of projection formation at 0 h B. Reorientation assay Direction of projection formation at 4 h rsr spa WT * All strains were examined at 67 nm pheromone in the pipet. Data are the average time in minutes of projection initiation (0 h) or of the cosine of the angle formed between the tip of the pipet to the tip of the mating projection and the line formed by the actual direction of projection growth (average standard error of the mean). For perfect orientation, cosine equals one; random orientation, cosine equals zero. Zero hours is the time at which the projection is first visible as a slight rounding on the cell surface and 4his4hafter projection initiation. A: Spatial gradient assay. B: Reorientation assay. See text and Materials and Methods for details. rsr1, ABY355; spa2, Y602; WT, (see Table I for complete genotypes). N, number of cells analyzed. N N those cells that initiated their projection growth greater than 45 away from the pheromone source (26 of 65 cells) for their ability to improve directional growth toward the pheromone source (a process that we call reorientation). We found that after 4 h, 92% of this subset of wild-type cells reoriented toward the pheromone source with an orientation cosine of (see Table IIB). Therefore, wild-type cells are able to reorient efficiently toward the pheromone source. Bud Site Selection Genes Are Dispensable For Mating Projection Orientation in an -Factor Gradient Many genes have been identified that are necessary for the successful and efficient mating of two yeast partners. However, the role of each identified gene product is just now beginning to be understood. We first examined whether proteins that had been implicated in various aspects of vegetative polarity or mating were necessary for the production and orientation of the mating projections using the SGA. Strains carrying representative mutations in bud site selection genes, structural element genes, signal transduction genes, the -factor receptor gene STE2, and those mutants that have poor or altered projection formation were analyzed. We analyzed two representative mutants defective in aspects of vegetative polarized cell growth for their ability to orient projections in the SGA. Mutants carrying a spa2 mutation display a bud site selection defect in older diploid mothers [Madden and Snyder, 1992; Snyder, 1989], a defect in low density mating [Gehrung and Snyder, 1990] and are defective in the default mating pathway [Dorer et al., 1995]. Mutants carrying a rsr1 (bud1 ) mutation are defective in haploid and diploid bud site selection, resulting in a random budding pattern [Bender and Pringle 1989; Chant and Herskowitz 1991]. We found that both MATa rsr1 /bud1 and MATa spa2 mutant cells are able to orient mating projections along an -factor pheromone gradient in the SGA (Table IIA). rsr1 /bud1 and spa2 cells initiate projection formation at min and min, respectively, which is similar to wild-type cells ( min). After 4 h, rsr1 mutant cells form and orient projections as well as wild type (average cosine of projection orientation after 4 h and , respectively). spa2 cells are also able to orient a mating projection toward a mating partner (average cosine at 4 h ), although slightly less well than either rsr1 or wild-type cells. Thus, the mating defect seen in spa2 mutants is unlikely to be due to problems in mating projection orientation. In summary, Rsr1p and Spa2p do not play critical roles in mating projection orientation, and the mechanism used in directing projection growth during mating appears to require different or additional components or mechanisms than those controlling budding patterns. We also tested whether these cells were able to reorient toward the pheromone source. From the set of cells subjected to the spatial gradient assay, we analyzed the subset that initiated mating projection formation on the cell surface at an angle greater than 45 away from the pheromone source for their ability to orient their projections toward the source. We found that MATa rsr1 and MATa spa2 mutant cells are able to effi-

5 Ste2p C-Terminus Role in Mating Polarity 255 ciently reorient mating projections along an -factor pheromone gradient as well as wild-type MATa cells (average cosine of projection orientation after 4 h for rsr1, spa2, and for WT; see Table IIB); as before, the values for spa2 mutants are slightly lower than wild type (0.66 vs. 0.77) (Table IIB). Spa2p shares 30% sequence identity with Sph1p over each of three 100 amino acid domains [Arkowitz and Lowe, 1997; Roemer et al., 1998]. Mating morphology defects for sph1 mutants are similar but less severe than those observed for spa2 mutants; sph1 spa2 double mutants exhibit a more severe shmoo morphology defect than either single mutant. Therefore, we determined if the nearly wild-type levels of orientation in spa2 mutants might be due to the redundant function of Sph1p by examining a spa2 sph1 mutant using the SGA. We found that these double mutants do not display more severe orientation defects than spa2 mutants alone (data not shown). In addition, the sph1 mutant orients as well as wild type (data not shown), consistent with previous data showing little mating or morphology defect in this strain [Roemer et al., 1998]. Therefore the slight defect observed in spa2 mutants is not due to the redundant function of SPH1. We also examined two other mutants involved in the mating response, kss1 and afr1 [Elion et al., 1991; Konopka et al., 1995]. Kss1p is a protein involved in the G-protein coupled MAP-kinase cascade that is required for mating response induction. The AFR1 gene encodes a protein that is associated with the Ste2p -factor receptor and shows localization to the shmoo neck. Neither mutant displayed defects in projection orientation (data not shown). The Carboxyl Terminus of STE2p Is Required for Rapid Onset of Shmoo Formation and Projection Orientation The STE2 gene encodes the -factor receptor, a 431 amino acid protein that is a member of the seventransmembrane superfamily of G-protein coupled receptors; this family includes rhodopsin and the -adrenergic receptor [Bockaert and Pin, 1999; Burkholder and Hartwell, 1985]. Various truncations of the cytoplasmic tail of Ste2p cause increased hypersensitivity to -factor and poor or altered projection formation [Blumer et al., 1988; Konopka and Jenness 1991; Konopka et al., 1988]. To test whether the cytoplasmic carboxyl terminus of the Ste2p receptor participated in orientation, we examined a MATa ste2 T326 mutant (truncated at residue 326, which removes all but 30 amino acids of the carboxy terminal cytosolic domain) and a MATa STE2 isogenic wild-type strain in the SGA. We found that MATa ste2 T326 cells were defective in projection initiation, morphology, and orientation (Fig. 1). The time required to initiate projections and the ability to orient projections in the SGA was quantified for both mutant and wild-type cells. MATa ste2-t326 cells require almost twice as long for projection initiation as an isogenic wild-type strain (Table III) and the projections were much larger than wild type. Wild-type cells take min to initiate projection formation whereas ste2-t326 cells take min. At the ideal pheromone concentration for wild type (67 nm in the pipet) described by Segall [1993] (see Materials and Methods), ste2-t326 cells are able to establish the initial direction of projection growth similar in accuracy to wild-type cells: the cosine at0his compared to , respectively (standard deviation 0.52 for both wild-type and ste2-t326 cells). However, after 4 h, ste2-t326 cells exhibit a reduced ability compared to wild-type cells to improve the orientation of their projections along the pheromone gradient: the cosine at 4 h is for ste2-t326 compared to for wild type (standard deviation is 0.51 for ste2-t326 compared to 0.26 for wild type). The fact that cells carrying ste2-t326 do not exhibit improved orientation at 4 h is not due to the tenfold hypersensitivity of ste2-t326 mutants to pheromone [Konopka et al., 1988], which would cause a perceived saturation of receptors on the cell surface. When ste2-t326 mutant cells were tested in the SGA using a tenfold lower pheromone concentration (6.7 nm), orientation at 4 h (cosine at 4 h ) was not significantly improved. Wild type cells are unable to respond to this pheromone concentration. Thus, the cytoplasmic carboxyl terminus is dispensable for initial orientation but is necessary for continued improvement of direction toward the mating partner. When the concentration of pheromone in the gradient is increased, we found that the ability of wild-type cells to orient toward a pheromone source was inversely proportional to the concentration of the pheromone. One possibility is that receptor occupation by pheromone becomes more uniform over the surface of the cell as the pheromone concentration in the gradient increases and thus affects the ability of the cell to detect the pheromone source. This is consistent with the possibility that a difference between receptor occupancy on the stimulus side of the cell and the opposite side of the cell is necessary for that cell to orient toward the stimulus [Segall, 1993]. At the highest concentration tested (333 nm), the orientation of wild-type cells at 4 h in the SGA bears a striking similarity to that seen for the ste2-t326 cells (compare wild type at 333 nm pheromone: cosine at 4h (standard deviation equals 0.60) to ste2-t326 at 6.7 nm: cosine at 4 h (standard deviation 0.55).

6 256 Vallier et al. Fig. 1. ste2-t326 mutants are defective in mating projection formation and orientation. Wild-type (top) and ste2-t326 (bottom) MATa cells were photographed at 0 h (left) and at 5 or 8 h, respectively (right), after being in a diffusion gradient of pheromone emanating from a micropipet (out-of-focus object at the right in all panels) at 30 C. In wild-type cells, mating projections grow and orient along the pheromone source; ste2-t326 cells take longer to initiate projection formation and the responding cells have a greater volume with projections frequently seen growing away from the gradient (direction of growth for representative cells is marked with arrow). Wild-type strain is and ste2-t326 strain is Concentration of pheromone in the pipet is 67 nm for both strains. Scale bar 10. The reduction in the ability of ste2-t326 cells to orient may be due to the fact that cells that initiate growth in an unfavorable direction are unable to improve their orientation. We, therefore, examined those cells that initiate their projections not directly in line with the pheromone source for their ability to improve directional growth toward the pheromone source. Cells that initiated their projections greater than 45 away from the pheromone source were examined for projection reorientation. We found that the subset of ste2-t326 cells that initiated their projections at an angle greater than 45 from the pheromone source (i.e., cosine at0hisless than 0.71) were slightly more impaired in their overall ability to orient in the gradient (compare Table IIIA and B) as compared to wild-type cells. At the optimal pheromone concentration for ste2-t326 (6.7 nm), the improvement over the 0-h time point is somewhat enhanced but not to the extent of wild-type cells at their ideal pheromone concentration (67 nm) (ste2-t326 cosine at4h compared to wild-type cosine at 4 h, which equals ). Thus, ste2-t326 cells that are not initially oriented toward the pheromone also are partially impaired in reorienting toward the pheromone gradient. ste2-t326 Cells Frequently Lose Orientation Toward the Pheromone Source The modest defect observed in the average cosine values for orientation of the ste2-t326 cells (see Table

7 TABLE III. ste2-t326 Mutants Are Defective in the Onset of Projection Formation and the Improvement of Projection Orientation* -factor (nm) Cosine at 0 h WT Cosine at 4 h A. Ability to orient in an -factor gradient ste2-t326 Time to initiate projection N Cosine at 0 h Cosine at 4 h Time to initiate projection 6.7 nd nd nd factor (nm) Cosine at 0 h WT Cosine at 4 h B. Ability to reorient in an -factor gradient Ste2p C-Terminus Role in Mating Polarity 257 ste2-t326 Time to initiate projection N Cosine at 0 h Cosine at 4 h Time to initiate projection 6.7 nd nd nd * All strains were examined at the concentration of pheromone indicated in the first column. Data are the average the standard error of the mean. Cosines are determined from the angle formed between the tip of the pipet to the tip of the mating projection and the line formed by the actual direction of projection growth. Time is the minutes after exposure to pheromone for projection formation to occur (0 h). Zero hours is the time at which the projection is first visible as a slight rounding on the cell surface and4his4hafter projection initiation. A: Spatial gradient assay. B: Reorientation assay. See Material and Methods and text for further details. WT, ; ste2-t326, (see Table I for complete genotypes). nd, not determined; N, number of cells analyzed. N N III) do not seem to reflect the defect seen in the visual record (see Fig. 1). We hypothesized that the ste2-t326 average cosine values might not be representative of the visual record because some of the ste2-t326 cells lose orientation at various times during the assay. Consistent with this interpretation, at a concentration of 6.7 nm pheromone in the pipet, the standard deviation for ste2-t326 cells at 4 h was almost twice as high as that observed for wild type (see Table IIIA and B). To determine if this large standard deviation was due to the loss of orientation in mutant cells, we measured how many cells analyzed in Table IIIB failed to maintain or improve orientation by comparing values at 4 h to those at 0 h (Fig. 2). We found that 15% of STE2 wild-type cells lose orientation, whereas ste2-t326 cells lose orientation almost three times more frequently than wild-type cells at their respective ideal pheromone concentration. Wildtype cells lose orientation with greater frequency as the -factor concentration is increased. In contrast, ste2-t326 cells consistently exhibit a high rate of loss of orientation at all pheromone concentrations. Reorienting cells display similar characteristics for both wild type and ste2-t326 (data not shown). Thus, MATa cells carrying the ste2-t326 allele appear to be defective in maintaining orientation. Fig. 2. ste2-t326 cells lose orientation in a pheromone gradient at increased frequency. Data from Table IIIA were examined to determine the percentage of wild-type and ste2-t326 cells that fail to maintain orientation in a pheromone gradient. The percentage of cells that lose orientation (more than 10 away from the pheromone source) as a function of pheromone concentration tested is shown. Wild-type cells lose orientation at a low frequency and this frequency increases as the pheromone concentration is increased; ste2-t326 cells exhibit a rate of orientation loss almost threefold higher than wild type.

8 258 Vallier et al. TABLE IV. ste2-337a,345stop Mutants Are Defective in the Reorientation Assay* -factor (nm) Cosine at 0 h ste2-345stop Cosine at 4 h Ability to reorient in an -factor gradient ste2-337a,345stop Time to initiate projection N Cosine at 0 h Cosine at 4 h Time to initiate projection 24 nd nd nd * Cells whose projections at 0 h initiate at a point on the cell surface greater than 45 away from the pheromone source were analyzed for the ability to reorient their projections toward the pheromone source. Data are the time of projection initiation at 0 hs and the average cosine of the angle formed between the tip of the pipet to the tip of the mating projection and the line formed by the actual direction of projection growth (average standard error of the mean). ste2-345stop, RH1860; ste2-337a,345stop, RH1906 (see Table I for complete genotype); time is in minutes; nd, not determined; N, number of cells analyzed in reorientation assay. N Endocytosis of the Receptor Is Needed for Reorientation The carboxy-terminal tail of the oligomerized Ste2p receptor is important for endocytosis of the pheromoneactivated receptor [Jenness and Spatrick, 1986; Overton and Blumer, 2000; Yesilaltay and Jenness, 2000]. A domain beginning at residue 331 (SINNDAKSS) within the tail that is necessary for Ste2p endocytosis has been identified previously [Rohrer et al., 1993]. Because the ste2-t326 mutation truncates the receptor protein prior to this domain, it is possible that endocytosis of the activated receptor contributes to the ability to track the gradient. Two strains were tested: one carrying a mutation in STE2 that truncates the gene product at residue 345 (ste2-345stop), and the other carrying the same truncation at residue 345 with a second mutation of Lysine to Alanine at residue 337 within the endocytosis domain (ste2-337a,345stop) [Rohrer et al., 1993]. The mutant carrying only the ste2-345stop allele displays proper endocytosis of the receptor while the ste2 337A,345Stop mutant abolishes endocytosis [Rohrer et al., 1993]. We examined these two mutants in the SGA and the reorientation assay for their ability to orient in a pheromone gradient. In the reorientation assay, the ste2-345stop mutant was able to improve its orientation in the gradient whereas the ste2 337A,345Stop mutant failed to improve projection orientation (Table IV). We note that under SGA conditions, both ste2-345stop and ste2-337a,345stop mutants were unable to maintain projection orientation (data not shown). We also note that cells carrying the wild-type STE2 allele at the same ectopic site do not show improved orientation in the SGA 4 h after projection initiation (data not shown). The reorientation data suggest that endocytosis is an important mechanism for the reorientation of mating projections and that an intact SINNDAKKS domain in the Ste2p carboxyl terminus is necessary. Although in the reorientation assay ste2-337a,345stop mutants display a more severe reorientation defect than ste2-t326 mutants, ste2-337a,345stop mutants do not completely mimic the spectrum of phenotypes observed in ste2-t326 mutants: the timely initiation of projection formation is unimpaired and the morphology of the projection appears wild type. Thus, endocytosis is likely to be an important but not exclusive mechanism in the orientation or reorientation of mating projections but is not involved in projection initiation or morphology. Mutants Lacking the Entire Carboxy-Terminal Tail of STE2p Are Unable to Efficiently Form Projections in an -Factor Gradient Because the Ste2-T326p mutant receptor retains 30 amino acids at the carboxyl terminus that are still present in the cytoplasm, we determined whether the residual orientation is due to the presence of these residues. A receptor mutant truncated at residue 296 (ste2-296) that removes the entire cytoplasmic tail of STE2 [Blumer et al., 1988] was constructed and integrated at the ura3-52 locus. In parallel, we integrated the wild-type STE2 gene with its full-length tail at the same locus in the identical background (see Materials and Methods). We examined the ability of these mutants to orient in the spatial gradient assay. Cells carrying the ste2-296 mutation have been reported to be 100-fold hypersensitive to pheromone [Blumer et al., 1988] and both wild-type and mutant constructs are in a bar1 background, which increases sensitivity to pheromone [Segall, 1993]. Therefore, we lowered the concentration of pheromone to compensate for this increased sensitivity. Surprisingly, we found that although ste2-296 cells arrested budding, they failed to initiate projections and grew isotropically in diameter, thereby forming larger round cells (data not shown). We then raised the pheromone concentration to see if these cells could form projections with higher amounts

9 Ste2p C-Terminus Role in Mating Polarity 259 Fig. 3. ste2 296 mutants rarely initiate projection formation in a pheromone gradient but form projections in an isotropic pheromone assay. A: STE2 (left) and ste2 296 mutant (right) strains were photographed after 20 or 15 h, respectively, in a pheromone gradient at C. Pipet containing -factor is indicated by black arrowhead. Pheromone concentrations in the pipet are 7 nm for the STE2 strain and 5 nm for the ste2 296 strain. White arrows mark the direction of growth. Scale bar 10. B: STE2 and ste2 296 strains were photographed after treatment with isotropically added -factor pheromone (5 mg/ml; see Materials and Methods). Scale bar 10. STE2, Y1200; ste2 296, Y971. of pheromone. We found that as the pheromone concentration increased, ste2-296 mutants displayed an increased, although still extremely diminished, ability to form shmoos (Fig. 3A). Lack of projection initiation in these cells was not due to insufficient pheromone concentration because the cells still underwent G1 arrest. We quantified the ability to form projections at varying concentrations of pheromone in Table VA. Wild-type cells formed projections efficiently at the expected pheromone concentration. We found that ste2-296 cells were unable to form projections efficiently over a broad range of pheromone concentrations in the gradient (Table VA). At the highest concentration tested, less than half of the cells responded; at concentrations closer to ideal for ste2-296 arrest ( nm), projection initiation drops to less than 10%. In contrast, almost all wild-type cells showed projection initiation. Therefore, the 30 residues between amino acid 296 and 326 in Ste2p seem to be important for projection initiation but not for cell cycle arrest. Because neither the Ste2-T326p nor the Ste2 296p mutant receptors are internalized, we determined whether those few projections formed by the ste2-296 cells had a similar or more severe orientation defect than that

10 260 Vallier et al. TABLE V. ste2 296 Cells Are Able to Make Projections Only Rarely and Are Unable to Reorient Them in a Pheromone Gradient* -factor (nm) A. Projection formation -factor (nm) WT ste2-296 (%) 0.2 nd 4/55 (7) 0.5 nd 6/48 (13) 0.75 nd 2/25 (8) 1.0 nd 10/41 (24) /109 (97%) 45/113 (40) Cosine at 0 h WT Cosine at 4 h Number of shmoos/ total number of cells scored B. Reorientation assay Ability to reorient in an -factor gradient ste2-296 Time to initiate projection N Cosine at 0 h Cosine at 4 h Time to initiate projection nd nd nd * A: The cells able to initiate projection formation were counted at each pheromone concentration and were expressed as a fraction of the total number of cells retaining the plasmid during the experiment. Percentage is in parentheses. B: Ability of cells to reorient in a pheromone gradient. Data are the average cosine of all cells that formed and maintained projections for 4 or more hours and the average time to initiate projection formation (average the standard error of the mean). Cells that initiated a projection in A but did not maintain it for at least 4 h due to the termination of the experiment or loss of plasmid construct were not analyzed in B. ste2-296, Y971; STE2, Y1200 (see Table I for complete genotypes); nd, not determined; N, number of cells analyzed in the reorientation assay. N observed in ste2-t326 cells. We found that those ste2 296 cells able to form a projection initiated them more slowly than ste2-t326 cells and over twice as slow as the STE2 control cells (Table VB). For ste2-296 mutants, the average time of projection initiation was 761 min (ranging from 666 to 1,230 min; n 35) whereas for ste2-t326 mutants, the average time of onset was 425 min (ranging from 370 to 473 min; n 215) as seen in Tables IIIB and VB. In comparison, wild-type cells in this strain background initiated projection orientation with an average time of 347 min (n 43) (Table VB). Wild-type cells arrested growth and then produced mating projections in 88% of the cells; 12% of the cells budded with an average doubling time of 3.5 h (and presumably lost the construct in the non-selective medium). In contrast, 23% of the ste2-296 cells arrested growth and formed mating projections (ranging from 6 to 39% depending on concentration) (see Table VA); 55% of the cells budded (ranging from 39 to 71%) with an average budding time of 9.3 h (ranging from 5.3 to 12.5 h) (data not shown); the remainder (22%) arrested and swelled but neither budded or formed projections. However, the budding by the ste2-296 mutants in a pheromone gradient does not seem likely to be due to loss of the ste2-296 construct because ste2-296 cells tend to bud at a slower rate than that observed for cells without the construct, which are unable to respond to a pheromone gradient. We note that ste2-296 cells are unimpaired for vegetative growth. Therefore, at this time it is unclear what role ste2-296 may have in promoting this slow budding. Additionally, ste2-296 cells are defective in the reorientation assay (average cosine after 4 h is vs at onset of projection initiation); in contrast, STE2 cells exhibit marked improvement in the reorientation assay (average cosine after 4his vs at onset of projection of initiation) (Table VB). Thus, mating projection initiation and reorientation in an -factor gradient is partially dependent on residues 296 to 326 in Ste2p. ste2-296 Mutants Form Projections in an Isotropic -Factor Assay In a standard shmoo assay, MATa cells are treated with a vast excess of -factor pheromone (5 g/ml) for a short period of time (2 h). Log-phase wild-type and ste2-296 cells were treated identically with -factor for 2 h and their response was photographed. Both wild-type and ste2-296 cells were able to form normal projections in isotropic pheromone at a high concentration (Fig. 3B). The ability of the ste2-296 mutant to form normal projections indicates first that the mutant is capable of forming mating projections and, second, that the mating response pathway is intact. DISCUSSION In this study, we have examined the function of carboxy-terminus of the Ste2p -factor receptor in mat-

11 Ste2p C-Terminus Role in Mating Polarity 261 ing polarized cell growth using the spatial gradient assay, an assay that approximates physiological conditions for mating projection formation in MATa yeast cells [Segall, 1993]. We presented evidence indicating that the formation of mating projections is a complex, separable process involving the selection of the site of projection formation, and the formation and orientation of the mating projection toward the pheromone of the mating partner. By analyzing mutations within the cytoplasmic carboxy-terminal tail of the Ste2p -factor receptor, we have shown definitively that the cytoplasmic tail is necessary for both the initiation and the maintenance of orientation of the mating projection toward a mating partner. We further present data that endocytosis of the activated receptor contributes to reorientation of the projection toward a pheromone source. Budding Polarity Components Are Not Required for Mating Polarity We have shown that components required for polarized cell growth during vegetative growth are not necessary for polarized cell growth during the mating response when the Ste2p receptor is intact. Mutants lacking RSR1 (BUD1) appear to orient projections as well as wild type. Thus, a subset of proteins is specifically dedicated to vegetative polarity function. This is not surprising because of the different nature of the signals: vegetatively growing cells use an intrinsic, internal signal to mark the site of the next budding event while cells responding to a mating partner need to utilize a more adaptable way to organize growth components to form a projection toward the projection of the partner [for reviews see Madden and Snyder, 1998; Roemer et al., 1996]. However, the process may be more complex because Nern and Arkowitz [1999] showed that in the absence of FAR1-CDC24-mediated morphological changes, RSR1/BUD1 was essential for pheromone-induced polarized cell growth. Mutants lacking Spa2p, which is required for polarized growth in budding diploid cells, exhibit a minor but consistent reduction in the ability to orient projections. One possibility is that the Spa2p homolog, Sph1p, has a redundant function with Spa2p. [Arkowitz and Lowe, 1997; Roemer et al., 1998]. In the spatial gradient assay, mutants deleted for SPH1 do not show any reduction in the ability to orient mating projections and the double mutant, spa2 sph1, does not further enhance the weak spa2 defect. Thus, SPA2 and SPH1 are not required for projection orientation but may assist in the process presumably by concentrating growth at the tip [Sheu et al., 2000]. ste2-t326 Mutants Show Defects in Mating Orientation Previous data had shown that the shmoo morphology was blunted in ste2-t326 mutants in a shmoo assay but its role in projection orientation had not been assessed [Konopka et al., 1988]. We have shown that this mutant is defective for several aspects of mating projection initiation and orientation. Mutants carrying the ste2-t326 allele require a longer time to initiate projection formation; the projections have a larger volume and often turn away from the pheromone gradient. These results are in agreement with a previous analysis of Ste2p in which ste2-t326 cells exposed isotropically to pheromone for 6 h exhibit abnormal shmoo morphology [Konopka et al., 1988]. However, our analysis reveals new functional defects in these mutants. Analysis of ste2-t326 mutant cells in the spatial gradient assay using increasing concentrations of -factor pheromone revealed that regardless of concentration, the initial direction of projection formation is equivalent to that in wildtype cells. However, cells carrying these mutant receptors tend to lose orientation in a pheromone gradient, such that some cells are oriented toward the pheromone gradient then turn away from the gradient. Wild-type cells rarely lose orientation at a normal concentration of pheromone but as the concentration of -factor increases, loss of orientation increases, consistent with loss of wild-type mating efficiency in saturating concentrations of -factor [Dorer et al., 1995]. In contrast, almost half of the ste2-t326 mutant cells lose orientation at all pheromone concentrations. The hypersensitivity to pheromone exhibited in cells carrying the ste2-t326 allele is unlikely to be the reason for the loss of orientation because when the concentration of pheromone is lowered to compensate for the hypersensitivity, the defect remains: mutant cells continue to lose orientation at a similar frequency; at the same concentration of pheromone, wild-type cells do not arrest growth. Thus, this work demonstrates for the first time that the cytoplasmic carboxy-terminal tail of the Ste2p receptor appears to be required for efficient initiation as well as for the orientation of mating projections in yeast. The Role of Endocytosis The carboxyl terminus of Ste2p is predicted to contain 135 amino acids located intracellularly [Burkholder and Hartwell, 1985; Cartwright and Tipper, 1991]. The Ste2p tail has many potential phosphorylation sites and an endocytosis motif, which contribute to the internalization of the receptor [Chen and Konopka, 1996; Hicke et al., 1998; Rohrer et al., 1993]. The endocytosis motif, 331 SINNDAKSS 339, is required for both constitutive and ligand-mediated internalization of the pher-

12 262 Vallier et al. omone receptor, which is signaled by the ubiquitination of the lysine at residue 337 [Hicke and Riezman 1996]. The ste2-t326 mutation truncates the last 105 amino acids of the receptor, which removes the endocytosis motif; 30 amino acids still remain in the cytoplasm [Konopka et al., 1988]. To test whether endocytosis of the receptor plays a role in projection orientation, we examined two alleles of the STE2. Cells carrying the ste2-345stop allele have normal internalization of the receptor while cells carrying the additional alteration of the lysine to alanine at residue 337 in the endocytosis motif (ste2-337a,345stop) lack internalization of the receptor [Rohrer et al., 1993]. When the cells were subjected to the reorientation assay, we found that the ste2-337a,345stop mutants were more defective in reorienting than the ste2-345stop cells (Table IV). Thus, endocytosis of the activated receptor contributes to reorientation of the mating projection. In contrast to ste2-t326 cells, the time of projection initiation of the ste2-337a,345stop cells was similar to that seen for the ste2-345stop cells as well as for cells carrying STE2 at its endogenous locus. This suggests that the cause for the delayed onset of projection initiation in ste2-t326 cells may be the loss of the amino acids between 326 and 345. We note that in the spatial gradient assay, both the ste2-345stop and the ste2-337a,345stop strains showed a defect in orientation. Tailless Receptor Mutants Are Defective in Projection Formation Both ste2-296 and ste2-t326 mutants are defective in the endocytosis of activated pheromone receptors [Blumer et al., 1988; Konopka and Jenness, 1991; Rohrer et al., 1993]. However, the phenotype of ste2-296 is much more severe than ste2-t326: when the residues after amino acid 296 are removed, cells can no longer efficiently establish projection formation and those rare projections that do occur are misshapen and are not directed toward the pheromone source. These results are consistent with the drop in mating efficiency in ste2-296 mutants compared to ste2-t326 mutants [Konopka et al., 1988; Reneke et al., 1988]. Therefore, other mechanisms besides endocytosis must contribute to projection formation and orientation. The distal half of the carboxy-terminus of the pheromone receptor has been shown to form preactivation complexes with its G-proteins [Docil et al., 2000]. Also, Cdc24p, Far1p, and Ste4p (G-beta) form a complex as gauged by the two-hybrid assay and localization of Cdc24p to the site of receptor activation occurs in the absence of the other factors in the complex [Nern and Arkowitz, 1999; Toenjes et al., 1999]. Furthermore, shuttling of the Cdc24p-Far1p complex by Msn1p from the nucleus to the site of polarized cell growth occurs in response to mating pheromones [Blondel et al., 1999; Butty et al., 1998; Shimada et al., 2000]. These varied mechanisms could rely on the Ste2p carboxy-terminus to localize the growth components to the site of activated pheromone receptors and together provide a timely and efficient response to mating pheromone. In a pheromone gradient, most ste2-296 mutants arrest growth or bud at an extremely slow rate and continue to swell. The slow budding may reflect the weakening or loss of a budding checkpoint that is maintained during mating through a component that is associated directly or indirectly with the Ste2p cytoplasmic tail. Alternatively, it is possible that this is a secondary effect due to the increased cell volume diluting the concentration of a critical repressor of cell division. Finally, the slow budding kinetics of ste2 296 cells might reflect a differential progression to a signaling threshold in which adaptation mechanisms to the pheromone signal are activated. Surprisingly, we found that ste2-296 cells are able to form a seemingly normal mating projection in an isotropic shmoo assay at high concentrations of -factor. This is probably due to the scavenging of bud site selection machinery already in place for the next cell division that is seen for wild-type cells [Madden and Snyder, 1992]. Madden and Snyder [1992] previously demonstrated that in an isotropic shmoo assay at high concentrations of -factor, cells use the previous site of cytokinesis to form the projection. The defect observed in ste2-296 cells in the spatial gradient assay might be due to the inability to select a site of projection formation (shmoo site selection) after the initial loss or masking of the bud site tags, which occurs when the cell is exposed to lower physiological concentrations of pheromone [Roemer et al., 1996]. This would explain why cells exposed to a pulse of high pheromone concentration can form a mating projection while those exposed to a physiological mating pheromone gradient can not. We favor this possibility. We note that the defect seen in the spatial gradient assay does not seem to be a secondary consequence of the low concentrations of pheromone used to compensate for the hypersensitivity of this mutant because in these assays ste2-296 cells arrest comparably to wild-type cells, thus indicating that the G-protein coupled MAP kinase cascade is functional in these mutants. However, it does not rule out that these cells may arrest normally but require a higher concentration of pheromone to form a projection. Another possibility is that upon severely truncating the receptor, its conformation might be altered such that proteins normally associating with other parts of the receptor that are required for mating polarized cell growth now can no longer associate with it. This does not