Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

THE yeast mating response is an excellent model system for the study of receptor-activated cell differentiation in eukaryotes. Upon encountering appropriate mating pheromones, haploid yeast cells follow a programmed pattern of cell differentiation in preparation for later events of mating such as cell and nuclear fusion (Cross et al., 1988; Sprague and Thorner, 1992; Kurjan, 1993; Herskowitz, 1995). Vegetative cells exposed to pheromone stop their progression through the cell cycle and undergo polarized cell growth to form a specialized structure termed a mating projection. Polarized mating cells signal one another through their projections, and thereby direct growth to a mutual site of cell contact and fusion. Cell fusion usually occurs at the tips of the projections, forming a conjugation tube or bridge. Nuclear congression and fusion then take place within the conjugation bridge and the zygote enters the vegetative cell cycle, dividing the diploid nucleus between itself and its bud. Although the cytological events of yeast mating have been well described, the molecular components and mechanisms important for mating cell morphogenesis, cell fusion, and nuclear fusion are not well understood.

At the molecular level, mating differentiation is initiated by the activation of a receptor-coupled signal transduction cascade. Pheromones are bound by the STE2 and STE3 gene products, which are seven transmembrane segment receptors located on the surface of MATa and MAT cells, respectively. These receptors are coupled to a heterotrimeric G protein complex and a cytoplasmic mitogen-activated protein (MAP)¹ kinase cascade (Sprague and Thorner, 1992). Transduction of the signal by the MAP kinase cascade leads to activation of the transcription factor Ste12p, which, in turn, promotes the transcription of a set of genes involved in mating-specific functions. These functions include cell cycle arrest in G1, polarized morphogenesis, agglutination, cell fusion, karyogamy, and adaptation to the pheromone signal (Sprague and Thorner, 1992). Many components of the mating MAP kinase cascade, including the Ste12p transcription factor, have also been shown to be required in both haploid and diploid cells for the transition from the normal yeast form of growth to filamentous forms stimulated by nutrient deprivation conditions (Liu et al., 1993; Roberts and Fink, 1994). These filamentous forms of polarized growth and unipolar budding have been proposed to be a mechanism by which cells forage for more favorable nutrient-rich environments (Gimeno et al., 1992; Kron et al., 1994).

The mating projection produced by cells exposed to pheromone serves two important purposes. First, the projection allows the nonmotile yeast cell to extend towards its mate. The position of the mate is perceived through pheromone gradients emanating from mating partners. This perception, or partner selection, is accomplished through the differential activation of mating pheromone receptors on the surface of the mating cell (Jackson and Hartwell, 1990; Jackson et al., 1991; Segall, 1993). Second, growth of the mating projection is an actin-dependent process that has been shown to depend on several proteins that also participate in polarized growth during budding (e.g., Spa2p, Pea2p, Bem1p, Tpm1p, and Cdc42p) (Herskowitz et al., 1995; Pringle et al., 1995; Roemer et al., 1996). Recent studies have demonstrated a physical association between Cdc24p (the GTP exchange factor for Cdc42p), Bem1p, actin, and the heterotrimeric G proteins associated with the pheromone receptors, suggesting a mechanism for linking pheromone pathway activation to localized cell polarization (Leeuw et al., 1995). However, because these interactions are independent of the state of activation of the pheromone pathway, the specific mechanism of polarization to sites of pheromone receptor activation remains obscure (Leeuw et al., 1995; Roemer et al., 1996).

The second role of the mating projection is to concentrate components involved in cell adhesion (agglutinins), signaling (pheromones and pheromone receptors), and fusion (Fus1p and Fus2p) to the area of intended cell contact and fusion. High levels of mating pheromone are required for normal cell fusion, and several proteins that function specifically in these processes (a-factor, -factor, Ste2p, Fus1p, and Fus2p) are all highly localized to projections or their tips (Trueheart and Fink, 1989; Jackson et al., 1991; Sprague and Thorner, 1992; Elion et al., 1995). Many cell polarity genes also function in the cell fusion pathway as indicated by the increase in cell fusion defects observed for mutants in a number of such genes (e.g., SPA2, PEA2, BNI1, RVS161) (Dorer et al., 1997). These observations suggest that efficient cell fusion is likely to depend on proper cell polarity to affect localization of the signaling and cell fusion components to the projection tip.

In contrast to our extensive knowledge of the components of the mating signal transduction cascade and their interactions, relatively few proteins are known to be specifically involved in the various downstream events of the mating process (Sprague and Thorner, 1992; Brill et al., 1994; Choi et al., 1994; Stevenson et al., 1995). For example, most of the polarity components known to affect mating cell shape and growth also participate in vegetative functions (Herskowitz et al., 1995; Pringle et al., 1995; Roemer et al., 1996). Thus, it is likely that certain mating-specific components remain undescribed that link general polarity proteins to specified sites of cell growth during mating. Some of these components would be expected to help direct the growth and shape of the mating projection. Understanding the downstream events of the mating process, including cell polarization, cell fusion, and nuclear fusion, is of general importance to elucidating these processes in higher eukaryotic cells. The limited number of downstream genes currently identified as functioning in these processes suggested that a search for new pheromone-regulated genes might yield additional components of the mating pathway, and thereby help determine the molecular and cellular mechanisms involved in mating cell differentiation.

We describe the results of an extensive screen for pheromone-regulated genes. The screen uses a recently developed method of random transposon tagging of yeast genes to monitor gene expression and investigate mutant phenotypes (Burns et al., 1994). From an initial bank of 189 pheromone-regulated transposon insertions, 45 new pheromone-regulated genes were identified. Among these 45 genes, 30 represent novel genes and 15 encode genes whose expression was previously unknown to be affected by pheromone. Furthermore, we find that a subset of pheromone-induced genes are also induced by conditions of nitrogen deprivation, suggesting a set of target genes is shared between the mating and pseudohyphal pathways. Four novel pheromone-induced genes designated Factor- Induced Gene FIG1, FIG2, KAR5/FIG3, and FIG4 were determined to be required for different steps of mating cell differentiation, including the control of mating cell polarity, cell fusion, and nuclear fusion.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Yeast Strains and General Methods

The yeast strains used in this study are listed in Table VIII. All strains are derivatives of Y800 (Burns et al., 1994) and in the S288c background. Y1406, the diploid strain used in the screen, was constructed by transforming strain Y1400 MATa-cry1 ura3-52 leu2-98 his3-200 trp1-1 with a PCR fragment (Baudin et al., 1993) containing the BAR1 gene, in which the entire protein coding sequence was substituted with the sequence of the HIS3 gene. The resulting strain, Y1402, was used to construct Y1405 (MATa-cry1/MAT-CRY1 bar1::HIS3/bar1::HIS3) through backcrossing. A MATa-cry1/MATa-cry1 mitotic recombinant Y1406 was selected from Y1405 by growth on plates containing crytopleurorine and then confirmed to be a diploid by transformation of a MAT plasmid, sporulation, and tetrad analysis. Y1411, the MATa bar1 haploid strain used for screening is an ascospore segregant derived in the construction of Y1405. General cloning procedures are described in Sambrook et al. (1989). Yeast media and methods are presented in Rose et al. (1990) and Sherman (1986).

Identification of Pheromone-regulated Genes

The plate assay for detection of pheromone-induced genes was first optimized using two Y1406 strains: one containing a cik1::lacZ fusion carried on a YCp50 plasmid and the other a FUS1::lacZ fusion carried on a 2 µ-based plasmid (Trueheart et al., 1987; Page and Snyder, 1992). Cells were grown on yeast extract/peptone/dextrose medium (YPD) plates and then replica-plated to two 1-mm filters (Whatman Inc., Clifton, NY). The duplicate filters were incubated for an additional 6 h on YPD medium and then transferred to petri dishes containing 0.8 ml of liquid YPD medium, one of which contained 5 µg/ml of -factor (Sigma Chemical Co., St. Louis, MO). After incubation at 30°C, the filters containing cells were exposed to chloroform vapors to permeabilize the cells, and then the filters were incubated on plates containing X-gal as described previously (Xie et al., 1993). In initial optimization experiments, cells were incubated for 6, 8, 10, and 12 h at 30°C, and then processed for -gal activity. Optimal signals were observed using pheromone incubation times of 10-12 h, which were used in screening experiments.

A yeast lacZ fusion library (Burns et al., 1994) was transformed into strains Y1406 and Y1411. 55,000 Y1406 and 36,200 Y1411 Leu⁺ transformants were patched on 90-mm petri plates containing synthetic complete (SC) medium lacking leucine (100 transformants/plate). After growth for 2 or 3 d at 30°C, the cells were replica-plated to two filters and incubated on YPD plates for an additional 6-12 h. Filters were processed as described above and strains containing potential pheromone-regulated fusions were identified. Individual strains were then retested as single colonies to identify strains that contained reproducibly pheromone-regulated fusions. 14% of yeast strains expressed -gal after vegetative growth for a total of ~13,000 strains. Since there are ~6,500 yeast genes in yeast (Mewes et al., 1997), this corresponds to 2.0 genome equivalents screened. Therefore, 158 out of 189 pheromone-regulated fusions corresponds to 1.7 genome equivalents analyzed.

The yeast sequence adjacent to the mTn3::lacZ insertion was determined using plasmid rescue procedures described previously (Burns et al., 1994, 1996). Briefly, either YIp5 or pRSQ plasmids were integrated into the mTn3 insertion, and the yeast sequences adjacent to lacZ were recovered as plasmids in Escherichia coli. A primer complementary to the end of the lacZ sequence was used to determine the sequences of the yeast DNA adjacent to the mTn3 insertions. The yeast sequences were compared to those in the GenBank database using the BLAST program (Altschul et al., 1990). These sequences (see Table I) are accessible in GenBank by a search using keywords Pheromone and the fusion number of interest (e.g., P158). Pheromone response element (PRE) sites were identified by searching sequences using the Fitconsensus program of the UWGCG package (Devereaux et al., 1984) and the sites described in (Kronstad et al., 1987).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Examples of pheromone-regulated lacZ fusions. Seven yeast strains containing the lacZ fusions indicated were incubated in YPD medium in either the absence (left) or presence (right) of pheromone for 12 h. Examples are shown of strains with fusions in genes whose expression is dependent upon mating pheromone (FUS2, FIG1, FIG2, and FIG4), enhanced by pheromone (CIK1, KAR5/FIG3), or repressed by pheromone (FOX2).

Quantitative -galactosidase (-gal) Assays

Cells of the indicated strains were grown to midlogarithmic phase (OD₆₀₀ = 0.4) in SC medium lacking leucine and then divided into two 10-ml aliquots. To determine pheromone induction levels, cells were treated for 2 h in YPD or YPD + 5 µg/ml of -factor, after which cell lysates were prepared and -gal activities were measured. Low nitrogen induction levels were measured by comparison of activities in cell lysates of cells grown for 12 h in SC lacking leucine or SC lacking both leucine and (NH₄)₂SO₄. Cells were harvested by centrifugation, washed once in Z buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, 50 mM -mercaptoethanol, pH 7.0) and the cell pellet frozen at 70°C until use. Cells were prepared for lysis by resuspending the frozen cell pellet in 0.5 ml of Z buffer, and followed by the addition of 250 µg zymolyase 100T to spheroplast cells (30 min at 30°C). Lysates were made by the addition of ~50 µl of glass beads (model G-8772; Sigma Chemical Co.), 15 µl of 100 mM PMSF, 7.5 µl of 20% SDS, and 25 µl of chloroform followed by vortexing for 2 min. Assays of -gal activity were performed by the addition of 200 µl of the lysate to 0.8 ml of Z buffer and 200 µl of 4 mg/ml O-nitrophenol--D-galactopyranoside. Reactions were stopped with 250 µl of 2 M Na₂CO₃, and then the activities were determined as a function of sample absorbance at 420nm, reaction time, and protein concentration (determined by Bradford assays).

Disruption of FIG1, FIG2, KAR5/FIG3, and FIG4

Complete deletions of the FIG1, FIG2, KAR5/FIG3, and FIG4 genes were made using a PCR disruption procedure (Baudin et al., 1993). Oligonucleotides containing the 55 bp immediately upstream of the ATG and downstream of the termination codon of each gene were synthesized with ends corresponding to sequences A and B below, respectively. Sequences A and B are complementary to regions that flank the URA3 gene of pRS316 (Sikorski and Hieter, 1989). URA3 fragments containing FIG flanking sequences were amplified by PCR and then transformed into the diploid strain Y800. Strains containing the correct substitution at the genomic locus were identified by PCR analysis. The resulting heterozygotes were sporulated and then haploid segregants were analyzed for vegetative growth and mating defects. Growth rates of all fig strains were identical to those of wild-type strains at 16, 30 and 37°C. (A) 5-...AGGCGCGTTTCGGTGATGACGGTG; (B) 5-...AGGGTGATGGTTCACGTAGTGGGC.

Localization of Fig1::-gal and Fig2::-gal Proteins

MATa cells of the indicated strains were grown to midlogarithmic phase (OD₆₀₀ = 0.3-0.4) in YPD, divided into 10-ml cultures, and then incubated for 2 h in either the presence or absence of 5 µg/ml -factor. Cells were harvested, fixed, and processed for immunofluorescence as described (Gehrung and Snyder, 1990; Pringle et al., 1991). To visualize -gal fusion proteins, a rabbit anti--gal primary antibody (Cappel Laboratories, Malvern, PA) was used at 1:12, followed by a CY3-conjugated sheep anti-rabbit secondary antibody (Sigma Chemical Co.) used at 1:200. All antibodies were preadsorbed against fixed and spheroplasted yeast cells before use.

Analysis of Yeast Mating Defects

Haploid strains containing the lacZ fusion insertion mutations were recovered by transforming the heterozygous MATa/MATa diploid strain with a YCp50 plasmid containing the MAT gene (gift of F. Cross, Rockefeller University, NY) followed by sporulation of the transformants. MATa segregants containing the transposon insertion mutation were recovered and mated to strain Y1402. MATa and MAT segregants were then obtained and tested for mating defects. Unilateral matings were carried out between MATa mutant strains and strain Y1408. Bilateral matings were performed between MATa and MAT segregants carrying either the same transposon insertion mutation or deletion. Diploids from the matings were selected on SC medium lacking histidine and tryptophan. Liquid mating reactions were carried out as described in Gehrung and Snyder (1990); agents such as polyethylene glycol (PEG)₃₃₅₀, EGTA, -factor, and polymyxin-B sulfate (Sigma Chemical Co.) were added to the tester strains immediately before addition of the strains whose mating efficiency was being measured. Relative mating efficiencies given in Table IV represent the mean of two separate assays and are normalized to wild-type levels (1.0% diploid formation). Filter mating assays were performed as described in Sprague (1991). Under these conditions, the wild-type frequency of diploid formation was 51.8 ± 1.3%; similar relative frequencies of mating were observed for fig1 and fig4 strains. fig2 strains exhibit a slight (1.5×) increase in cell number/OD₆₀₀ of cells. However, this cannot account for the increase in mating efficiency of fig2 mutants in unilateral and bilateral matings under liquid conditions: experiments in which the density of cells in wild-type control matings similarly increased, or fig2 cells decreased, did not show comparable increases in mating efficiency (data not shown).

Projection formation was analyzed by two methods. To assess projection formation in the presence of isotropic pheromone, cultures of mutant strains were grown to OD₆₀₀ = 0.3-0.5 and -factor was then added to final concentrations of 0.5, 1.0, 2.5, and 5 µg/ml. Second, after incubation for 1, 2, and 4 h, cell morphologies were examined by phase-contrast microscopy. Under these conditions in our strain background, cell cycle arrest is observed at the lowest pheromone concentration and polarized cells with broad projections are formed at intermediate concentrations, whereas sharp mating projections are formed at the highest concentration.

Polarized projection formation and zygote morphologies were also analyzed for the fig strains by quantitation of different cell types present in mating mixes. For these assays, cultures of mutant strains were grown to OD₆₀₀ = 0.5, and then 2 ml of each were mixed and pelleted by low-speed centrifugation. Mating cells were then resuspended in 5 ml of fresh YPAD and allowed to mate for either 8 h at 30°C or 16 h at 16°C without shaking. After incubation, cells were fixed by the addition of formaldehyde to a final concentration of 3.7% for 1 h, sonicated briefly to disperse cells and zygotes, and then washed and stored in 1× PBS, 1 M sorbitol. Cell mixtures were prepared for microscopy by pelleting an aliquot of the mating mix and resuspending in mounting solution containing 4,6-diamidino-Z-phenylindole (DAPI) (70% glycerol, 30% PBS, 2% wt/vol n-propyl gallate, 0.0225 µg/ml DAPI). The scoring of cell type (round, small, or large polarized) was done by placing an aliquot of the fixed cells in a haemocytometer to facilitate counting. Round cells were scored as unpolarized; polarized cells contained projections and were counted as small-medium (with an overall length less than that of a typical zygote), or large (equal to or larger in length than a typical zygote). Quantitation of projection tip shape was determined by scoring medium to large cells, as these cells have longer projections, the shape of which (pointed or blunt) are most clearly differentiated. When indicated, staining of cytoplasmic membranes and lipids was done after fixation by addition of FM4-64 (Molecular Probes, Inc., Eugene, OR) to 33 µM final concentration, followed by incubation of the cells on ice for 30 min. Cells were then washed once in 1× PBS, 1 M sorbitol before resuspending in mounting solution containing DAPI. Under these conditions (i.e., in formaldehyde-fixed cells) FM4-64 uniformly stains the cytoplasm and nucleus, but is absent from cell wall material as judged by both the reduced diameter of the staining region relative to the cell outline (as viewed by differential interference-contrast microscopy [DIC] optics), and by the absence of staining at sites of cytokinesis in budded cells. Measurements of zygotes comparing fusion bridge width to the mean width of the parental pair were performed using an intraocular micrometer at a magnification of 1600×. 50 zygotes were measured for each strain.

Electron microscopy of thin sections through zygotes was as described in Byers and Goetsch (1975), with the following differences. Zygotes were prepared by mating cells as described above; they were pelleted, washed once in 2 ml 0.1 M cacodylate buffer, pH 7.4, and then fixed in 0.1 M cacodylate, pH 7.4, containing fresh 3% glutaraldehyde for 30 min at room temperature. Cells and zygotes were then washed once in 0.1 M cacodylate buffer and then stained with 4% KMnO₄, followed by dehydration through a series of ethanol washes, and then embedded in LR-White acrylic resin (Polysciences Inc., Warrington, PA) before sectioning. Sections were stained with uranyl acetate and lead citrate and then viewed on an electron microscope (model EM-109; Carl Zeiss, Inc., Thornwood, NY) at magnifications of 8-40,000×. Pheromone sensitivity of the different strains was assayed by the halo method using strains Y1450 and Y1451 (Sprague, 1991).

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

Isolation of Pheromone-regulated Genes

To identify genes specifically regulated during yeast mating, a random lacZ insertional mutagenesis scheme was used. This method uses a library of yeast DNA fragments containing mini-Tn3::lacZ::LEU2 insertions (Burns et al., 1994). The lacZ gene lies near one end of the insertion and lacks an ATG initiator methionine codon; therefore, expression in yeast is primarily expected to occur because of in-frame insertion into yeast genes to produce yeast protein::-gal fusions. The library was introduced into either a diploid MATa/MATa leu2/leu2 bar1/bar1 or a haploid MATa leu2 bar1 yeast strain, and then transformants that exhibited enhanced or reduced expression of -gal in the presence of the -factor mating pheromone were identified. The use of a diploid strain allows for the isolation of pheromone-regulated genes that are essential for vegetative growth, whereas the use of strains that lack the Bar1 protease degrades -factor increases the responsiveness of the cells to pheromone under our screening conditions. To facilitate screening large numbers of transformants, an X-gal plate assay for identifying pheromone regulated -gal fusions was developed and then optimized using two yeast strains expressing -gal fusions with known pheromone-induced proteins, Fus1p and Cik1p (refer to Materials and Methods; Trueheart et al., 1987; Page and Snyder, 1992).

55,000 transformants of a diploid strain and 36,200 transformants of a haploid strain were screened for -gal expression in the presence and absence of -factor. 186 strains were identified that reproducibly exhibited increased -gal activity after pheromone treatment; three strains displayed decreased activity after treatment. Examples of the pheromone regulated--gal expression levels observed for lacZ fusions in the four novel FIG genes further characterized in this study, and an example of the class of pheromone-repressed genes are presented in Fig. 1.

To determine the identity of the pheromone-regulated yeast genes producing the -gal fusion proteins, the yeast genomic DNA adjacent to the lacZ insertions was plasmid-rescued into E. coli and then sequenced for 158 fusion strains (Burns et al., 1994). A summary of these results and the relative levels of vegetative and pheromone-induced (or -repressed) expression for the different pheromone-regulated genes identified in this study is presented in Table I. Based on the combined criteria of expression pattern and sequence identity, the fusions occur in genes that can be classified into five major categories: (a) known pheromone-induced genes; (b) previously characterized genes not reported to be induced by pheromone; (c) novel pheromone-induced genes; (d) pheromone-repressed genes; and (e) pheromone- and nitrogen-regulated genes.

Comparison of the number of genes identified by our screen to the total number of reported pheromone- induced genes (~22, Sprague and Thorner, 1992; Table I), along with the observation that many genes are represented by only one or two transposon fusions, indicates that our screen is not yet saturated. However, many genes are represented by multiple independent insertions. Extrapolating from the number of different genes identified, 54, and the 1.7 genome equivalents screened and analyzed (refer to Materials and Methods), we estimate there are ~67 different pheromone-regulated genes in yeast. This number is probably an underestimate because our transposon mutagenesis procedures have certain biases as shown by the overrepresentation of fusions to SPO11 and HOG1 (Burns et al., 1994). A larger and probably more accurate figure of 132 genes is obtained if we extrapolate from the number of pheromone-induced genes identified in our screen, nine, with those already known. Thus, we conclude there are ~67-132 pheromone-induced genes in yeast, thereby comprising 1-2% of all yeast genes.

Several Types of Genes Respond to Mating Pheromone

65 insertions reside in nine known pheromone-induced genes including STE6, FUS2, PCL2, CIK1, AFR1, KAR4, and Ty elements (see Table I for references). Ty1, Ty2, and Ty3 were previously known to be pheromone-induced (Boeke and Sandmeyer, 1991; Sprague and Thorner, 1992; Kurihara et al., 1996); our study indicates that the expression of Ty5 elements is also induced. Ty elements and their long terminal repeats (LTRs) are abundant in the genome (Olson, 1991), and comprise a large fraction (50 out of 158) of the pheromone-induced fusions identified in this screen. Additionally, some of the genes identified in this study are located adjacent to known pheromone-induced genes (see Table I). Examples include fusion P313B, which lies in an open reading frame (ORF) adjacent to AFR1, and the fusions in YFL027c (P28) and the HOG1 gene (P423A), which lie next to STE2 and a Ty LTR delta sequence, respectively. It is likely that the nearby regulatory sequences affect the expression of these genes as documented previously for Ty elements (Van Arsdell et al., 1987; Company et al., 1988). Some of these cross-regulated genes may also perform functions in the mating pathway.

In addition to known pheromone-induced genes, many genes (13) had been identified previously, but were not known to be pheromone-induced (Table I B). These include SPO11, HOG1, CKI3/YCK3, and RVS161. SPO11 is a sporulation-induced gene required in the early steps of meiosis. HOG1 is a MAP kinase homologue that regulates the osmotic stress response (Brewster et al., 1993). CKI3/ YCK3 is a homologue of the yeast casein kinase I-related genes YCK1, YCK2, and HRR25, and has recently been identified as a high copy suppressor of gcs1 mutants, which are defective in exit from stationary phase (Wang et al., 1996). RVS161, previously characterized as playing a role in actin cytoskeletal functions and cell polarity, has recently been described as important for efficient cell fusion and mating under certain conditions (Crouzet et al., 1991; Dorer et al., 1997). The induced expression of these genes during pheromone response suggests that many of these genes may function in the mating process.

A surprising subset of the pheromone-induced genes identified in this study include genes which are known, or can be expected to participate in pseudohyphal growth and/or in nitrogen metabolism, a determinant of pseudohyphal growth (Gimeno et al., 1992; Ljungdahl et al., 1992). These genes include PHD1, YFL056c, GAP1, AMD1, DUR1,2, and potentially YGR111w. PHD1 was originally isolated as a gene that, when present in multiple copies, promotes pseudohyphal growth (Gimeno and Fink, 1994). YFL056c encodes a protein with 57% amino acid identity over the first 174 of its 212 residues to an aryl alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium. In that organism, the gene is induced by nitrogen starvation conditions, and its product is implicated in lignin degradation (Reiser et al., 1994). The degradation of lignins, an important constituent of plant cell walls, facilitates fungal invasion into host plant tissues. GAP1 and AMD1 encode a general amino acid permease and AMD1 encodes a putative amidase. YGR111w encodes a probable lysine N6-acetyltransferase, an enzyme involved in the degradation of lysine. DUR1,2 encodes a urea amidolyase that converts urea to ammonia. The functions of these last four genes are likely to permit the efficient use of alternative nitrogen sources such as those provided by amino acids. PHD1, GAP1, AMD1, and DUR1,2 (Table I E) are each induced by nitrogen starvation (Table III), as has been shown previously for DUR1,2 and GAP1 (Jauniaux and Grenson, 1990; Stanbrough and Magasanik, 1995).

Another class of pheromone-regulated genes display decreased expression in pheromone-treated cells. The three pheromone-repressed genes we identified include: PHO81, FOX2, and a novel gene, QOR1 (refer to Fig. 1 for the pheromone-dependent repression of FOX2 expression). PHO81 encodes a repressor of the Pho85 CDK-G1 kinase complex (Ogawa et al., 1995), FOX2 functions in peroxisome biogenesis (Kunau and Hartig, 1992), and QOR1 has strong similarity to quinone oxidoreductases, suggesting a function in oxidative respiration in mitochondria. The relatively limited number of pheromone-repressed genes identified may be the result of the long half-life (~20 h) of -gal in yeast (Bachmair et al., 1992); this could make many pheromone-repressed genes difficult to identify in the 12-h pheromone incubation used in our screen. Surprisingly, the FOX2 and QOR1 -gal fusions are not in-frame. However, it is likely that these out-of-frame fusions reflect the normal regulation patterns of these genes. In a separate study, we have prepared an in-frame fusion in the QOR1 gene (Minehart, S., S. Erdman, and M. Snyder, unpublished data). Although the absolute levels of expression for the original out-of-frame fusion strain were lower, as expected, both the in- and out-of-frame fusions exhibited similar relative levels and kinetics of -gal induction (expression of QOR1 is induced by carbon source changes at the diauxic shift) and pheromone repression. Interestingly, each of the pheromone-repressed genes is likely to be subject to glucose repression; possible mechanisms to explain their regulation by the pheromone pathway are presented in the Discussion.

A large number of novel genes was also identified, and further characterization of four of these genes, FIG1-4, is presented below. Some novel genes encode proteins that have homologues in higher eukaryotes, whereas others are predicted to encode proteins that lack extensive homology to other known proteins in the databases (Table I C). Nonetheless, many of the unique proteins have distinctive sequence features. For example, many of the novel pheromone-regulated proteins contain regions predicting their insertion into, or association with, cellular membranes (examples include Fig1p, Fig2p, Yar027wp, andYpl156cp).

Finally, in several cases the lacZ fusion resided either in short ORFs, out-of-frame, reverse orientation, or in regions flanking genes (although most fusions were found to be in-frame with ORFs). These results indicate that sequences in addition to long ORFs can be expressed as protein in vivo, and are corroborated both by our previous study that found that short ORFs outside of predicted coding sequences are often expressed, and by recent analyses of the yeast transcriptome using SAGE techniques (Burns et al., 1994; Velculescu et al., 1997). For two genes, FUS2 and GAP1, out-of-frame fusions were found in addition to several in-frame fusions. For both genes, in- and out- of-frame fusions were regulated similarly. Many of the insertions obtained in the HOG1 region are either out-of-frame, in reverse orientation, or in flanking regions; nonetheless, all exhibit similar levels of pheromone induction consistent with responses to the same regulatory elements in each case. Thus, we presume that in many, if not most, cases, the regulation that is observed for any particular lacZ fusion reflects the expression of the transcript for the ORF into which the lacZ is inserted, an interpretation supported by our studies with QOR1 insertions. One mechanism to account for the expression of out-of-frame fusions is translational frameshifting.

Pheromone-regulated Gene Expression

Based on the results of quantitative -gal assays presented in Table II, the levels of induced expression upon pheromone treatment are ~1.3->700-fold for most of the pheromone-induced fusions. In cases where these levels have been measured, the figures reported here agree closely with those found previously (e.g., FUS2 and CIK1; Page and Snyder, 1992; Elion et al., 1995).

An upstream regulatory element termed the PRE has been identified as mediating the pheromone-induced transcription of a number of genes involved in the mating response (e.g., FUS2, CSH1, MFA2, STE6, STE2, BAR1, Ty elements and CIK1) (Van Arsdell et al., 1987). These sequences represent potential binding sites for Ste12p, the transcription factor that mediates pheromone-induced transcription, and are generally found upstream of pheromone-induced protein coding sequences (Kronstad et al., 1987; Errede and Ammerer, 1989; Page and Snyder, 1992). We searched the regions immediately upstream of the four novel FIG genes characterized in this study and found sequences matching the PRE consensus (Fig. 2). Since several of these genes are pheromone dependent for their expression yet contain only PRE sites that differ from the consensus, these results indicate that variant PRE sites are likely to be important for Ste12p-dependent regulation of some genes (e.g., FIG1 and FIG4, Fig. 2). An additional search for Mcm1p binding sites, which can be found near PRE sites of a subset of pheromone-induced genes such as FUS1 (Herskowitz et al., 1992), failed to identify sequences in the upstream regions of the FIG1-4 genes closely matching the consensus binding site.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Sequences similar to the consensus Ste12p binding site in the upstream regions of the FIG1, FIG2, KAR5/FIG3, and FIG4 genes. Sites preceded by an asterisk indicate they occur on the opposite strand. Underlined sequences represent exact matches to the PRE consensus sequence.

Mating pheromone treatment of cells causes cell cycle arrest in G1, and it has been proposed that this arrest may influence the expression of some genes that would be indirectly controlled by activation of the mating pathway (Stetler and Thorner, 1984; Price et al., 1991). We tested whether the pheromone-induced expression of the four FIG genes characterized in the present study is a consequence of direct or indirect regulation by the pheromone- response pathway. MATa strains carrying lacZ fusions of the four FIG genes were crossed to a MAT cdc28-1 strain, and MATa cdc28-1 fig::lacZ progeny were tested for induction of gene expression after cell cycle arrest in the absence of mating pheromone treatment (cdc28-1 strains shifted to the restrictive temperature arrest in G1). No increase in gene expression was observed for any of the four genes in the absence of pheromone treatment, nor was any expression observed in a/ cells (data not shown). In addition, mating-induced expression of the four genes was observed in both a and  cell types as monitored by the mating of strains of either cell type carrying lacZ fusions in these genes to yeast strains of the opposite mating type. These data, combined with the presence of upstream sites similar to the PRE consensus sequence in the four FIG genes, strongly suggest that the pheromone-induced expression of these genes in haploid cells of both mating types is because of direct regulation by Ste12p.

Four Novel Pheromone-induced Genes Are Important for Yeast Mating

To begin the characterization of the pheromone-regulated genes identified from our screen, the mating phenotypes of 20 haploid mutant strains carrying different transposon insertions were analyzed (Table I). Haploid strains containing the lacZ insertions were derived from MATa/MATa diploid parental insertion strains and examined for defects in (a) viability; (b) cell cycle arrest and polarized growth in response to pheromone; (c) pheromone sensitivity and adaptation; (d) pheromone production in each cell type; and (e) mating efficiency in both unilateral and bilateral matings (i.e., a lacZ insertion strain ×  wild-type or a lacZ insertion strain ×  lacZ insertion strain, respectively). No defects in viability, cell cycle arrest, polarized projection formation, adaptation, or pheromone production were detected for the strains that were examined. Evaluation of mating efficiencies under conditions of reduced cell densities, however, did identify three mutant strains, fig1::lacZ, fig2::lacZ, and fig3::lacZ that were each altered in mating efficiency relative to a wild-type strain.

The roles of FIG1, FIG2, and KAR5/FIG3 in yeast mating were investigated in detail using a variety of mating conditions. Because of its striking pheromone-induced expression pattern and its homology to the yeast Sac1p, a known effector of actin cytoskeletal dynamics (Cleves et al., 1989; Novick et al., 1989), the role of FIG4 in mating was also examined. Although initial studies failed to reveal a mating defect in fig4::lacZ strains, it is possible that the transposon insertion allele that was tested, P403A-2, may encode a fusion protein that retains some level of Fig4p activity, as it contains 90% of the Sac1p homology domain (see below). To ensure that null phenotypes were analyzed, strains in which the entire protein coding sequence of each of these genes was substituted with URA3 were constructed by PCR (Baudin et al., 1993). The fig1, fig2, kar5/fig3, and fig4 strains grew at rates identical to those of wild-type cells, and no vegetative growth defects were apparent at 16°, 25°, 30°, and 37°C.

As observed with the transposon insertion alleles, fig1, fig2, kar5/fig3, and fig4 mutants appeared normal for cell cycle arrest and recovery, pheromone sensitivity, and projection formation at all pheromone concentrations tested (Fig. 3 for mating projection results; refer to Materials and Methods). However, the fig strains each exhibited quantitative mating defects, and the severity of the defect differed depending upon the mating condition (Table IV). At 30°C, absence of Fig1p, Kar5/Fig3p, or Fig4p results in a bilateral mating defect that reduces mating efficiency 2.5-, 77.4-, and 2.9-fold, respectively, relative to that of a wild-type strain. In contrast, loss of Fig2p reproducibly increases the mating efficiency 3.2-7.2-fold in both unilateral and bilateral matings. Increased mating efficiency through the loss of a gene product in otherwise wild-type cells is a novel phenotype for a gene that functions in mating. The increased mating efficiency for fig2 strains is likely because of their enhanced agglutination relative to wild-type cells (see below). The mating phenotypes of the fig1, fig2, and kar5/fig3 strains were the same as their respective transposon insertion mutants. We also tested the relative mating efficiencies of fig1, fig2, and fig4 mutants using mating conditions that concentrate cells on filters (Sprague, 1991). Under these conditions, the relative mating efficiencies of fig 1 and fig4 were similar to those observed by liquid conditions. The increased mating efficiency of fig2 strains was no longer observed; instead we observed a 6.6-fold decrease in mating efficiency relative to wild-type strains. We presume that in contrast to liquid mating conditions that require cells to agglutinate to mate efficiently (Kurjan, 1993), the close packing of cells caused by collection on filters reduces or eliminates the need for agglutination in the filter-mating assays. As noted below, the increased mating efficiency of fig2 strains in liquid assays is likely due to the hyperagglutination activity of these cells; this activity is no longer expected to be important in filter-mating assays.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Mating projection formation by MATa wild-type and fig cells in the presence of isotropic mating pheromone. Cells shown were treated for 2 h with -factor mating pheromone at a concentration of 5 µg/ml. fig cells treated with reduced concentrations of pheromone and/or examined at additional timepoints (i.e., 1, 4, 6 h after treatment) also appeared normal in the shape, number, and rate of appearance of mating projections relative to wild-type cells.

We also investigated the effects of different conditions on the mating efficiencies of fig 1, fig2, and fig4 mutants (Table IV); the severe effect of the kar5/fig3 mutation on mating efficiency precluded its accurate measurement under these conditions. At 16°C, the mating efficiencies of both fig1 and especially fig2 bilateral matings are impaired relative to wild-type strains (1.4- and 18-fold, respectively). The bilateral matings involving fig1 and fig2 mutants are also inhibited more strongly than wild type by polymyxin B sulfate, a membrane-disrupting agent. The effects of PEG and EGTA on the mutant matings revealed additional differences between the fig1 and fig2 strains. While PEG is a potent (5.2-7.6-fold) enhancer of mating efficiency for wild-type, fig1, and fig4 strains, it has a much smaller effect on the mating efficiency of fig2 strains. Interestingly, the mating efficiency of fig1 bilateral matings is more sensitive to EGTA, exhibiting a 3.1-fold decrease relative to wild-type strains. The relative mating efficiency of fig4 mutants was affected to similar degree as the mating efficiency of wild-type strains by the different conditions. In summary, the differing effects of the conditions of cold temperature, PEG, and EGTA on the mating efficiencies of fig 1, fig2, and fig4 strains suggest that Fig1p, Fig2p, and Fig4p play distinct roles in mating, and may provide insights into their molecular functions (see Discussion).

fig2 and kar5/fig3 Mating Cells Hyperagglutinate and Form Small Colonies, Respectively

After the discovery that fig mutants exhibit altered mating efficiencies, we sought to determine the phenotypic basis of these effects. Two macroscopic phenotypes were observed in matings involving fig2 and kar5/fig3 mutants. During mating, wild-type cells gather into clusters through agglutination. fig2 strains exhibit a hyperagglutination phenotype in which mating cells aggregate to a greater extent than wild-type cells. This phenotype is observed by both uni- and bilateral crosses using settling assays (Fig. 4 A), and microscopic examination of mating cells (data not shown). Hyperagglutination caused by the fig2 mutation is an interaction specific to mixtures of mating cells; fig2 mutant strains of either mating type do not aggregate during vegetative growth or when mixed with cells of the same mating type. Hyperagglutination of fig2 strains during mating was observed at both 30° and 16°C, indicating that the cold sensitivity of fig2 mutant matings is caused by a defect independent of agglutination.

View larger version (75K): [in this window] [in a new window]   Fig. 4.   Hyperagglutination and small colony phenotypes observed in matings of fig2 and kar5/fig3 strains. (A) Unilateral and bilateral matings involving fig2 strains cause hyperagglutination, observable as rapid sedimentation of extensive cell clusters. Depicted are bilateral matings of wild-type and fig strains. (B) Bilateral matings of MATa kar5/fig3 and MAT kar5/ fig3 cells often produce very small colonies. The left panels are magnifications of sections of the plates shown on the right. Plates were incubated for 36 h. The large colonies are visible after 1 d. Both large and small colonies from the kar5/fig3 matings contain abnormal cells as described in the text.

The second macroscopic mating phenotype occurs in bilateral crosses of kar5/fig3 mutants. Matings of wild-type and all other fig mutant strains gave rise to uniformly-sized diploid colonies after 1.5 d of incubation at 30°C. In contrast, matings of kar5/fig3 mutants produced many small, irregular colonies as shown in Fig. 4 B. The number of smaller colonies approximates that of the total number of colonies formed in matings involving wild-type cells. Cells from both large and small colonies were fixed and then stained with Hoechst to examine their nuclear contents. Budding cells, cells with mating projections, anucleate and multinucleate cells, and zygotes were observed in each case. Progeny from both classes of colonies mated with both MATa and MAT tester strains. These phenotypes are consistent with nuclear fusion failures in kar5/ fig3 prezygotes (see below). Such failures would be expected to lead to unstable heterokaryons, which, in turn, produce haploid progeny.

fig1, fig2, and fig4 Strains Exhibit Defects in Mating Cell Morphology

The mating properties of the fig mutant strains were investigated further by examining the morphology and distribution of nuclei in cells and zygotes in wild-type and bilateral fig mating mixtures (Fig. 5). Cell shape and degree of polarization (unpolarized, small-medium polarized, and large polarized cells and zygotes) were quantified (Table V). Three of the fig mutations, fig1, fig2, and fig4, each alter the morphologies of mating projections and zygotes in distinct ways.

View larger version (82K): [in this window] [in a new window]   Fig. 5.   Mating mixtures of wild-type and fig cells reveal cell polarization and zygote formation defects. Bilateral matings are shown. The inset shows a typical polarized mating cell. Note the fig 1 prezygote has a septum indicative of a failure in cell fusion. fig2 zygotes have a narrow bridge. kar5 zygotes have two unfused nuclei in close proximity.

fig1, and to a lesser extent fig4, mating mixtures have fewer medium and large polarized cells than wild-type or fig3 matings (Fig. 5; Table V). Many of the fig1 and fig4 cells that are polarized possess mating projections with tips that are broader and less focused than those of wild-type cells; for these strains the percentage of large cells with pointed projections was less than half that of wild-type cells or other fig mutants (Fig. 5, insets; Table V). In addition, in the case of fig4 cells, we often observe multiple bumps around the cell periphery of unpolarized but enlarged cells, suggestive of failures in the intial establishment of mating cell polarity. We also examined the distribution of actin in these strains by rhodamine conjugated-phalloidin staining (Fig. 6). The pattern of actin staining at the mating projection tip is typically less intense and more dispersed in both fig1 and fig4 cells compared to that of wild-type cells, whereas actin polarization remains normal in fig2 cells. Thus, whereas FIG1 and FIG4 are dispensible for forming normal projections in isotropic levels of mating pheromone, in mating mixtures these genes are important both for the execution of cell polarization and the development of mating projection shape (see Discussion). Although the effects of the fig 1 and fig4 mutations on cell polarization are similar, differences in zygote morphologies between these two mutants suggest they perform different functions in the mating process; fig 1, but not fig4, zygotes display cell fusion defects (Fig. 5, and see below).

View larger version (99K): [in this window] [in a new window]   Fig. 6.   Actin distribution in wild-type, fig1, fig2 and fig4 polarized cells containing mating projections. Cells shown are derived from mating mixtures stained with rhodamine-conjugated phalloidin after fixation. fig1 and fig4 cells typically contain less actin at the growing tip of the mating projection, whereas actin distribution in fig2 appears similar to that of wild-type cells.

The morphological alterations in mating projection formation caused by the fig2 mutation are distinct from those generated by the fig1 and fig4 mutations. fig2 cells form hyperpolarized mating projections that are often narrower and longer than those of wild-type cells (Fig. 5). A consequence of the hyperpolarization of the fig2 mating projection is the formation of zygotes possessing narrow fusion bridges (the central portion of zygotes formed by fusion between the polarized tips of mating cells) (Fig. 5). Measurement of the ratio of fusion bridge width/average parental cell pair width for 50 wild-type, fig1, and fig2 zygotes supports this observation; for wild-type and fig1 zygotes these ratios are 0.52 and 0.51, respectively, whereas for fig2 zygotes the value is 0.30. Thus, FIG2 is important for mating cell projection shape and conjugation bridge diameter.

While preparing this manuscript, we learned that FIG3 corresponds to the previously identified KAR5 gene, whose molecular characterization has not been reported. Analysis of cell polarization and zygote formation in fig3 mutant cells indicated that cell polarization and zygote morphology is normal, unlike that of fig1, fig2, and fig4 mating cells. Instead, kar5/fig3 zygotes displayed nuclear fusion defects in which nuclei lie within close proximity but fail to fuse (Fig. 5). This result is consistent with that reported previously for kar5 mutant alleles (Kurihara et al., 1994; Fig. 5, this study).

FIG1 and FIG2 Function in Cell Fusion and Nuclear Migration

To help understand the functions of FIG1 and FIG2 in the differentiation of wild-type mating cells, we examined the cell morphologies and nuclear positions of prezygotes and zygotes formed by wild-type, fig1, and fig2 strains mated at 16°C; this condition enhances the mating defects of the mutant strains. As observed for fig1 strains at 30°C, fig1 and fig2 zygotes formed at 16°C display cell fusion defects. These defects were quantified by examining prezygotes and zygotes using DIC microscopy, DAPI staining (to examine nuclear fusion and morphology), and staining with the lipophilic dye FM4-64 which decorates lipids and membranes, but not cell wall material (Fig. 7). As shown in Table VI, the incidence of partial and complete failures in cell fusion is increased markedly in fig1 zygotes (ninefold), and more modestly in fig2 zygotes (approximately twofold).

View larger version (68K): [in this window] [in a new window]   Fig. 7.   Cell fusion and nuclear morphology defects in fig1 and fig2 zygotes incubated at 16°C. Left panels are zygotes as viewed by DIC. Center panels show the same zygotes stained with the lipophilic dye FM4-64; the dye stains lipids and membrane, but not cell wall material. Panels at right show DAPI staining of nucleic acids within zygotes. fig1 zygotes often display complete (top and bottom rows) or partial (middle row) fusion defects. fig2 zygotes have narrow conjugation bridges, and often have cell fusion defects (top two rows) or nuclear migration/segregation defects (bottom two rows).

When fig2 strains mate at both 30° and 16°C, a high frequency (84%) of zygotes show the hyperpolarization/ narrow fusion bridge phenotype. As shown in Fig. 7, a number of defects appear to be caused by the narrow fusion bridge phenotype of fig2 mutants. The most prevalent phenotype, observed in ~80% of fig2 zygotes, is a novel nuclear morphology that suggests a failure to comple the late steps of nuclear fusion. Normally, nuclear fusion proceeds by the microtubule-dependent congression of nuclei, followed by nuclear membrane fusion (Kurihara et al., 1994). The fused haploid nuclei then form a contiguous, elliptical or quasispherical diploid nucleus. In wild-type zygotes possessing a bud, the nucleus is often located near the site of bud emergence, or can be seen to be segregating or to have segregated between the zygote and bud (Fig. 7; top two rows). In fig2 zygotes, the newly fused nucleus nearly always has an abnormal shape, and in zygotes possessing a bud it is frequently observed to lie in abnormal positions, suggesting difficulties in nuclear migration to the bud site or in subsequent segregation events (Fig. 5; Fig. 7, bottom two rows; Table VI). fig2 zygotes appear delayed in rounding up of the nucleus, as judged by the presence of contiguous DAPI staining material across the fusion bridge region (Fig. 7, Table VI). In the majority of these nuclear configurations, two interconnected DAPI staining regions are observed on either side of the fusion bridge, whereas less frequently a single DAPI staining reg