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
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::β-gal fusions. The library was introduced into either a diploid MATa
or a haploid MATa leu2Δ bar1Δ
yeast strain, and then transformants that exhibited enhanced or reduced expression of β-gal in the presence of the α-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 α-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 β-gal fusions was developed and then optimized using two yeast strains expressing β-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 β-gal expression in the presence and absence of α-factor. 186 strains were identified that reproducibly exhibited increased β-gal activity after pheromone treatment; three strains displayed decreased activity after treatment. Examples of the pheromone regulated–β-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. .
Figure 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 (more ...)
To determine the identity of the pheromone-regulated yeast genes producing the β-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 . 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 ), 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
(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
, and Ty elements (see Table 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 ). 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
, 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
is a homologue of the yeast casein kinase I–related genes YCK1
, 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
, 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
, and potentially YGR111w. PHD1
was originally isolated as a gene that, when present in multiple copies, promotes pseudohyphal growth (Gimeno and Fink, 1994
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
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 ), as has been shown previously for DUR1,2
(Jauniaux and Grenson, 1990
; Stanbrough and Magasanik, 1995
Induction of Gene Expression by Low Nitrogen Medium
Another class of pheromone-regulated genes display decreased expression in pheromone-treated cells. The three pheromone-repressed genes we identified include: PHO81
, and a novel gene, QOR1
(refer to Fig. for the pheromone-dependent repression of FOX2
encodes a repressor of the Pho85 CDK–G1 kinase complex (Ogawa et al., 1995
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 β-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
β-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 β-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
, 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 β-gal assays presented in Table , 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
; Page and Snyder, 1992
; Elion et al., 1995
Induction of Gene Expression by α-Factor
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
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. ). 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
, Fig. ). 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.
Figure 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 (more ...)
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
strain, and MATa cdc28-1 fig
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/α cells (data not shown). In addition, mating-induced expression of the four genes was observed in both a and α 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 ). 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 × α wild-type or a lacZ insertion strain × α 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
, and KAR5
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
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 fig1Δ
, and fig4Δ
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, fig1Δ
, and fig4Δ
mutants appeared normal for cell cycle arrest and recovery, pheromone sensitivity, and projection formation at all pheromone concentrations tested (Fig. for mating projection results; refer to Materials and Methods). However, the figΔ
strains each exhibited quantitative mating defects, and the severity of the defect differed depending upon the mating condition (Table ). 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 fig2Δ
strains is likely because of their enhanced agglutination relative to wild-type cells (see below). The mating phenotypes of the fig1Δ
, and kar5Δ
strains were the same as their respective transposon insertion mutants. We also tested the relative mating efficiencies of fig1Δ
, and fig4Δ
mutants using mating conditions that concentrate cells on filters (Sprague, 1991
). Under these conditions, the relative mating efficiencies of fig 1Δ
were similar to those observed by liquid conditions. The increased mating efficiency of fig2Δ
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 fig2Δ
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.
Figure 3 Mating projection formation by MATa wild-type and figΔ cells in the presence of isotropic mating pheromone. Cells shown were treated for 2 h with α-factor mating pheromone at a concentration of 5 μg/ml. figΔ cells (more ...)
We also investigated the effects of different conditions on the mating efficiencies of fig 1Δ, fig2Δ, and fig4Δ mutants (Table ); the severe effect of the kar5Δ/fig3Δ mutation on mating efficiency precluded its accurate measurement under these conditions. At 16°C, the mating efficiencies of both fig1Δ and especially fig2Δ bilateral matings are impaired relative to wild-type strains (1.4- and 18-fold, respectively). The bilateral matings involving fig1Δ and fig2Δ 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 fig1Δ and fig2Δ strains. While PEG is a potent (5.2–7.6-fold) enhancer of mating efficiency for wild-type, fig1Δ, and fig4Δ strains, it has a much smaller effect on the mating efficiency of fig2Δ strains. Interestingly, the mating efficiency of fig1Δ bilateral matings is more sensitive to EGTA, exhibiting a 3.1-fold decrease relative to wild-type strains. The relative mating efficiency of fig4Δ 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 1Δ, fig2Δ, and fig4Δ strains suggest that Fig1p, Fig2p, and Fig4p play distinct roles in mating, and may provide insights into their molecular functions (see Discussion).
fig2Δ and kar5/fig3Δ Mating Cells Hyperagglutinate and Form Small Colonies, Respectively
After the discovery that figΔ mutants exhibit altered mating efficiencies, we sought to determine the phenotypic basis of these effects. Two macroscopic phenotypes were observed in matings involving fig2Δ and kar5/fig3Δ mutants. During mating, wild-type cells gather into clusters through agglutination. fig2Δ 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. A), and microscopic examination of mating cells (data not shown). Hyperagglutination caused by the fig2Δ mutation is an interaction specific to mixtures of mating cells; fig2Δ mutant strains of either mating type do not aggregate during vegetative growth or when mixed with cells of the same mating type. Hyperagglutination of fig2Δ strains during mating was observed at both 30° and 16°C, indicating that the cold sensitivity of fig2Δ mutant matings is caused by a defect independent of agglutination.
Figure 4 Hyperagglutination and small colony phenotypes observed in matings of fig2Δ and kar5Δ/fig3Δ strains. (A) Unilateral and bilateral matings involving fig2Δ strains cause hyperagglutination, observable as rapid sedimentation (more ...)
The second macroscopic mating phenotype occurs in bilateral crosses of kar5/fig3Δ mutants. Matings of wild-type and all other figΔ mutant strains gave rise to uniformly-sized diploid colonies after 1.5 d of incubation at 30°C. In contrast, matings of kar5/fig3Δ mutants produced many small, irregular colonies as shown in Fig. 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 MATα tester strains. These phenotypes are consistent with nuclear fusion failures in kar5Δ/ fig3Δ prezygotes (see below). Such failures would be expected to lead to unstable heterokaryons, which, in turn, produce haploid progeny.
fig1Δ, fig2Δ, and fig4Δ Strains Exhibit Defects in Mating Cell Morphology
The mating properties of the figΔ mutant strains were investigated further by examining the morphology and distribution of nuclei in cells and zygotes in wild-type and bilateral figΔ mating mixtures (Fig. ). Cell shape and degree of polarization (unpolarized, small–medium polarized, and large polarized cells and zygotes) were quantified (Table ). Three of the figΔ mutations, fig1Δ, fig2Δ, and fig4Δ, each alter the morphologies of mating projections and zygotes in distinct ways.
Figure 5 Mating mixtures of wild-type and figΔ cells reveal cell polarization and zygote formation defects. Bilateral matings are shown. The inset shows a typical polarized mating cell. Note the fig 1Δ prezygote has a septum indicative of a (more ...)
Morphologies of Cells in Wild-type and figΔ Mutant Mating Mixtures
fig1Δ, and to a lesser extent fig4Δ, mating mixtures have fewer medium and large polarized cells than wild-type or fig3Δ matings (Fig. ; Table ). Many of the fig1Δ and fig4Δ 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 figΔ mutants (Fig. , insets; Table ). In addition, in the case of fig4Δ 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. ). The pattern of actin staining at the mating projection tip is typically less intense and more dispersed in both fig1Δ and fig4Δ cells compared to that of wild-type cells, whereas actin polarization remains normal in fig2Δ 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 1Δ and fig4Δ mutations on cell polarization are similar, differences in zygote morphologies between these two mutants suggest they perform different functions in the mating process; fig 1Δ, but not fig4Δ, zygotes display cell fusion defects (Fig. , and see below).
Figure 6 Actin distribution in wild-type, fig1Δ, fig2Δ and fig4Δ polarized cells containing mating projections. Cells shown are derived from mating mixtures stained with rhodamine-conjugated phalloidin after fixation. fig1Δ and (more ...)
The morphological alterations in mating projection formation caused by the fig2Δ mutation are distinct from those generated by the fig1Δ and fig4Δ mutations. fig2Δ cells form hyperpolarized mating projections that are often narrower and longer than those of wild-type cells (Fig. ). A consequence of the hyperpolarization of the fig2Δ 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. ). Measurement of the ratio of fusion bridge width/average parental cell pair width for 50 wild-type, fig1Δ, and fig2Δ zygotes supports this observation; for wild-type and fig1Δ zygotes these ratios are 0.52 and 0.51, respectively, whereas for fig2Δ 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 fig3Δ
mutant cells indicated that cell polarization and zygote morphology is normal, unlike that of fig1Δ
, and fig4Δ
mating cells. Instead, kar5Δ
zygotes displayed nuclear fusion defects in which nuclei lie within close proximity but fail to fuse (Fig. ). This result is consistent with that reported previously for kar5
mutant alleles (Kurihara et al., 1994
; Fig. , 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, fig1Δ, and fig2Δ strains mated at 16°C; this condition enhances the mating defects of the mutant strains. As observed for fig1Δ strains at 30°C, fig1Δ and fig2Δ 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. ). As shown in Table , the incidence of partial and complete failures in cell fusion is increased markedly in fig1Δ zygotes (ninefold), and more modestly in fig2Δ zygotes (approximately twofold).
Figure 7 Cell fusion and nuclear morphology defects in fig1Δ and fig2Δ 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 (more ...)
Cell Fusion and Nuclear Morphology Defects in Wild-type, fig1Δ and fig2Δ Zygotes
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. , a number of defects appear to be caused by the narrow fusion bridge phenotype of fig2Δ
mutants. The most prevalent phenotype, observed in ~80% of fig2Δ
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. ; top two rows
). In fig2Δ
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. ; Fig. , bottom two rows
; Table ). fig2Δ
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. , Table ). 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 region is observed to be contiguous with nuclear material remaining in the fusion bridge (Table ). For each of these cases, the majority of these altered nuclear configurations occur in fig2Δ
zygotes displaying the narrow bridge phenotype shown in Figs. and .
To further examine the cell and nuclear fusion defects visualized by light microscopy, we performed electron microscopic analysis on thin section preparations of wild-type, fig1Δ, and fig2Δ zygotes (Fig. ). Inspection of micrographs of the fig1Δ zygotes confirms the presence of undissolved cell wall materials and membrane causing both partial and complete fusion defects (Fig. , B and C; this is particularly evident in higher magnification micrographs; data not shown). Moreover, examination of the partial fusion defects by both fluorescent microscopic techniques and electron microscopy indicates that nuclear fusion is a robust process, capable of being executed through very small regions of cytoplasmic continuity (for example, Fig. , fig1Δ center panel; Fig. C; and Table , partial fusion defect column). Analysis of fig2Δ zygotes revealed elongated nuclear morphologies consistent with those visualized by DAPI staining of whole zygotes. In summary, these different data demonstrate that fig1Δ and fig2Δ zygotes exhibit both cell fusion and nuclear morphology defects.
Figure 8 Electron micrographs of thin sections through zygotes formed from bilateral matings. (A) wild-type; (B) fig1Δ mutant, complete fusion defect; (C) fig1Δ mutant, partial fusion defect; and (D) fig2Δ mutant, note narrow fusion (more ...)
FIG1, FIG2, and FIG4 Function in at Least Two Different Mating Cell Differentiation Pathways Required for Cell Shape and Polarity
The different effects of nonoptimal mating conditions on the mating efficiencies of fig1Δ, fig2Δ, and fig4Δ strains suggested that these mutants are defective in different pathways involved in mating cell differentiation (Table ). To investigate this further, we examined the epistatic relationships of the figΔ mutations by characterizing the mating cell projection and zygote morphologies of double mutant strains mated at 30° and 16°C; bilateral matings of MATa and MATα fig1Δfig2Δ, fig1Δfig4Δ, and fig2Δfig4Δ mutant strains were examined (Fig. ). For most of the double mutant strains, the phenotype of any single mutation was never completely epistatic to that of another (Table ). All double mutants carrying the fig1Δ mutation displayed reductions in the fraction of cells producing pointed mating projections and increases in the rate of cell fusion defects. Similarly, double mutants involving the fig2Δ mutation displayed a narrow conjugation bridge and the aberrant nuclear morphology phenotypes; these mutants also hyperagglutinated at both 30° and 16°C. All double mutants involving the fig4Δ mutation displayed a reduction in the percentage of cells with pointed projections. Thus, the morphological phenotypes of the fig1Δfig2Δ and fig2Δfig4Δ double mutants represent a combination of those observed in each of the corresponding single mutants, suggesting that Fig2p functions in a distinct pathway from that of either Fig1p or Fig4p (Table ).
Figure 9 Phenotypes of double figΔ mutants affecting mating projection and zygote morphology. A typical polarized cell (Mating Projection) and zygote from bilateral matings of fig1Δ fig2Δ cells (top), fig1Δ fig4Δ cells (more ...)
Morphologies of Cells in Wild-Type and figΔ Double-Mutant Mating Mixtures
There are exceptions to these independent epistasis relationships. The fig1Δ and fig4Δ mutations did not produce additive effects in cell polarization, suggesting that these mutants may function in the same or significantly overlapping pathways for this particular process (Table ; however, see Discussion). In addition, the fraction of cells with a pointed projection tip in the fig1Δfig2Δ and fig2Δfig4Δ mutants was reduced relative to that of fig2Δ mutants alone (Table ). This suggests that hyperpolarization caused by the absence of Fig2p function may partly require the function of the polarization pathway(s) in which Fig1p and Fig4p function. We are cautious, however, in interpreting this relationship as one that applies to normal mating cell polarization, since hyperpolarization is a consequence of the loss of FIG2 function and not a polarization event normally occurring in wild-type mating cells. In summary, these results indicate that the FIG1, FIG2, and FIG4 genes encode proteins that are components of at least two distinct mating cell differentiation pathways required for projection shape and polarity.
Fig1p and Fig2p Localize to the Cell Periphery
To gain further insight into the function of the different pheromone-regulated genes, the subcellular localizations of β-gal fusion proteins in 14 strains carrying lacZ
fusions to different genes, including fig1::lacZ
, and fig4::lacZ
, were analyzed using anti–β-gal antibodies and indirect immunofluorescence. Fusion proteins encoded by fig3::lacZ
, along with those from 10 other strains, failed to localize in a discrete pattern or at a level above background. Presumably, some of these fusion proteins may lack sequences required for their stability or subcellular localization. Two strains, fig1::lacZ
, were, however, found to exhibit strong β-gal staining at discrete sites in pheromone-treated cells (Fig. ). In each case the β-gal fusion proteins appeared to be abundant, based on staining intensity, and localized to the cell periphery in 100% of the cells (n
> 400). The Fig1::β-gal and Fig2::β-gal fusion proteins were often slightly polarized toward the projection tips, but did not appear to be as sharply concentrated at the tips as reported previously for the Fus1 and Fus2 proteins (Trueheart et al., 1987
; Elion et al., 1995
). For both Fig1::β-gal and Fig2::β-gal fusions (~10%), in a small fraction of cells perinuclear staining was observed (Fig. ). Such staining was also observed in rare cells (<3%) that were not treated with pheromone and the staining was very weak (Fig. ). This perinuclear staining may represent low levels of the fusion proteins contained in the endoplasmic reticulum. The localization of Fig1p and Fig2p suggests they may perform their functions at the cell periphery.
Figure 10 Localization of Fig1::β-gal and Fig2::β-gal fusion proteins. Cells were incubated in the absence or presence of mating pheromone for 2 h and then stained with anti-β::gal antibodies by indirect immunofluorescence. Staining was (more ...)
The FIG1, FIG2, KAR5/FIG3, and FIG4 Proteins Contain Distinct Sequence Features
The four genes characterized in detail in this study, FIG1, FIG2, KAR5/FIG3, and FIG4, are predicted to encode proteins of 298-, 1609-, 504-, and 879-amino acids, respectively. Each of these proteins is predicted to contain domains suggestive of a structure, localization, or function of the proteins. (Fig. A).
Figure 11 (A) Predicted structural features of the Fig1, Fig2, Kar5/Fig3, and Fig4 proteins. Putative transmembrane domains (TMDs) are indicated by vertical wavy lines. Potential N-linked glycosylation sites are indicated by circles. These occur within a predicted (more ...) FIG1
are predicted to encode membrane- associated proteins. Fig1p contains four predicted transmembrane (TM) domains with a loop between the first and second TM segments that is expected to be extracellular and contain several potentially glycosylated residues (Fig. A
). The protein has several features in common with members of the four transmembrane (4TM) superfamily of proteins (Wright and Tomlinson, 1994
), including the transmembrane segments, the potential extracellular glycosylated loop, and the location of polar and charged residues at conserved points within two of the TM domains (N23 in TMD1 and D255 in TMD4) (Wright and Tomlinson, 1994
). Fig2p contains a predicted signal peptide at its amino terminus and potential glycosyl phosphatidylinositol (GPI) anchor sequence at its carboxy terminus. The protein is serine/threonine rich (44.5% serine or threonine) as are many extracellular proteins, and contains many potential N
-linked glycosylation sites (Klis, 1994
; Cid et al., 1995
The sequence of KAR5/FIG3 is predicted to encode a protein capable of containing several long coiled-coil domains (i.e., α helical regions with heptad repeats of hydrophobic residues) in the center of the protein. A protein of 577 amino acids in length and possessing limited sequence, but high structural similarity is also present in the Schizosaccharomyces pombe genome (EMBL/GenBank/DDBJ accession number D87337).
encodes a protein that belongs to a family of proteins characterized by a domain that is similar in sequence to the yeast Sac1 protein. Mutations in SAC1
affect actin cytoskeletal and secretory functions, possibly through alterations in phospholipid metabolism (Cleves et al., 1989
; Novick et al., 1989
; Whitters et al., 1993
). The alignment of the protein sequences of selected members of this multigene family is shown in Fig. B.
In S. cerevisiae
there are five genes encoding proteins that belong to this multigene family, SAC1
, and YIA2
, and several members also exist in other organisms (Majerus, 1996
; McPherson et al., 1996
; Mewes et al., 1997). Fig4p and Sac1p share 25% identity over 530 amino acids, including three highly conserved domains of 14/17, 9/13, and 10/12 identical amino acids (Fig. B
). Interestingly, Fig4p displays highest sequence identity within the Sac1 domain (29–34%) to a subgroup of the Sac1 domain–containing proteins that includes a Caenorhabditis elegans
and human homologue of Fig4p. This subgroup of proteins is distinguished, in part, by a 19–amino acid extension of the region of homology shared by the proteins past the carboxy-terminal border of the domain of Sac1p similarity (Fig4p residues 596–614, Fig. B
). Since each of the proteins contains a unique carboxy terminus, it is possible that the carboxy-terminal domains confer different specificities on the proteins toward the execution of their cellular functions.