Schizosaccharomyces pombe Ste7p Is Required for Both Promotion and Withholding of the Entry to Meiosis

Corresponding author: Masayuki Yamamoto, Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan., myamamot{at}ims.u-tokyo.ac.jp (E-mail)

Communicating editor: A. P. MITCHELL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The fission yeast ste7 mutant cannot mate and undergo meiosis, but shows no defect in vegetative growth. We cloned and characterized the ste7 gene. The deduced ste7 gene product (Ste7p) was a protein of 569 amino acids with no significant similarity to other proteins. Transcription of ste7 was induced by nutrient starvation via the function of the transcription factor Ste11p. Disruption of the ste7 gene blocked both conjugation and meiosis, showing that Ste7p plays a positive role in these two processes, probably activating the pheromone signal pathway. Unexpectedly, overexpression of ste7⁺ promoted conjugation but inhibited meiosis in wild-type cells. The temperature-sensitive pat1-114 mutant underwent ectopic conjugation at the semirestrictive temperature when its genetic background was ste7⁺, whereas the same mutant initiated haploid meiosis when its genetic background was ste7. Two-hybrid analysis suggested that Ste7p interacts physically with both Pat1p and Mei2p, which together constitute the major switch to initiate meiosis. Ste7p tagged with green fluorescent protein accumulated in haploid cells under nutrient starvation until they completed conjugation, but this protein disappeared when they were to enter meiosis. These observations suggest that Ste7p may have a function to suppress the onset of meiosis until the conjugation process has been duly completed.

CELLS of fission yeast Schizosaccharomyces pombe normally grow as haploids, taking one of the two mating types termed h⁺ and h^-. Cells of the opposite mating types undergo conjugation to form diploid zygotes under depletion of nutrients. This process involves mating pheromone signaling. Zygotes perform meiosis and sporulation if they are kept under starved conditions. Many genes are required to accomplish these complex processes of sexual development. Conjugation and meiosis occur consecutively under starved conditions in fission yeast, although they are two separate processes in the budding yeast Saccharomyces cerevisiae. Reflecting this trait, S. pombe appears to employ the same regulatory machinery in controlling conjugation and meiosis, at least in part. This is illustrated by the fact that a considerable number of genes are required specifically for both conjugation and meiosis. Especially, a high-mobility-group-family protein encoded by ste11, which is allelic with aff1 (SIPICZKI 1988 ), serves as a key transcription factor regulating both conjugation and meiosis (SUGIMOTO et al. 1991 ). S. pombe uses a G-protein-coupled receptor system and a mitogen-activated protein (MAP) kinase cascade for the mating pheromone signaling, as S. cerevisiae does, but unlike S. cerevisiae, the pheromone signaling is essential for meiosis in addition to conjugation in fission yeast (KITAMURA and SHIMODA 1991 ; OBARA et al. 1991 ; TANAKA et al. 1993 ).

The pheromone signaling pathway, involving a MAP kinase cascade and associated with the Ras1 protein, has been worked out by analyses of sterile (ste) mutants: ste1(byr1) and ste8(byr2) encode a MAP kinase kinase and a MAP kinase kinase kinase, respectively, which are involved in the transduction of mating pheromone signaling (NADIN-DAVIS and NASIM 1988 ; WANG et al. 1991 ); ste5 is allelic to ras1 and encodes a homolog of mammalian Ras, which regulates the Ste8p-Ste1p-Spk1p MAP kinase cascade (NADIN-DAVIS and NASIM 1990 ; GOTOH et al. 1993 ; NEIMAN et al. 1993 ; MASUDA et al. 1995 ); and ste6 encodes a guanine nucleotide exchange factor for Ras1p/Ste5p (HUGHES et al. 1990 ).

To date, 17 different ste genes (ste1–ste16 and ste20), including those aforementioned, have been described in S. pombe. Conjugation and meiosis involve organized morphological and cytological changes of the cell, in addition to the mating pheromone signaling, and much is left unknown about how the mating and meiotic process is completed. Analyses of the ste genes must provide insight into this complex process. Indeed, recent studies have led to an intriguing observation that ste9(srw1) encodes a WD-repeat protein that belongs to the fizzy-related family and regulates the anaphase-promoting complex (YAMAGUCHI et al. 1997 ; KITAMURA et al. 1998 ; KOMINAMI et al. 1998 ).

While the fission yeast ste mutations define cellular factors required for conjugation, the pat1 (also called ran1) mutation encodes a factor that can promote conjugation when inactivated (BEACH et al. 1985 ). The pat1 gene encodes a protein kinase that represses initiation of sexual development, and complete loss of its activity results in ectopic meiosis, even in haploid cells (IINO and YAMAMOTO 1985 ; NURSE 1985 ; MCLEOD and BEACH 1986 ). It has been noted, however, that when the temperature-sensitive pat1-114 mutant is shifted to the semipermissive temperature, the cells undergo mating rather than haploid meiosis (BEACH et al. 1985 ). Contrarily, artificial overexpression of pat1⁺ suppresses conjugation (MCLEOD and BEACH 1988 ). These observations have led to a proposal that S. pombe cells are programmed to initiate conjugation when Pat1 kinase is partially inactivated and to enter meiosis when it is completely inactivated (NIELSEN and EGEL 1990 ). Although this hypothesis cannot be readily ruled out, it has not been proven either. When S. pombe cells are to enter meiosis physiologically, Mei3p inhibits the activity of Pat1 kinase (MCLEOD and BEACH 1988 ). Dephosphorylated Mei2p, which accumulates in the absence of Pat1 kinase activity, shifts the cell cycle from mitotic to meiotic (WATANABE et al. 1997 ). However, Mei3p is expressed only in diploid cells (MCLEOD et al. 1987 ) and hence cannot be an inhibitor of Pat1 kinase responsible for the initiation of conjugation. No sensible candidate for such an inhibitor has been found in haploid cells.

To facilitate analysis of the conjugation mechanisms, we set out to characterize the S. pombe ste7 gene, which had been excluded from previous studies. Cells defective in ste7 can neither conjugate nor undergo meiosis, indicating that Ste7p is a positive factor for both conjugation and meiosis. Curiously, however, we found that Ste7p serves as a negative factor for meiosis under certain conditions. In this article, we describe basic characterization of ste7 and Ste7p and will discuss a possible role for Ste7p as a factor for harmonized progression of conjugation and meiosis.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

S. pombe strains:
S. pombe strains used in this study are listed in Table 1. The original ste7 mutant was described by GIRGSDIES 1982 and subsequently by MICHAEL and GUTZ 1987 , and we refer to this ste7 allele as ste7-1 in this study. We isolated a weak allele of ste7 in a screen for mutants resistant to pheromone-induced growth arrest, details of which will be published elsewhere. This allele will be referred to as ste7-111. The ste7-111 mutant is partially sterile (see RESULTS). Allelism between ste7-1 and ste7-111 is evidenced also in RESULTS.

Media, genetic methods, and transformation of S. pombe:
General methods to handle fission yeast were as described (GUTZ et al. 1974 ). Complete medium YE (0.5% yeast extract, 2% glucose, 50 µg/ml adenine) and minimal SD medium (SHERMAN et al. 1986 ) were used for routine culture of S. pombe strains. Synthetic medium SSA (EGEL and EGEL-MITANI 1974 ) and SPA (GUTZ et al. 1974 ) were used to examine conjugation and sporulation proficiency. Minimal medium (MM; MORENO et al. 1990 ) and its nitrogen-free derivative MM(-N), slightly modified as described (ISSHIKI et al. 1992 ) and containing only 1% glucose, were used for nitrogen starvation experiments. For transformation of S. pombe cells, a high-efficiency protocol described previously (OKAZAKI et al. 1990 ) was employed with slight modifications, as briefly described in the following. Either exponentially growing or growth-saturated cells cultured in appropriate medium such as YE were used. Cells were collected by centrifugation and resuspended in 50 µl of 0.1 M lithium acetate, pH 5.0. Sample DNA, together with salmon sperm DNA and 150 µl of 50% (v/v) polyethylene glycol (PEG 4000), was added immediately to the cell suspension, and the mix was incubated at room temperature for at least 30 min. After addition of 20 µl of dimethyl sulfoxide, cells were heated at 42° for 15 min, spun down, resuspended in H₂O, and plated on appropriate selective medium.

Cell fusion:
To obtain a diploid product from two haploid strains that do not conjugate normally, we employed a cell fusion technique essentially as described by SIPICZKI and FERENCZY 1977 . Cells to be fused were grown separately to the mid-log phase in SD containing 0.5% glucose, harvested, and washed once with water and once with buffer A [1.2 M sorbitol, 50 mM citrate-phosphate (pH 6.0), 50 mM ß-mercaptoethanol]. Cell pellets were resuspended in buffer A containing 5 mg/ml NovoZym 234 (Novo Nordisk) at ~10⁸ cells/ml, and incubated at 30° for 1 hr. After confirming generation of spheroplasts, they were washed with buffer B (1.2 M sorbitol, 30 mM Tris-HCl [pH 7.5]). Spheroplasts were resuspended in buffer B and two samples to be fused were mixed thoroughly. The mixture was spun down, and the pellet was mildly resuspended in PEG buffer [30% PEG 4000, 10 mM Tris-HCl (pH 7.5), 10 mM CaCl₂]. After incubation at 25° for 30 min, cells were plated on adenine-depleted medium containing 1.2 M sorbitol and fusants were selected by intragenic complementation of the ade6-M210 and ade6-M216 alleles. When necessary, heterozygosity of the mating loci of a fusant was confirmed by PCR analysis.

Cloning of ste7⁺:
To clone the ste7 gene, we introduced an S. pombe genomic library based on the multicopy vector pDB248' (BEACH et al. 1982 ) into an h⁹⁰ ste7-1 strain (JY836). Approximately 30,000 transformants were screened by staining with iodine vapor. Eleven transformants gave dark brown colonies, suggesting that cells in these colonies might have resumed the ability to conjugate and sporulate. Plasmids were recovered from the transformants into Escherichia coli. Reintroduction of these plasmids into JY836 confirmed that they had the ability to rescue the sterility. Southern analysis and restriction mapping indicated that they were overlapped to each other, and one plasmid, named pFR503, was chosen as a representative for further characterization.

DNA sequence analysis:
A 3.2-kb SpeI-SpeI fragment carried on pFR503, which was sufficient to rescue the sterility of JY836 (Fig 1A), was subjected to nucleotide sequence analysis. Subclones for sequencing were unidirectionally deleted with exonuclease III and S1 nuclease (TaKaRa Shuzo), according to the method of HENIKOFF 1984 . Single-stranded template DNA was prepared by using the helper M13KO7 bacteriophage. Nucleotide sequencing was performed by the chain-termination method of SANGER et al. 1977 by using a DNA sequencer model 4000L (LI-COR). All parts of the sequence shown in Fig 1B have been determined in both directions at least once. The sequence of the ste7 locus was identical, except for one base substitution, with the sequence determined by the genome sequencing project at Sanger Centre and deposited under EMBL accession no. Z68887. The sequence data disclosed in this study are available from EMBL/GenBank/DDBJ under accession no. AB036789.

View larger version (63K): In this window In a new window Download PPT slide   Figure 1. A restriction map of the ste7 locus and the nucleotide and deduced amino acid sequences of the ste7 gene. (A) Schematic illustration of the insert of pFR503, which carries the ste7 gene. The arrow indicates the position and orientation of the ste7 ORF. The open triangle indicates a frameshift at the NdeI site. Restriction sites are abbreviated as follows: B, BamHI; EV, EcoRV; H, HindIII; K, KpnI; Nc, NcoI; Nd, NdeI; R, EcoRI; Sc, ScaI; Sp, SpeI. Restriction fragments of the original insert were subcloned, and the ability of the subclones to rescue conjugation deficiency of the ste7 mutant was tested. The ability (+) or inability (-) of each subclone is indicated on the right. The construct used for disruption of the chromosomal ste7 gene is shown underneath. (B) The nucleotide and deduced amino acid sequences of ste7. The nucleotide sequence of a 3.5-kb BamHI-EcoRV fragment is shown. Numbering starts at the putative initiation codon for both nucleotide and amino acid residues. Underlined nucleotide sequences represent putative TR-boxes. Underlined amino acid sequences represent possible target sites of Pat1 kinase (LI and MCLEOD 1996 ; WATANABE et al. 1997 ). The 5' end and the poly(A) addition site of a cDNA clone are indicated by boldface type (C and T, respectively). The amino acid residue altered in the ste7-1 mutant is also indicated by boldface type (Q38).

Disruption of the ste7 gene:
A 2.0-kb HindIII-HindIII fragment was eliminated from the cloned ste7 open reading frame (ORF) and an S. pombe ura4⁺ cassette was inserted instead (GRIMM et al. 1988 ). A SpeI-SpeI fragment carrying the disrupted ste7 ORF was used to transform homothallic haploid ura4 strain JY878. Ura⁺ transformants were obtained efficiently by this procedure. Stable transformants were selected and proper replacement of the wild-type ste7 allele with the disrupted construct was confirmed by PCR and Southern blot analysis of their genomic DNA. For PCR analysis, two ste7-specific primers, namely, ste7pro [5'-CCAAGATTTAGTGTTTAGTGTG-3'] and ste7term [5'-TGCCGAGTTATTGACTCCAG-3'], were used.

Southern and Northern blot analysis:
The probe for Southern analysis of ste7 mRNA was prepared using a 0.5-kb EcoRV-HindIII fragment downstream of the EcoRV site (Fig 1) and a randomly primed DNA labeling kit (Amersham Corp., Piscataway, NJ). For preparation of RNA, S. pombe cells were grown in MM(+N) medium to a cell density of 5 x 10⁶/ml. A portion of the culture was sampled, and the rest was transferred to MM(-N) and subjected to nitrogen starvation for various time spans. Total cellular RNA was prepared from each sample by disrupting the cells with glass beads and following a standard extraction protocol (ELDER et al. 1983 ). For Northern blot analysis, 4 µg of each RNA preparation was denatured with formamide, separated by formaldehyde gel electrophoresis (SAMBROOK et al. 1989 ), and blotted to a membrane (GeneScreen plus, Dupont, Wilmington, DE). A 1.6-kb NcoI-NcoI fragment was used as a probe to detect transcripts of the ste7 gene.

Plasmids:
Deletion and frameshift derivatives of the original ste7 clone pFR503 were constructed in pDB248' (BEACH et al. 1982 ). To produce a plasmid that could overexpress ste7 ectopically, an NdeI site was created at the initiation codon of the ste7 ORF using an oligonucleotide designated STE7 [5'-CCGTACCCCATATGTTTTT-3']. A 1.9-kb NdeI-NdeI fragment containing the entire ste7 ORF was then inserted into the expression vector pREP1, which has the thiamine-repressible nmt1 promoter (MAUNDRELL 1990 ). pR3C-ste7 was isolated from an S. pombe cDNA library in a screen for plasmids that could rescue the sterility of ste7-111 mutant cells. This plasmid was a derivative of pREP3, but it had lost the TATA element for the nmt1 promoter (BASI et al. 1993 ) and expressed the cloned cDNA only weakly.

Fluorescence microscopy of GFP-tagged Ste7 protein:
We constructed a mutant version of green fluorescent protein (GFP, with Ser⁶⁵ substituted by Cys; HEIM et al. 1995 ) by localized mutagenesis (KUNKEL 1985 ) using an oligonucleotide designated S65C [5'-CACTACTTTCTGTTATGGTGTTCAATG-3']. We then connected an XhoI-XhoI fragment carrying the ORF for this mutated GFP to the C terminus of the ste7 ORF on a plasmid. To create an XhoI site at the terminus of the ste7 ORF, we used an oligonucleotide designated CX7 [5'-ACAGTCTCGAGAATTATTT-3']. The resulting fusion protein (Ste7p-GFP) was expressed in host cells from the authentic ste7 promoter. GFP fluorescence images in living cells were taken by a cooled CCD camera (Hamamatsu Photonics) attached to a Zeiss Axiophot microscope and stored digitally using the Fish Imaging software (Hamamatsu Photonics) program. Cells were counterstained with Hoechst 33342 to visualize nuclei.

Yeast two-hybrid interaction assay:
We performed yeast two-hybrid assay essentially as described by DURFEE et al. 1993 . An NcoI-ScaI fragment carrying C-terminal 80% of the ste7 ORF was fused in frame to the GAL4-activating domain in pACTII. A BamHI-BglII fragment carrying the C-terminal half of the mei2 ORF was fused in frame to the LexA DNA-binding domain in pBTM116, and the entire mei2 ORF was fused to the GAL4-activating domain in pACTII. An NdeI-BamHI fragment carrying the entire pat1 ORF was fused to the LexA DNA-binding domain in pBTM116 and to the GAL4-activating domain in pACTII. As a positive control pair, a plasmid expressing the LexA DNA-binding domain fused to Ras and a plasmid expressing the VP16-activating domain fused to Raf were employed. S. cerevisiae strain L40, which carried multimerized LexA-binding sequences and a reporter lacZ gene, was transformed with various combinations of plasmids, and the ß-galactosidase activity was assayed by a coloring reaction.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Characterization of the ste7 gene:
We isolated S. pombe genomic clones that could rescue the sterility of the ste7-1 mutant JY836, as described in MATERIALS AND METHODS. Because the gene that was carried on all of these clones and was responsible for the complementation appeared to be ste7 itself rather than a multicopy suppressor of ste7 (see below), we call it ste7 hereafter. One of the original ste7 clones, named pFR503, contained a 4.5-kb-long insert, and subcloning analysis indicated that a 3.2-kb-long SpeI-SpeI fragment was sufficient to rescue the sterility of JY836 (Fig 1A). Nucleotide sequence analysis revealed an uninterrupted ORF encoding 569 amino acid residues as a candidate for ste7 (Fig 1B). Analysis of deletion derivatives supported that this ORF was ste7 (Fig 1A). A database search revealed no particular functional motif in the deduced ste7 gene product. Ste7p was 26% identical with pig mucin over 536 amino acids, 29% with a subunit of soybean RNA polymerase II over 278 amino acids, 28% with Drosophila deformed over 118 amino acids, and 21% with Drosophila neuralized over 192 amino acids. It was also similar to certain ORFs identified in the genome project in Caenorhabditis elegans and S. pombe. Although significance of these observed similarities remains to be examined, the Ste7p homologs mentioned above have little similarity to each other, except that they are relatively rich in serine and proline. It is hence possible that none of them is a functional homolog of Ste7p. Ste7p was found to carry two possible phosphorylation sites by Pat1 kinase (see DISCUSSION).

We constructed a null allele of ste7 by gene disruption, as described in MATERIALS AND METHODS. A HindIII-HindIII fragment that covered most of the ste7 ORF, including the initiation codon, was replaced by the ura4⁺ cassette (Fig 1A). The resulting ste7 haploid strain exhibited no growth defect, but was completely sterile, as was the original ste7-1 mutant (Table 2).

To confirm that the disrupted gene was indeed ste7⁺, we constructed a diploid strain by crossing the ste7-1 mutant JY836 and the disruptant, using a cell fusion technique. The diploid strain thus obtained was sporulation defective, like the ste7-1/ste7-1 diploid, suggesting that they were likely to be allelic (Table 2). However, because this strain produced no progeny spores, linkage of the disrupted gene to ste7-1 could not be demonstrated. To make the linkage analysis possible, we employed a weak ste7 allele termed ste7-111. As shown in Table 2, the ste7-111 mutant was only partially sterile and could generate spores, though inefficiently, when crossed with ste7-1 by cell fusion. No completely fertile progeny appeared among these spores, indicating that ste7-1 and ste7-111 were allelic. When we crossed the disruptant and the ste7-111 mutant by cell fusion, the resulting diploid strain sporulated at nearly the same rate as ste7-1/ste7-111, and it produced no completely fertile progeny. Thus, the disrupted gene was tightly linked to ste7. In addition, the original clone pFR503 could complement the sterility of ste7-111. These results supported strongly that the disrupted gene was ste7 itself.

The allelism of ste7 and ste7-1 was confirmed further by sequence analysis. The ste7 locus was isolated from the ste7-1 mutant using PCR and sequenced. The ste7-1 allele contained a single substitution (C to T) at position 106 (Fig 1B), which resulted in generation of a stop codon. Taken together, we conclude that the gene given in Fig 1 is ste7⁺.

Expression of ste7⁺:
Using Northern blot analysis, we examined the expression pattern of ste7 mRNA. Only a very small amount of ste7 mRNA was detected in mitotically growing cells. Strong induction of ste7 expression occurred in response to nitrogen starvation, regardless of the ploidy and the mating type of the cell (Fig 2A). Two TR-boxes were found in the promoter region of ste7 (see Fig 1B), which are known to serve as the binding site for Ste11p transcription factor (SUGIMOTO et al. 1991 ). Because a number of mating- and meiosis-related genes expressed under nutrient starvation carry one or more TR-boxes and are regulated by Ste11p, we examined whether transcription of ste7 was also dependent on ste11⁺. As shown in Fig 2B, ste7 mRNA was expressed scarcely in ste11-defective cells and was expressed strongly in cells transformed with pREP1-ste11⁺, a plasmid that could provide a large amount of Ste11p. These results suggest strongly that expression of ste7 is regulated directly by Ste11p.

View larger version (42K): In this window In a new window Download PPT slide   Figure 2. Northern analysis of ste7 mRNA. (A) Cells of JY333 (h-), JY334 (h+), JY450 (h90), and JY362 (h+/h-) were grown to the mid-log phase in MM. A portion of each culture was harvested (0 hr), and the remainder was transferred to MM(-N). Sampling was done at 2, 4, 6, and 8 hr after the transfer. Total RNA was extracted from each sample and analyzed with hybridization probe for ste7. Ribosomal RNAs stained with ethidium bromide are shown in the bottom panel to confirm that nearly the same amount of RNA was loaded in each lane. (B) Expression of ste7 in ste11-defective and ste11-overexpressing strains. RNA prepared from either JY450 (ste11+), JZ396 (ste11), or JY450 transformed with a ste11-overexpressing plasmid (SUGIMOTO et al. 1991 ; ste11 o.e.) was analyzed as above (top). For each strain, RNA was prepared from vegetatively growing cells (+) and cells starved of nitrogen for 4 hr. Expression of ste11 was checked as described previously (SUGIMOTO et al. 1991 ; bottom). The open arrowhead indicates chromosomal ste11 transcripts, and the filled arrowhead indicates truncated ste11 transcripts from the plasmid. Loading of nearly the same amount of RNA in each lane was confirmed by ethidium bromide staining of rRNA.

Requirement of Ste7p for promotion of meiosis:
The original ste7-1 mutant and the ste7 disruptant constructed above were both defective in sporulation in addition to conjugation (Table 2). Diploid cells defective in ste7 do not form spores because they cannot enter meiosis, indicating that Ste7p is a positive factor essential for meiosis (GIRGSDIES 1982 ; data not shown). Our analysis showed that both haploid pat1-114 ste7 (JX636) and diploid pat1-114/pat1-114 ste7/ste7 (JX860) strains underwent uncontrolled meiosis at 32° as efficiently as their ste7⁺ counterparts (JX606 and JW266), indicating that Ste7p is required for the promotion of meiosis at a stage prior to inactivation of Pat1p kinase (see DISCUSSION).

ste7⁺ jams uncontrolled haploid meiosis in pat1-114 cells:
A negative role of ste7⁺ for entry to meiosis was noticed when we combined ste7 with the pat1-114 temperature-sensitive mutation. Haploid cells carrying the pat1-114 allele initiate unconditional lethal meiosis on nutrient medium when shifted to the restrictive temperature (IINO and YAMAMOTO 1985 ; NURSE 1985 ). However, the h⁹⁰ pat1-114 strain initiates conjugation rather than meiosis when the temperature is raised to a semipermissive temperature, 30°, for instance (BEACH et al. 1985 ; Fig 3). A previous genetic study examined the combination of pat1-114 and ste7-1, and it was concluded that pat1-114 did not suppress the mating deficiency of ste7-1 at the semipermissive temperature, although pat1-114 could suppress some sterile mutations such as ste1, ste3, or ste8 (SIPICZKI 1988 ). Our reinvestigation of the combination of pat1-114 and ste7 led to an interesting observation. We constructed an h⁹⁰ pat1-114 ste7 strain (JX636) and grew it to the exponential phase at the permissive temperature, together with the wild-type and the pat1-114 ste7⁺ strains. The temperature was then shifted to 29.5° to induce pat1-114-dependent hypermating. As shown in Fig 3B, pat1-114 ste7⁺ cells initiated conjugation at this temperature and few of them underwent lethal haploid meiosis. In contrast, pat1-114 ste7 cells initiated haploid meiosis and did not undergo conjugation at this temperature (Fig 3C). One may assume that pat1-114 ste7 cells underwent meiosis because the mating pathway was blocked by loss of ste7 function. However, h^- pat1-114 ste7⁺ cells did not initiate haploid meiosis at the same temperature (<1%), even though they did not perform mating, whereas h^- pat1-114 ste7-111 cells underwent meiosis at a considerable frequency (23%). These results suggest that Ste7p is likely to lead cells to conduct conjugation, suppressing meiosis, when cell physiology is potentially suitable for both.

View larger version (15K): In this window In a new window Download PPT slide   Figure 3. ste7+ is required for withholding cells from entry to meiosis in pat1-114-induced ectopic conjugation. Cells of h90 wild-type (A), h90 pat1-114 (B), and h90 pat1-114 ste7 (C) were grown at 24.5° to a density of 2 x 106 cells/ml and shifted to 29.5°. The percentage of zygotes formed and that of haploid cells containing either one/two or three/four nuclei was determined chronologically in each culture.

Negative role for ste7⁺ in physiologically induced meiosis:
To examine whether ste7⁺ could be deleterious for the progression of physiologically induced meiosis as well, we overexpressed ste7⁺ from the strong nmt promoter in h⁺/h^- wild-type diploid cells (JY362). As shown in Fig 4A, these cells showed a reduced ability to produce spores compared to the control cells carrying the vector. We performed a similar experiment using h⁹⁰/h⁹⁰ diploid cells as the host. In addition to performing meiosis and sporulation, h⁹⁰/h⁹⁰ wild-type cells are known to initiate conjugation at a low frequency upon nutrient starvation and to form tetraploid zygotes, which eventually generate asci mainly composed of either four diploid spores or eight haploid spores (GUTZ 1967 ). In the current experiment, we used a mutant h⁹⁰/h⁹⁰ diploid strain (JW234; genotype imp1/imp1) that completes meiosis but is defective in spore packaging (Y. AKIYOSHI, Y. WATANABE and M. YAMAMOTO, unpublished results), thus preventing spore release and making calculation of the number of tetraploid zygotes easier. JW234 was transformed with two kinds of ste7-overproducing plasmids, one of which carried the strong nmt1 promoter (pREP1-ste7), whereas the other carried the weak nmt1 promoter (pR3C-ste7). Like JY362, JW234 cells overexpressing ste7⁺ exhibited a reduced ability to carry out meiosis in a dose-dependent manner (Fig 4B). In contrast, these cells underwent conjugation more frequently as the expression of ste7⁺ was elevated (Fig 4B), indicating that overexpression of ste7⁺ was not inhibitory for mating. To examine the effect of ste7⁺ overexpression on conjugation of haploid cells, we analyzed an h⁹⁰ strain carrying pR3C-ste7. This strain exhibited a higher conjugation efficiency compared to the control strain carrying the vector (Fig 4C). These observations suggest that overproduced Ste7p supports conjugation but inhibits meiosis.

View larger version (20K): In this window In a new window Download PPT slide   Figure 4. Inhibitory effects of ste7+ on meiosis. (A) h+/h- diploid cells transformed with either vector pREP1 or pR1-ste7 overexpressing ste7+ were streaked on sporulation medium SSA, and the percentage of sporulated cells was determined after incubation for 3 days at 30°. Average scores obtained from at least three independent colonies are given. Each error bar represents a standard deviation. (B) Profiles of mating and meiosis in a h90/h90 diploid strain carrying either pREP1, pR3C-ste7, or pR1-ste7 were examined as in A. The number of cells that underwent sporulation was determined as opposed to the number of cells that underwent conjugation. (C) Conjugation frequency of a homothallic (h90) wild-type strain carrying either pREP3 or pR3C-ste7 was examined as in A.

Interaction of Ste7p with Pat1p and Mei2p in yeast two-hybrid assay:
One possible hypothesis is that Ste7p may suppress progression of meiosis by affecting the Pat1p-Mei2p regulatory system (WATANABE et al. 1997 ). To examine whether Ste7p physically interacts with either Pat1p or Mei2p, we carried out the yeast two-hybrid assay as detailed in MATERIALS AND METHODS. As shown in Fig 5, Ste7p was judged to interact with both Pat1p and Mei2p, reinforcing the possibility that Ste7p may control initiation of meiosis through the Pat1p-Mei2p system.

View larger version (19K): In this window In a new window Download PPT slide   Figure 5. Yeast two-hybrid assay. The GAL4-activating domain fused to either Ste7p, Pat1p, or Mei2p was coexpressed with the LexA DNA-binding domain fused to either Mei2p or Pat1p in the reporter S. cerevisiae strain L40. The VP16-activating domain fused to Raf and the LexA DNA-binding domain fused to Ras were also included in the analysis, coexpression of which served as a positive control. Two transformants were examined for each pair of fusion proteins. The ß-galactosidase activity was assayed by blue coloring of X-gal.

Ste7p disappears when conjugation is completed:
We examined intracellular localization of Ste7p by conjugating it with jellyfish GFP, as described in MATERIALS AND METHODS. The ste7-GFP fusion gene was expressed from the authentic ste7 promoter on the multicopy vector pSP1 (COTTAREL et al. 1993 ). The sterility of ste7 cells could be rescued by transforming them with this plasmid, indicating that the Ste7p-GFP fusion protein was functionally similar to authentic Ste7p (data not shown). For comparison, we constructed a plasmid in which the ste7 ORF was removed and only the GFP ORF was expressed from the ste7 promoter. We transformed a homothallic h⁹⁰ haploid strain (JY450) with these two plasmids. The transformants were shifted from nitrogen-rich to nitrogen-free medium (MM to MM-N) to allow conjugation and meiosis/sporulation. Very little fluorescence, if any, was detected in both transformants when they were vegetatively growing (Fig 6A and Fig B), as was consistent with the results of Northern blot analysis. Fluorescence of Ste7p-GFP appeared in response to nitrogen starvation, uniformly throughout the cell (Fig 6A). Interestingly, however, we could not detect Ste7p-GFP fluorescence in cells that had completed conjugation and became zygotes (Fig 6A). The control strain expressing GFP also became fluorescent in response to nitrogen starvation, but it continued to emit fluorescence even after conjugation was completed (Fig 6B). These results suggest that Ste7p accumulates prior to conjugation but undergoes degradation soon after the completion of conjugation. This was directly demonstrated by time-lapse microscopy. As shown in Fig 7, JY450 expressing Ste7p-GFP emitted green fluorescence while they were performing conjugation, but the fluorescence gradually diminished during karyogamy and disappeared almost completely when nuclear fusion was finished. Essentially the same results were obtained with an h⁹⁰ ste7 haploid strain (JX600) expressing Ste7p-GFP (data not shown), declining the possibility that authentic Ste7p might persist and perform an essential function after nuclear fusion in the above experiments. Thus, we conclude that Ste7p undergoes degradation between completion of conjugation and initiation of meiosis.

View larger version (50K): In this window In a new window Download PPT slide   Figure 6. Ste7p is present in unconjugated cells but disappears in zygotes. Live observation of cells expressing GFP-tagged Ste7p. h90 wild-type cells (JY450) carrying either pSP1-ste7GFP, which expressed GFP-tagged Ste7p, or pSP1-ste7::GFP, which expressed GFP alone from the ste7 promoter, were grown in minimal SD medium. They were transferred to sporulation medium SPA and incubated at 30°. Cells right after the shift (+N) and cells incubated on SPA for 6 hr (-N) were examined for GFP fluorescence. Cells were counterstained with Hoechst 33342 for observation of their nuclei, and their cell morphology was examined by DIC microscopy. Bar, 10 µm.

View larger version (43K): In this window In a new window Download PPT slide   Figure 7. A pair of conjugating cells was chronologically examined for the fate of fluorescence emitted from GFP-tagged Ste7p. Cells of JY450 carrying pSP1-ste7GFP were incubated on SPA for 6 hr at 30°, resuspended in MM(-N), and subjected to DIC and fluorescence microscopy. Images of the same pair were taken successively at an interval of 5 min. Nuclei were stained with Hoechst 33342. Bar, 10 µm.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We identified the nucleotide sequence of S. pombe ste7 in this study. The deduced ste7 gene product appeared to be unique, and its amino acid sequence provided little information concerning its molecular function. ste7⁺ is essential for mating and meiosis. However, our analysis revealed that overproduction of ste7⁺ is inhibitory to meiosis, but not to conjugation, in the wild-type genetic background. More remarkably, overproduction of ste7⁺ caused frequent induction of mating with simultaneous suppression of meiosis in h⁹⁰/h⁹⁰ diploid cells. Contrarily, deletion of ste7 hampered hyperconjugation of pat1-114 cells at the semipermissive temperature and led them to haploid meiosis. These observations suggest that Ste7p has a role in blocking meiosis under certain situations, in addition to the positive function of promoting it. How can S. pombe cells reconcile these apparently contradictory roles? We speculate the following as a possible explanation.

First, let us consider the positive role of Ste7p in sexual development. A few lines of evidence support that Ste7p functions in an early step of development. Previous studies have demonstrated that ste7-deficient cells cannot activate pheromone-regulated events, such as transcription of the mating-type genes (AONO et al. 1994 ). Consistently, we have found that ste7-defective cells do not undergo pheromone-induced G₁ arrest (our unpublished data). In addition, ste7 does not block ectopic meiosis of pat1-114 in either haploid or diploid cells, indicating that Ste7p normally functions before the meiotic repressor Pat1p is inactivated. Thus, we speculate that Ste7p is most likely to be involved in the establishment of mating pheromone signaling. This explains the requirement of Ste7p for both conjugation and meiosis, because mating pheromone signaling is essential for not only conjugation but also meiosis in fission yeast (KITAMURA and SHIMODA 1991 ; OBARA et al. 1991 ; TANAKA et al. 1993 ).

Then, what does Ste7p do to block meiosis? It is presumable that Ste7p withholds the initiation of meiosis so that its function to establish the mating pheromone signaling and other relevant processes can take place appropriately during conjugation. Our two-hybrid analysis has suggested that Ste7p interacts with both Pat1p and Mei2p. This implies that Ste7p is likely to control initiation of meiosis through the Pat1p-Mei2p regulatory system (WATANABE et al. 1997 ). One possibility is that Ste7p binds to Pat1p and fixes its activity to a reduced level in haploid cells. This reduction may facilitate mating reactions, as postulated by NIELSEN and EGEL 1990 , but the remaining activity may still be sufficiently high to block the entry to meiosis. Alternatively, Ste7p may primarily affect either activation or function of Mei2p, so that full activation of Mei2p is restricted until conjugation has been completed and Ste7p degraded. In either case, the discussed function of Ste7p is still largely speculative and should await more rigorous proof.

Ste7p appears to continue to suppress the onset of meiosis until conjugation has been properly accomplished. Successful conjugation may generate a signal that leads to degradation of Ste7p. One obvious way that a zygote recognizes completion of conjugation is coexpression of the heterozygous mating-type genes, namely, mat1-P and mat1-M, which indeed has been shown to activate production of Mei3p, an inhibitor of Pat1 kinase (MCLEOD et al. 1987 ; WILLER et al. 1995 ; VAN HEECKEREN et al. 1998 ). A signal leading to degradation of Ste7p may stem from the coexpression of mat1-P and mat1-M as well. To perform its function to block meiosis, Ste7p may be modified by being phosphorylated, for instance. Because Ste7p appears to interact with Pat1p kinase and carries two possible phosphorylation sites by this kinase (Fig 1B), it is even presumable that Ste7p persists while it is phosphorylated by Pat1p but becomes susceptible to degradation once it is dephosphorylated. Alternatively, more structural traits reflecting the fusion of two partners may generate a signal to degrade Ste7p. Certainly, it will be interesting to identify factors responsible for the degradation of Ste7p and signaling pathways involved in this degradation.

	FOOTNOTES

¹ Present address: Institute of Medical Science, University of Tokyo, Takanawa, Tokyo 108-8639, Japan.

	ACKNOWLEDGMENTS

We thank Yuji Akiyoshi for the imp1 strain. This work was supported by a Grant-in-Aid for Specially Promoted Research from the Ministry of Education, Science, Sports and Culture of Japan.

Manuscript received November 30, 1999; Accepted for publication January 31, 2000.

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