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Most sexually reproducing organisms have the ability to recognize individuals of the same species. In ascomycete fungi including yeasts, mating between cells of opposite mating type depends on the molecular recognition of two peptidyl mating pheromones by their corresponding G-protein coupled receptors (GPCRs). Although such pheromone/receptor systems are likely to function in both mate choice and prezygotic isolation, very few studies have focused on the stringency of pheromone receptors. The fission yeast Schizosaccharomyces pombe has two mating types, Plus (P) and Minus (M). Here, we investigated the stringency of the two GPCRs, Mam2 and Map3, for their respective pheromones, P-factor and M-factor, in fission yeast. First, we switched GPCRs between S. pombe and the closely related species Schizosaccharomyces octosporus, which showed that SoMam2 (Mam2 of S. octosporus) is partially functional in S. pombe, whereas SoMap3 (Map3 of S. octosporus) is not interchangeable. Next, we swapped individual domains of Mam2 and Map3 with the respective domains in SoMam2 and SoMap3, which revealed differences between the receptors both in the intracellular regions that regulate the downstream signaling of pheromones and in the activation by the pheromone. In particular, we demonstrated that two amino acid residues of Map3, F214 and F215, are key residues important for discrimination of closely related M-factors. Thus, the differences in these two GPCRs might reflect the significantly distinct stringency/flexibility of their respective pheromone/receptor systems; nevertheless, species-specific pheromone recognition remains incomplete.

Most sexually reproducing organisms have the ability to recognize individuals of the same species. In ascomycetes including yeasts, potential mating between cells of opposite mating-type depends on the molecular recognition of two peptidyl mating pheromones by their corresponding G-protein coupled receptors (GPCRs). Although such pheromone/receptor systems are likely to function in both mate choice and prezygotic isolation, very few studies have focus on the differences in pheromone/receptor system between mating types. The fission yeastSchizosaccharomyces pombehas two mating types (sexes), Plus (P) and Minus (M). Here we investigated the specificity of the two GPCRs, Mam2 and Map3, for their respective pheromones, P-factor and M-factor, in fission yeast. First, we switched GPCRs betweenS. pombeand the closely related speciesSchizosaccharomyces octosporus, which showed that SoMam2 (Mam2 ofS. octosporus) is partially functional inS. pombe, whereas SoMap3 (Map3 ofS. octosporus) is not interchangeable. Next, we swapped individual domains of Mam2 and Map3 with the respective domains in SoMam2 and SoMap3, which revealed differences between the receptors both in the intracellular regions that regulate the downstream signaling of pheromones and in the molecular recognition for pheromone binding. In particular, we demonstrated that two amino acid residues of Map3, F214 and F215, are essential for the specificity of M-factor recognition. Thus, the differences in these two GPCRs are likely to reflect the significantly distinct specificities of their respective pheromone/receptor systems; that is, the specificity of Map3 is more stringent than that of Mam2. We speculate that this sexual asymmetry might allow ascomycete fungi to generate novel prezygotic barriers within a population, while maintaining strong mate choice. Our genetic analyses also contribute to our understanding of the receptors that comprise the Class D GPCRs belonging to the fungal pheromone receptor family.Author summaryCourtship signals play a key role in the fertilization processes of living beings from animals to microorganisms and, in particular, sex pheromones are involved in differentiating among species. On the other hand, changes in a pheromone/receptor system might also alter species-specificity during the selection of a mating partner, which is likely to facilitate prezygotic isolation. Here we demonstrate a distinct difference in the specificity of GPCRs for pheromones between cells of opposite mating-type in the fission yeastSchizosaccharomyces pombe, by conducting a comprehensive genetic analysis and comparison of these proteins between closely related fission yeast species. Our finding of sexual asymmetry due to the relative strictness of receptors inS. pombesuggests that it might be one of the driving forces behind the formation of new species. Such differences in pheromone/receptor interactions between the sexes may occur in a variety of life-forms such as insects and amphibians; hence, the findings of this study might be extended to other organisms. In addition, this study ofS. pombepheromone receptors sheds light on the functions of the unique Class D GPCRs.

Many species, from mammals to microorganisms, release sex pheromones to attract a potential partner of the opposite sex. The combination of a pheromone and its corresponding receptor determines the species-specific ability of males and females to recognize each other, and therefore causes reproductive isolation. This barrier, which has arisen to restrict gene flow between mating pairs, might facilitate reproductive isolation leading to incipient speciation, but how do new combinations of pheromone and receptor evolve? Our recent study demonstrated an “asymmetric” pheromone recognition system in the fission yeast Schizosaccharomyces pombe: among the two pheromone/receptor pairs in this yeast, recognition between one pair is stringent, while that between the other pair is rather relaxed. We speculate that the asymmetric properties of these pheromone recognition systems are beneficial for gradual evolution resulting in reproductive isolation in yeasts.

In the fission yeast Schizosaccharomyces pombe, the mating reaction is controlled by two mating pheromones, M-factor and P-factor, secreted by M- and P-type cells, respectively. M-factor is a C-terminally farnesylated lipid peptide, whereas P-factor is a simple peptide. To examine whether this chemical asymmetry in the two pheromones is essential for conjugation, we constructed a mating system in which either pheromone can stimulate both M- and P-cells, and examined whether the resulting autocrine strains can mate. Autocrine M-cells responding to M-factor successfully mated with P-factor-lacking P-cells, indicating that P-factor is not essential for conjugation; by contrast, autocrine P-cells responding to P-factor were unable to mate with M-factor-lacking M-cells. The sterility of the autocrine P-cells was completely restored by expressing the M-factor receptor. These observations indicate that the different chemical characteristics of the two types of pheromone, a lipid and a simple peptide, are not essential; however, a lipid peptide might be required for successful mating. Our findings allow us to propose a model of the differential roles of M-factor and P-factor in conjugation of S. pombeThis article has an associated First Person interview with the first author of the paper.

In fungi, mating between partners depends on the molecular recognition of two peptidyl mating pheromones by their respective receptors. The fission yeast Schizosaccharomyces pombe (Sp) has two mating types, Plus (P) and Minus (M). The mating pheromones P-factor and M-factor, secreted by P and M cells, are recognized by the receptors mating type auxiliary minus 2 (Mam2) and mating type auxiliary plus 3 (Map3), respectively. Our recent study demonstrated that a few mutations in both M-factor and Map3 can trigger reproductive isolation in S. pombe. Here, we explored the mechanism underlying reproductive isolation through genetic changes of pheromones/receptors in nature. We investigated the diversity of genes encoding the pheromones and their receptor in 150 wild S. pombe strains. Whereas the amino acid sequences of M-factor and Map3 were completely conserved, those of P-factor and Mam2 were very diverse. In addition, the P-factor gene contained varying numbers of tandem repeats of P-factor (4–8 repeats). By exploring the recognition specificity of pheromones between S. pombe and its close relative Schizosaccharomyces octosporus (So), we found that So-M-factor did not have an effect on S. pombe P cells, but So-P-factor had a partial effect on S. pombe M cells. Thus, recognition of M-factor seems to be stringent, whereas that of P-factor is relatively relaxed. We speculate that asymmetric diversification of the two pheromones might be facilitated by the distinctly different specificities of the two receptors. Our findings suggest that M-factor communication plays an important role in defining the species, whereas P-factor communication is able to undergo a certain degree of flexible adaptation–perhaps as a first step toward prezygotic isolation in S. pombe., An asymmetric pheromone/receptor system in the fission yeast Schizosaccharomyces pombe might allow flexible adaptation of pheromones to mutational changes while maintaining stringent recognition for mating partners, perhaps as a first step toward prezygotic mating isolation., Author summary The emergence of a new species might occur when two groups can no longer mate. Although such reproductive isolation is considered a key evolutionary process, the mechanisms by which it actually occurs have been confined to conjecture. The two sexes (Plus [P] and Minus [M]) of S. pombe each secrete a pheromone (P-factor and M-factor), which binds to a corresponding receptor (mating type auxiliary minus 2 [Mam2] and mating type auxiliary plus 3 [Map3]) on cells of the opposite sex. The interaction between a pheromone and its receptor is essential for successful mating. Here, we explored conservation of the mating pheromone communication system among 150 wild S. pombe strains of different geographical origins and the closely related species S. octosporus. We found that 1) the M-factor/Map3 interaction was completely conserved, whereas the P-factor/Mam2 interaction was very diverse in the strains investigated, and 2) most of the P-factor variants were functional across species. Thus, we have revealed an asymmetric pheromone/receptor system in fungal mating: namely, whereas M-factor communication operates extremely stringently, P-factor communication has the flexibility to create variations, perhaps facilitating prezygotic isolation in S. pombe.

The diversification of sex pheromones is regarded as one of the causes of prezygotic isolation that results in speciation. In the fission yeast Schizosaccharomyces pombe, the molecular recognition of a peptide pheromone by its receptor plays an essential role in sexual reproduction. We considered that molecular coevolution of a peptide-mating pheromone, M factor, and its receptor, Map3, might be realized by experimentally diversifying these proteins. Here, we report the successful creation of novel mating-type pairs by searching for map3 suppressor mutations that rescued the sterility of M-factor mutants that were previously isolated. Several strong suppressors were found to also recognize WT M factor. The substituted residues of these Map3 suppressors were mapped to F204, F214, and E249, which are likely to be critical residues for M-factor recognition. These critical residues were systematically substituted with each of the other amino acids by in vitro mutagenesis. Ultimately, we successfully obtained three novel mating-type pairs constituting reproductive groups. These novel mating-type pairs could not conjugate with WT maters. Furthermore, no flow of chromosomally integrated drug-resistance genes occurred between the novel and the WT mating pairs, showing that each experimentally created reproductive group [e.g., M factor(V5H) and Map3(F214H)] was isolated from the WT group. In conclusion, we have succeeded in creating an artificial reproductive group that is isolated from the WT group. In keeping with the biological concept of species, the artificial reproductive group is a new species.

The outermost layer of fission yeast spore is mainly composed of the lsp3 protein. Isp3-deleted spores show decreased tolerance to heat, digestive enzymes, and ethanol. Thus Isp3 constitutes the spore coat, thereby conferring resistance to various environmental stresses., The spore is a dormant cell that is resistant to various environmental stresses. As compared with the vegetative cell wall, the spore wall has a more extensive structure that confers resistance on spores. In the fission yeast Schizosaccharomyces pombe, the polysaccharides glucan and chitosan are major components of the spore wall; however, the structure of the spore surface remains unknown. We identify the spore coat protein Isp3/Meu4. The isp3 disruptant is viable and executes meiotic nuclear divisions as efficiently as the wild type, but isp3∆ spores show decreased tolerance to heat, digestive enzymes, and ethanol. Electron microscopy shows that an electron-dense layer is formed at the outermost region of the wild-type spore wall. This layer is not observed in isp3∆ spores. Furthermore, Isp3 is abundantly detected in this layer by immunoelectron microscopy. Thus Isp3 constitutes the spore coat, thereby conferring resistance to various environmental stresses.

Calmodulin in Schizosaccharomyces pombe is encoded by the cam1 + gene, which is indispensable for both vegetative growth and sporulation. Here, we report how Cam1 functions in spore formation. We found that Cam1 preferentially localized to the spindle pole body (SPB) during meiosis and sporulation. Formation of the forespore membrane, a precursor of the plasma membrane in spores, was blocked in a missense cam1 mutant, which was viable but unable to sporulate. Three SPB proteins necessary for the onset of forespore membrane formation, Spo2, Spo13, and Spo15, were unable to localize to the SPB in the cam1 mutant although five core SPB components that were tested were present. Recruitment of Spo2 and Spo13 is known to require the presence of Spo15 in the SPB. Notably, Spo15 was unstable in the cam1 mutant, and as a result, SPB localization of Spo2 and Spo13 was lost. Overexpression of Spo15 partially alleviated the sporulation defect in the cam1 mutant. These results indicate that calmodulin plays an essential role in forespore membrane formation by stably maintaining Spo15, and thus Spo2 and Spo13, at the SPB in meiotic cells.

During yeast sporulation, a forespore membrane (FSM) initiates at each spindle-pole body and extends to form the spore envelope. We used Schizosaccharomyces pombe to investigate the role of septins during this process. During the prior conjugation of haploid cells, the four vegetatively expressed septins (Spn1, Spn2, Spn3, and Spn4) coassemble at the fusion site and are necessary for its normal morphogenesis. Sporulation involves a different set of four septins (Spn2, Spn5, Spn6, and the atypical Spn7) that does not include the core subunits of the vegetative septin complex. The four sporulation septins form a complex in vitro and colocalize interdependently to a ring-shaped structure along each FSM, and septin mutations result in disoriented FSM extension. The septins and the leading-edge proteins appear to function in parallel to orient FSM extension. Spn2 and Spn7 bind to phosphatidylinositol 4-phosphate [PtdIns(4)P] in vitro, and PtdIns(4)P is enriched in the FSMs, suggesting that septins bind to the FSMs via this lipid. Cells expressing a mutant Spn2 protein unable to bind PtdIns(4)P still form extended septin structures, but these structures fail to associate with the FSMs, which are frequently disoriented. Thus, septins appear to form a scaffold that helps to guide the oriented extension of the FSM.

Syntaxin is a component of t-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), which is responsible for docking membrane vesicles at the target membrane and is highly conserved among eukaryotes. In the fission yeast Schizosaccharomyces pombe, the psy1(+) gene encoding a syntaxin 1 homolog was originally isolated as a multicopy suppressor of the sporulation-deficient mutant, spo3, but little is known about the way Psy1 is involved in sporulation. Here we report the isolation of a sporulation-defective mutant, psy1-S1, generated by random PCR mutagenesis. psy1-S1 also exhibited temperature sensitivity in growth. In psy1-S1 cells, assembly of the forespore membrane (FSM) initiated near the spindle pole bodies during meiosis II, but subsequent expansion of the membrane was severely impaired. Overproduction of the cognate SNARE proteins, Syb1 and Sec9, suppressed both the temperature sensitivity and sporulation defects of psy1-S1. These results indicate that Psy1 plays an essential role in FSM formation coordinated by Syb1 and Sec9.

Both farnesyl diphosphate synthase (FPS) and geranylgeranyl diphosphate synthase (GGPS) are key enzymes in the synthesis of various isoprenoid-containing compounds and proteins. Here, we describe two novel Schizosaccharomyces pombe genes, fps1+and spo9+, whose products are similar to FPS in primary structure, but whose functions differ from one another. Fps1 is essential for vegetative growth, whereas, a spo9 null mutant exhibits temperature-sensitive growth. Expression of fps1+, but not spo9+, suppresses the lethality of a Saccharomyces cerevisiae FPS-deficient mutant and also restores ubiquinone synthesis in an Escherichia coli ispA mutant, which lacks FPS activity, indicating that S. pombe Fps1 in fact functions as an FPS. In contrast to a typical FPS gene, no apparent GGPS homologues have been found in the S. pombe genome. Interestingly, although neither fps1+nor spo9+expression alone in E. coli confers clear GGPS activity, coexpression of both genes induces such activity. Moreover, the GGPS activity is significantly reduced in the spo9 mutant. In addition, the spo9 mutation perturbs the membrane association of a geranylgeranylated protein, but not that of a farnesylated protein. Yeast two-hybrid and coimmunoprecipitation analyses indicate that Fps1 and Spo9 physically interact. Thus, neither Fps1 nor Spo9 alone functions as a GGPS, but the two proteins together form a complex with GGPS activity. Because spo9 was originally identified as a sporulation-deficient mutant, we show here that expansion of the forespore membrane is severely inhibited in spo9Δ cells. Electron microscopy revealed significant accumulation membrane vesicles in spo9Δ cells. We suggest that lack of GGPS activity in a spo9 mutant results in impaired protein prenylation in certain proteins responsible for secretory function, thereby inhibiting forespore membrane formation.

Myo1, a heavy chain of type I myosin of the fission yeast Schizosaccharomyces pombe, is essential for sporulation. Here we have analyzed the expression, localization and cellular function of the type I myosin light chain calmodulin, Cam2, encoded by cam2(+). Transcription of cam2(+) was constitutive and markedly enhanced in meiosis. The cam2 null mutant was viable and completed sporulation normally at 28 degrees C, but formed four-spored asci poorly at 34 degrees C. In those sporulation-defective cells, the forespore membrane was formed abnormally. A Cam2-GFP fusion protein accumulated at the cell poles in interphase cells and at the medial septation site in postmitotic cells, colocalizing with Myo1 and F-actin patches. During the mating process, a single Cam2-GFP dot was detected at the tip of the mating projection. During meiosis-I, the Cam2-GFP dots dispersed into the cell periphery and the cytoplasm. At metaphase-II, intense Cam2-GFP signals appeared near Meu14 rings which were formed at the leading edge of expanding forespore membranes. This localization of Cam2 was dependent upon Myo1; and sporulation defect of cam2Delta at 34 degrees C was alleviated by overexpressing Myo1DeltaIQ. These results suggest a close relationship between Cam2 and Myo1. In addition, both F-actin and Myo1 localized with Cam2 in the leading edge region. In summary, type I myosin and F-actin accumulate at the leading edge area of the forespore membrane and may play a pivotal role in its assembly.

Cullin‐4 forms a scaffold for multiple ubiquitin ligases. In Schizosaccharomyces pombe , the Cullin‐4 homologue (Pcu4) physically associates with Ddb1 and the COP9 signalosome (CSN). One target of this complex is Spd1. Spd1 regulates ribonucleotide reductase (RNR) activity. Spd1 degradation during S phase, or following DNA damage of G2 cells, results in the nuclear export of the small RNR subunit. We demonstrate that Cdt2, an unstable WD40 protein, is a regulatory subunit of Pcu4–Ddb1–CSN ubiquitin ligase. cdt2 deletion stabilises Spd1 and prevents relocalisation of the small RNR subunit from the nucleus to the cytoplasm. cdt2 + is periodically transcribed by the Cdc10/DSC1 transcription factor during S phase and transiently transcribed following DNA damage of G2 cells, corresponding to Spd1 degradation profiles. Cdt2 co‐precipitates with Spd1, and Cdt2 overexpression results in constitutive Spd1 degradation. We propose that Cdt2 incorporation into the Pcu4–Ddb1–CSN complex prompts Spd1 targeting and subsequent degradation and that Cdt2 is a WD40 repeat adaptor protein for Cullin‐4‐based ubiquitin ligase.

Protein O-glycosylation is an essential protein modification in eukaryotic cells. In Saccharomyces cerevisiae, O-mannosylation is initiated in the lumen of the endoplasmic reticulum by O-mannosyltransferase gene products (Pmt1p-7p). A search of the Schizosaccharomyces pombe genome database revealed a total of three O-glycoside mannosyltransferase homologs (ogm1+, ogm2+, and ogm4+), closely related to Saccharomyces cerevisiae PMT1, PMT2, and PMT4. Although individual ogm genes were not found to be essential, ogm1Delta and ogm4Delta mutants exhibited aberrant morphology and failed to agglutinate during mating. The phenotypes of the ogm4Delta mutant were not complemented by overexpression of ogm1+ or ogm2+, suggesting that each of the Ogm proteins does not have overlapping functions. Heterologous expression of a chitinase from S. cerevisiae in the ogm mutants revealed that O-glycosylation of chitinase had decreased in ogm1Delta cells. A GFP-tagged Fus1p from S. cerevisiae was specifically not glycosylated and accumulated in the Golgi in ogm4Delta cells. These results indicate that O-glycosylation initiated by Ogm proteins plays crucial physiological roles and can serve as a sorting determinant for protein transport of membrane glycoproteins in S. pombe.

A novel sporulation-deficient mutant, sev4-L5, was isolated in a genetic screen of a collection of temperature-sensitive mutants of Schizosaccharomyces pombe. The wild-type sev4 gene was identified as cta4+, which encodes a putative cation-transporting P-type ATPase. The sev4-L5 allele harbored a single missense mutation that caused replacement of Gly615 with a glutamate at the putative ATP-binding site. Similar to cta4-null mutants, sev4-L5 exhibited defects in growth at high and low temperatures, and sensitivity to high and extremely low concentrations of Ca2+. The cta4+ mRNA level was considerably enhanced during meiosis. When sev4-L5 cells were incubated in sporulation medium at the permissive temperature, meiotic nuclear divisions proceeded with normal kinetics, but spores were not formed. Structural alteration of the spindle pole body, which is prerequisite to construction of the forespore membrane in wild type, was incomplete. Consequently, formation of the forespore membrane was severely impaired. These observations show that perturbation of Ca2+ homeostasis by mutation of cta4/sev4 blocks sporulation mainly by interfering with forespore membrane assembly.

We report the identification of Schizosaccharomyces pombe mde10 + as a gene possessing a FLEX element, which forms a binding site for the meiosis-specific transcription factor Mei4. In fact, mde10 + is transcribed only in diploid cells that are induced to meiosis in a Mei4-dependent manner. Western blot analysis indicated that the epitope-tagged Mde10 protein accumulates transiently during meiosis and then rapidly decreases. Mde10 is a multidomain protein containing a metalloprotease catalytic domain, a disintegrin domain, a cysteine-rich domain, and membrane-spanning regions, all of which are shared by members of the mammalian ADAM family. A fusion protein of Mde10 and green fluorescent protein localized to the endoplasmic reticulum during meiosis and was located at the peripheral region of spores at the end of meiosis. An mde10 Δ deletion mutant showed no apparent defects in meiosis, sporulation, or spore germination. However, the mutant spores exhibited an aberrant surface appearance, in which the ragged outer spore wall was lost to a large extent. Furthermore, mde10 Δ spores were found to be less tolerant to ethanol and diethyl ether than were wild-type spores. The mutagenic replacement of the conserved glutamic acid in the putative protease active site with an alanine residue did not affect the surface morphology or the resistance of spores to environmental stress. Our observations indicate that Mde10 is important in the development of the spore envelope, although this function of Mde10 seems to be independent of its metalloprotease activity.

Stability of ribosomal RNA (rRNA) is not only essential for ribosome biogenesis but also crucial to the maintenance of proper translational level for cell viability. rRNA processing (maturation) is one of the key steps to derive functional rRNA, and to date, a large number of factors involved in this process have been identified. We investigated Rps0 binding proteins in fission yeast, Schizosaccharomyces pombe, and revealed that Rps0p is associated with Rps21 protein, similar to that of our previous observation in human cells. We demonstrated that both rps0(+)s and rps21(+) are essential genes for S. pombe analyzed by tetrad dissection assay. To study the functions of both genes, we established disruption strains transformed with inducible rescue plasmids. Using the strains our studies revealed that the loss of rps0(+)s or rps21(+) led to a deficiency of 40S ribosomal subunit formation. Additional functional studies indicate that this phenomenon is likely to be caused by insufficient 18S rRNA stability. The possible role of Rps0p and Rps21 that contribute to 18S rRNA maturation is further discussed.

Phosphatidylinositol (PI) 3-kinase (encoded by the pik3(+) gene) in Schizosaccharomyces pombe has been identified as a homologue of VPS34p, a protein required for proper vesicular protein sorting. The clone defective in this protein carries enlarged vacuoles and exhibits sensitivity to high temperature or high ion concentration. The effect of disruption of pik3(+) on sporulation of Sz. pombe was examined. The diploid cells underwent G(1) arrest and meiosis. However, the spores formed by the deltapik3 cells were not viable. Electron-microscopic analysis revealed that the growth of the forespore membrane of deltapik3 cells was not correctly orientated, failing to engulf the nucleus or forming extremely small spores, as was confirmed by the use of Spo3p-GFP and GFP-Psy1p, which are markers of the forespore membrane. The coating materials found along the forespore membrane of the wild-type were greatly reduced in these cells. PI 3-P, the product of Pik3p, was detected on the forespore membrane, suggesting that PI 3-P-dependent vesicle transport may take place in formation of the forespore membrane. Misshaped forespore membrane, accumulation of vesicles, formation of small non-viable spores, and suppression by over expression of Psy1p were the phenotypes commonly seen in deltapik3 and deltaspo3 cells, suggesting a relationship between the functions of Pik3p and Spo3p in formation of the forespore membrane in Sz. pombe.

Background: Phosphatidylinositol (3,5) bisphosphate, which is converted from phosphatidylinositol 3-phosphate by phosphatidylinositol 3-phosphate 5-kinase, is implicated in vacuolar functions and the sorting of cell surface proteins within endosomes in the endocytic pathway of budding yeast. A homologous protein, SpFab1p, has been found in the fission yeast Schizosaccharomyces pombe, but its role is not known. Results: Here we report that SpFab1p is encoded by ste12+ known as a fertility gene in S. pombe. The ste12 mutant grew normally under stress-free conditions, but was highly vacuolated and swelled at high temperatures and under starvation conditions. In nitrogen-free medium, ste12 cells were arrested in G1 phase, but partially defective in the expression of genes responsible for mating and meiosis. The ste12 mutant was defective both in the production of, and in the response to, mating pheromones. The amount of the pheromone receptor protein Map3p, was substantially decreased in ste12 cells. Map3p was transported to the cell surface, then internalized and eventually transported to the vacuolar lumen, even in the ste12 mutant. Conclusion: The results indicate that phosphatidylinositol(3,5)bisphosphate is essential for cellular responses to various stresses and for the mating pheromone signalling under starvation conditions.

Formation of the forespore membrane, which becomes the plasma membrane of spores, is an intriguing step in the sporulation of the fission yeast Schizosaccharomyces pombe. Here we report two novel proteins that localize to the forespore membrane.spo3+encodes a potential membrane protein, which was expressed only during sporulation. Green fluorescent protein (GFP) fusion revealed that Spo3 localized to the forespore membrane. The spo3 disruptant was viable and executed meiotic nuclear divisions as efficiently as the wild type but did not form spores. One of the spo3 alleles,spo3-KC51, was dose-dependently suppressed bypsy1+, which encodes a protein similar to mammalian syntaxin-1A, a component of the plasma membrane docking/fusion complex. psy1+was essential for vegetative growth, and its transcription was enhanced during sporulation. As expected, Psy1 localized to the plasma membrane during vegetative growth. Interestingly, Psy1 on the plasma membrane disappeared immediately after first meiotic division and relocalized to the forespore membrane as the second division initiated. In thespo3 null mutant, the forespore membrane was initiated but failed to develop a normal morphology. Electron microscopy revealed that membrane vesicles were accumulated in the cytoplasm of immaturespo3Δ asci. These results suggest that Spo3 is a key component of the forespore membrane and is essential for its assembly acting in collaboration with the syntaxin-like protein.

The Schizosaccharomyces pombe spo20-KC104 mutation was originally isolated in a screen for sporulation-deficient mutants, and the spo20-KC104 mutant exhibits temperature-sensitive growth. Herein, we report that S. pombe, spo20+is essential for fission yeast cell viability and is constitutively expressed throughout the life cycle. We also demonstrate that thespo20+gene product is structurally homologous to Saccharomyces cerevisiae Sec14, the major phosphatidylinositol transfer protein of budding yeast. This structural homology translates to a significant degree of functional relatedness because reciprocal complementation experiments demonstrate that each protein is able to fulfill the essential function of the other. Moreover, biochemical experiments show that, like Sec14, Spo20 is a phosphatidylinositol/phosphatidylcholine-transfer protein. That Spo20 is required for Golgi secretory function in vegetative cells is indicated by our demonstration that the spo20-KC104mutant accumulates aberrant Golgi cisternae at restrictive temperatures. However, a second phenotype observed in Spo20-deficient fission yeast is arrest of cell division before completion of cell separation. Consistent with a direct role for Spo20 in controlling cell septation in vegetatively growing cells, localization experiments reveal that Spo20 preferentially localizes to the cell poles and to sites of septation of fission yeast cells. We also report that, when fission yeasts are challenged with nitrogen starvation, Spo20 translocates to the nucleus. This nuclear localization persists during conjugation and meiosis. On completion of meiosis, Spo20 translocates to forespore membranes, and it is the assembly of forespore membranes that is abnormal in spo20-KC104 cells. In such mutants, a considerable fraction of forming prespores fail to encapsulate the haploid nucleus. Our results indicate that Spo20 regulates the formation of specialized membrane structures in addition to its recognized role in regulating Golgi secretory function.

Spore germination, a transition from the quiescent G0 phase to the proliferation cycle, is triggered by glucose in Schizosaccharomyces pombe. The role of cAMP/protein kinase A (PKA) signalling in germination is investigated. Gene disruption of cyr1+, pka1+ and gpa2+ encoding adenylate cyclase, PKA and the α-subunit of a trimeric GTP-binding protein, respectively, reduced the colony-forming efficiency of spores in minimal medium. Isolated spores of these null mutants did not germinate in minimal medium for up to 12 h, at which time wild-type spores had completed germination and formed germ projections. In wild-type spores, cortical actin patches randomly distributed in the early stage of outgrowth and then localized to one side of spores before the formation of projections. In contrast, the mutant spores exhibited no actin patches, but the cell surface was predominantly stained, like ungerminated spores of wild-type. Flow fluorocytometric analysis of propidium iodide-stained spores revealed a distinct 1C DNA peak after germination was completed. The fluorescent profile of the mutant spores, however, did not change during 12 h incubation in the minimal medium. These observations indicate that spores harbouring either cyr1Δ, pka1Δ or gpa2Δ are hardly triggered to germination. When wild-type spores were exposed to glucose, the intracellular cAMP level transiently increased in a few minutes, but gpa2Δ spores did not respond to glucose. We conclude that S. pombe spores initiate germination in response to glucose through the cyclic AMP–PKA pathway. Copyright © 2000 John Wiley & Sons, Ltd.

Background Sporulation of the fission yeast Schizosaccharomyces pombe is a cell differentiation process which accompanies meiosis. The spo6+ gene was identified as a sporulation-specific gene, whose transcription was regulated by the forkhead family transcription factor Mei4. Results spo6+ encodes a protein with sequence similarity to Saccharomyces cerevisiae Dbf4p, which is required for the initiation of DNA replication. However, doubling time and cell morphology of spo6 deletion mutants and spo6-cDNA over-expressing cells were indistinguishable from wild-type cells. Spliced mature mRNAs of spo6+ appeared when diploid cells committed to meiosis. Spo6p fused to green fluorescent protein (GFP) preferentially localized in a nucleus. Although spo6Δ diploids normally underwent premeiotic DNA replication and meiosis-I, approximately 80% of cells were blocked at the binucleate stage during meiosis and virtually no asci were formed. Anti-tubulin staining revealed that only 25% of the binucleate cells assembled spindle microtubules for meiosis-II. In a small number of tetranucleate cells, sister nuclei insufficiently separated and spindles were frequently fragmented. The meiosis-II arrest phenotype was exaggerated at low temperature and in the presence of caffeine. Conclusions These results indicate that Spo6p is a novel Dbf4-related nuclear protein, which is expressed during meiosis and is indispensable for normal progression of meiosis-II and sporulation.

The Schizosaccharomyces pombe mei4+ gene encoding a forkhead transcription factor is necessary for the progression of meiosis and sporulation. We searched for novel meiotic genes, the expression of which is dependent on Mei4p, since only the spo6+ gene has been assigned to its targets. Six known genes responsible for meiotic recombination were examined by Northern blotting, but none were Mei4 dependent for transcription. We determined the important cis-acting element, designated FLEX, to which Mei4p can bind. The S. pombe genome sequence database (The Sanger Centre, UK) was scanned for the central core heptamer and its flanking 3′ sequence of FLEX composed of 17 nucleotides, and 10 candidate targets of Mei4 were selected. These contained a FLEX-like sequence in the 5′ upstream nontranslatable region within 1 kb of the initiation codon. Northern blotting confirmed that 9 of them, named mde1+ to mde9+, were transcriptionally induced during meiosis and were dependent on mei4+. Most mde genes have not been genetically defined yet, except for mde9+, which is identical to spn5+, which encodes one of the septin family of proteins. mde3+ and a related gene pit1+ encode proteins related to Saccharomyces cerevisiae Ime2. The double disruptant frequently produced asci having an abnormal number and size of spores, although it completed meiosis. We also found that the forkhead DNA-binding domain of Mei4p binds to the FLEX-like element in the putative promoter region of mei4 and that the maximum induction level of mei4 mRNA required functional mei4 activity. Furthermore, expression of a reporter gene driven by the authentic mei4 promoter was induced in vegetative cells by ectopic overproduction of Mei4p. These results suggest that mei4 transcription is positively autoregulated.

The haploid cells of the fission yeast, Schizosaccharomyces pombe, are arrested in the G1-phase by nitrogen starvation and are committed to sexual reproduction (mating and sporulation). We isolated the sterile mutants which were defective in G1 arrest following nitrogen starvation. Genetic analysis of these mutants defined a single locus designated as ste16. The nucleotide sequence revealed that ste16+ encodes an 82-kDa protein containing eight WD40-repeats in its carboxy terminal half. The ste16 disruptant was viable, but arrested the cell cycle in the G2-phase after the nutritional down-shift. When transferred to fresh growth medium, the G2-arrested ste16Delta haploids resumed the mitotic cycle from the S-phase, resulting in diploidization. This diploidization phenomenon was completely suppressed by the null mutation of rum1 encoding the inhibitor of Cdc2 kinase. As the Rum1 protein level was remarkably elevated in the ste16Delta, the Ste16 protein negatively controls the Rum1 level. The loss of function of ste16 disturbs the cell-cycle progression and impairs the mechanism for the maintenance of ploidy.

Mating pheromone signaling is essential for conjugation between haploid cells of P-type (P-cells) and haploid cells of M-type (M-cells) in Schizosaccharomyces pombe. A peptide pheromone, M-factor, produced by M-cells is recognized by the receptor of P-cells. An M-factor-less mutant, in which the M-factor-encoding genes are deleted, is completely sterile. In liquid culture, sexual agglutination was not observed in the mutant, but it could be recovered by adding exogenous synthetic M-factor, which stimulated expression of the P-type-specific cell adhesion protein, Map4. Exogenous M-factor, however, failed to recover the cell fusion defect in the M-factor-less mutant. When M-factor-less cells were added to a mixture of wild-type P- and M-cells, marked cell aggregates were formed. Notably, M-factor-less mutant cells were also incorporated in these aggregates. In this mixed culture, P-cells conjugated preferentially with M-cells secreting M-factor, and rarely with M-factor-less M-cells. The kinetics of mating parameters in liquid culture revealed that polarized growth commenced from the contact region of opposite mating-type cells. Taken together, these findings indicate that M-factor at a low concentration induces adhesin expression, leading to initial cell-cell adhesion in a type of "distal pheromone action", but M-factor that is secreted directly in the proximity of the adhered P-cells may be necessary for cell fusion in a type of "proximal pheromone action".

The mating reaction is triggered by specific pheromones in a wide variety of organisms. Small peptides are used as mating pheromones in yeasts and fungi. In the fission yeast Schizosaccharomyces pombe, M-factor is a C terminally farnesylated nonapeptide secreted from M-cells, and its counterpart, P-factor, is a simple peptide composed of 23 amino acids. The primary structure requirements for the biological activity of pheromone peptides remain to be elucidated. Here, we conducted comprehensive substitution of each of the amino acids in M-factor peptide and inspected the mating ability of these missense mutants. Thirty-five sterile mutants were found among an array of 152 mutants with single amino acid substitutions. Mapping of the mutation sites clearly indicated that the sterile mutants were associated exclusively with four amino acid residues (VPYM) in the carboxyl-terminal half. In contrast, the substitution of four amino-terminal residues (YTPK) with any amino acid had no or only a slightly deleterious effect on mating. Furthermore, deletion of the three N-terminal residues caused no sterility, although truncation of a fourth residue had a marked effect. We conclude that a farnesylated hexapeptide (KVPYMCFar–OCH3) is the minimal M-factor that retains pheromone activity. At least 15 nonfunctional peptides were found to be secreted, suggesting that these mutant M-factor peptides are no longer recognized by the cognate receptor.

Transcription of the mat1-Pm gene of Schizosaccharomyces pombe controlling entry into meiosis is stimulated by the mating pheromone, M-factor. We have studied its expression by monitoring beta-galactosidase activity in cells carrying a plasmid-borne mat1-Pm/lacZ fusion construct. Stimulation required the M-factor receptor (Map3) and other proteins (Gpa1, Byr1, Byr2 and Spk1) thought to be involved in propagating the pheromone signal within the cell. Mutational activation of gpa1 encoding an alpha subunit of the receptor-coupled heterotrimeric G protein causes full expression of mat1-Pm even in the absence of pheromone, suggesting that Gpa1 is a key signal transmitter. Furthermore, an activated ras1val17 mutant exhibited a much stronger level of induction than wild-type cells, though full expression needs M-factor treatment. Deletion analysis of the mat1-Pm promoter region identified a stretch of 21 bp that is shown to play a critical role in controlling expression. This region lies just upstream of a TATA-like box and contains a TR-box (TTCTTTGTTY) motif which is the recognition site of a putative transcription factor Ste11. Point mutations in the TR-box motif abolished the expression of mat1-Pm/lacZ. Almost no expression of mat1-Pm was detected in a ste11 deletion mutant, whereas overproduction of Ste11 greatly increased the expression.

In order to characterize the morphological steps defined by sporulation (spo) genes during the formation of ascospores in the fission yeast Schizosaccharomyces pombe, we performed an electron microscopic study of the ultrastructure of the spindle pole body (SPB) and of the development of the forespore membrane during the second meiotic division (meiosis II) in sporulation-deficient (spo) mutants (spo4, spo5, spo14 and spo18). No difference was found in terms of the function and the structure of the SPB during the first meiotic division (meiosis I) between the four mutants and wild-type cells. However, during meiosis II, the spo4 and spo18 mutants underwent nuclear division but in neither case were the SPBs modified nor were forespore membranes formed. The SPBs of the spo18 mutant diminished in size after meiosis II and eventually disappeared after 18 h in sporulation medium. By contrast, the SPBs of the spo4 mutant remained unchanged even after an 18-h incubation. The outer plaques of SPBs of spo5 and spo14 mutants were sufficiently modified to allow them to initiate development of the forespore membrane, but the membrane had an abnormally expanded lumen and did not enclose the nuclei during meiosis II. The spo5 mutant produced anucleate spore-like bodies while the spo14 mutant formed unorganized structures with irregular peripheries which, presumably, contained spore-wall precursors, instead of anucleate spore-like bodies. We conclude that the modification of the SPB is essential for the formation of ascospores and at least two genes (spo5 and spo14) participate in the development of the forespore membrane. The defective phenotypes define discrete steps in the development of ascospores, which proceeds via steps defined by the mutant spo4, spo18, spo14 and spo5 genes respectively. Our observations provide further substantial evidence that the SPB plays a pivotal role in the normal development of ascospores in yeasts.

Fission yeast sporulation seems to accompany a dynamic alteration of membrane traffic pathways in which the destination of secretory vesicles changes from the plasma membrane to the developing spore membrane. Evidence shows that endocytosis is responsible for this alteration in traffic pathways via the relocalization of syntaxin 1., Syntaxin is a component of the target soluble N-ethylmaleimide–sensitive factor attachment protein receptor complex, which is responsible for fusion of membrane vesicles at the target membrane. The fission yeast syntaxin 1 orthologue Psy1 is essential for both vegetative growth and spore formation. During meiosis, Psy1 disappears from the plasma membrane (PM) and dramatically relocalizes on the nascent forespore membrane, which becomes the PM of the spore. Here we report the molecular details and biological significance of Psy1 relocalization. We find that, immediately after meiosis I, Psy1 is selectively internalized by endocytosis. In addition, a meiosis-specific signal induced by the transcription factor Mei4 seems to trigger this internalization. The internalization of many PM proteins is facilitated coincident with the initiation of meiosis, whereas Pma1, a P-type ATPase, persists on the PM even during the progression of meiosis II. Ergosterol on the PM is also important for the internalization of PM proteins in general during meiosis. We consider that during meiosis in Schizosaccharomyces pombe cells, the characteristics of endocytosis change, thereby facilitating internalization of Psy1 and accomplishing sporulation.

We analyzed parentage in a wild population of the Bull-headed Shrike (Lanius bucephalus) by DNA fingerprinting. Male Bull-headed Shrikes pair with a single female and defend a large all-purpose territory. Generally, both parents provide substantial parental care. Among 99 nestlings from 24 clutches, 10 (10%) nestlings from 4 (17%) clutches had extrapair paternity, but were offspring of the females. These results show that copulation frequency is not necessarily a good measure of reproductive success even in monogamous birds where no extrapair copulations are observed. The average band-sharing proportion between full siblings was the same as that between parent and offspring, but the variance was greater. This is likely the case because the band-sharing proportion reflects the gene-sharing proportion. Received 6 May 1991, accepted 9 February 1992. OvER 90% of birds are thought to be monogamous (Lack 1968), and males of these species employ mate guarding to avoid being cuckolded (M0ller 1985). Nevertheless, recent evidence from field observations and genetic analyses has clearly demonstrated that extrapair copulations (EPCs) and extrapair fertilizations (EPFs) occur in monogamous species. Among colonially breeding species of monogamous birds like Bank Swallows (Riparia riparia; Beecher and Beecher 1979) and Cattle Egrets (Bubulcus ibis; Fujioka and Yamagishi 1981), EPCs occur frequently. In Purple Martins (Progne subis; Morton et al. 1990) and Zebra Finches (Taeniopygia guttata; Birkhead et al. 1990), EPCs often result in fertilization, and intraspecific brood parasitism (ISBP) has been documented. In House Sparrows (Passer domesticus), which have a loosely colonial breeding system, up to 8% of the offspring were not fathered by their putative male parent (Wetton et al. 1987). EPCs have been recorded even in species with large all-purpose territories, where individuals are relatively difficult to observe. In Indigo Buntings (Passerina cyanea), EPCs often resulted in EPFs (Westneat 1987a, b), while in monogamous Willow Warblers (Phylloscopus trochilus), EPCs resulted in few EPFs (Gyllensten et al. 1990). These results show that copulation frequency is not necessarily a good measure of reproductive success. However, there are only a few genetic studies of strongly monogamous and territorial species; all have found low frequencies of EPFs. The Bull-headed Shrike (Lanius bucephalus) has a strongly monogamous breeding system and has a large all-purpose territory (radius ca. 100 m; Yamagishi 1982b). No EPCs have been observed (Yamagishi and Saito 1985). In monogamous birds for which no EPCs have been observed, there are few studies involving the determination of parentage; an exception is an analysis of Dunnocks (Prunella modularis) under a monogamous system (Burke et al. 1989). Their study showed that in monogamous pairs no EPFs occurred, so that the reproductive success of a pair was related to the number of fledged young. To test if this was also the case for Bull-headed Shrikes, we looked for evidence of EPFs and ISBP using DNA fingerprinting, which can be a powerful tool for determining parentage (Jeffreys et al. 1985b). STUDY AREA AND METHODS Study area and capture techniques.-The study was conducted from March to June 1989 in Oizumi city park, Osaka Prefecture, central Japan (34?34'N, 135?32'E). The park covers an area of about 70 ha, is about 20 m in elevation, and is planted mainly with oaks (Quercus). The incubating female and/or the female and the male that fed their nestlings were captured with a mist net, and were regarded as the putative father and mother of the brood. All males that produced extrapair offspring were captured during incubation or nestling period (i.e. when their female mate was not fertilizable, therefore, a capture of the male did not affect our results). Age determination was based on the coloration of the wing coverts (Yamagishi 1982a). DNA fingerprinting.-DNA was prepared from 100 ,ul blood samples collected by jugular venipuncture

Mei4 is a key sporulation-specific transcription factor in fission yeast. Ectopic expression of Mei4 in vegetative cells caused formation of nucleated membranous compartments, which shared common features with normal forespore membranes, thereby perturbing nuclear division. These results suggest why expression of development-specific transcription factors must be strictly controlled.

Small guanine triphosphatases (GTPases) of the Rab family are key regulators of membrane trafficking events between the various subcellular compartments in eukaryotic cells. Rab7 is a conserved protein required in the late endocytic pathway and in lysosome biogenesis. A Schizosaccharomyces pombe (S. pombe) homolog of Rab7, Ypt7, is necessary for trafficking from the endosome to the vacuole and for homotypic vacuole fusion. Here, we identified and characterized a second fission yeast Rab7 homolog, Ypt71. Ypt71 is localized to the vacuolar membrane. Cells deleted for ypt71(+) exhibit normal growth rates and morphology. Interestingly, a ypt71 null mutant contains large vacuoles in contrast with the small fragmented vacuoles found in the ypt7 null mutant. Furthermore, the ypt71 mutation does not enhance or alleviate the temperature sensitivity or vacuole fusion defect of ypt7Delta cells. Like ypt7Delta cells, overexpression of ypt71(+) caused fragmentation of vacuoles and inhibits vacuole fusion under hypotonic conditions. Thus, the two S. pombe Rab7 homologs act antagonistically in regulating vacuolar morphology. Analysis of a chimeric Ypt7/Ypt71 protein showed that Rab7-directed vacuole dynamics, fusion versus fission, largely depends on the medial region of the protein, including a part of RabSF3/alpha3-L7.

The fission yeast Schizosaccharomyces pombe has two mating-types, h+ (P) and h- (M). The mam2 mutant exhibits an h(-)-specific sterile phenotype. Nucleotide sequencing of the mam2 gene isolated from an S. pombe genomic library revealed an open reading frame composed of 348 amino acids. The deduced mam2 product is a hydrophobic protein of 39 kDa that has significant sequence similarity (26.3% for identical amino acids) with the transmembrane domains of the Saccharomyces cerevisiae STE2 product, the alpha-pheromone receptor. Hydropathicity analysis suggests that the Mam2 protein contains seven possible membrane-spanning domains and a carboxy-terminal hydrophilic region. The mam2 gene was disrupted and found to be non-essential for growth. An h- haploid strain harbouring this disrupted null allele failed to respond to the pheromone of h+ cells, P-factor. These observations imply that the mam2 gene encodes a receptor for P-factor. Transcription of mam2 was induced only when strains containing functional mat1-M allele were cultured under conditions of nitrogen starvation. The mam2 gene was also transcribed in h+/h- diploid strains. The fact that the map1/mam2 homozygous diploid cells are incapable of sporulation implies that the pheromone signalling system is necessary for sporulation in diploid cells.

A novel gene trs1 in the fission yeast Schizosaccharomyces pombe has been genetically defined. The trs1 mutant showed several intriguing phenotypes. Cells were sensitive to starvation and rapidly lost viability in the stationary phase; cells in the stationary phase were sensitive to heat shock. Some heat-shock proteins were not induced and the heat-shock response in log-phase cells was defective. These mutant phenotypes strongly suggest a vital function of the trs1 gene product for transition from the G1 to G0 phase on starvation and for the normal heat-shock response.

Sporulation in the fission yeast Schizosaccharomyces pombe is a unique biological process in that the plasma membrane of daughter cells is assembled de novo within the mother cell cytoplasm. A double unit membrane called the forespore membrane (FSM) is constructed dynamically during meiosis. To obtain a dynamic view of FSM formation, we visualized FSM in living cells by using green fluorescent protein fused with Psy1, an FSM-resident protein, together with the nucleus or microtubules. The assembly of FSM initiates in prophase II, and four FSMs in a cell expand in a synchronous manner at the same rate throughout meiosis II. After the meiosis II completes, FSMs continue to expand until closure to form the prespore, a spore precursor. Prespores are initially ellipsoidal, and eventually become spheres. FSM formation was also observed in the sporulation-deficient mutants spo3, spo14, and spo15. In the spo15 mutant, the initiation of FSM formation was completely blocked. In the spo3 mutant, the FSM expanded normally during early meiosis II, but it was severely inhibited during late and postmeiosis, whereas in the spo14 mutant, membrane expansion was more severely inhibited throughout meiosis II. These observations suggest that FSM expansion is composed of two steps, early meiotic FSM expansion and late and post meiotic FSM expansion. Possible regulatory mechanisms of FSM formation in fission yeast are discussed.

The spindle pole body (SPB) of Schizosaccharomyces pombe is required for assembly of the forespore membrane (FSM) during meiosis. Before de novo biogenesis of the FSM, the meiotic SPB forms outer plaques, an event referred to as SPB modification. A constitutive SPB component, Spo15, plays an indispensable role in SPB modification and sporulation. Here, we analyzed two sporulation-specific genes, spo13+and spo2+, which are not required for progression of meiotic nuclear divisions, but are essential for sporulation. Spo13 is a 16-kDa coiled-coil protein, and Spo2 is a 15-kDa nonconserved protein. Both Spo13 and Spo2 specifically associated with the meiotic SPB. The respective deletion mutants are viable, but defective in SPB modification and in the onset of FSM formation. Spo13 and Spo2 localized on the cytoplasmic side of the SPB in close contact with the nascent FSM. Localization of Spo13 to the SPB was dependent on Spo15 and Spo2; that of Spo2 depended only on Spo15, suggesting that their recruitment to the SPB is strictly controlled. Spo2 physically associated with both Spo15 and Spo13, but Spo13 and Spo15 did not interact directly. Taken together, these observations indicate that Spo2 is recruited to the SPB during meiosis and then assists in the localization of Spo13 to the outer surface of the SPB.

The mating reaction in the fission yeast, Schizosaccharomyces pombe, is induced by nitrogen deprivation. In order to understand the genetic control of cellular responses to starvation, sterile mutants defective in the mating reaction were isolated and analyzed. Tetrad analysis and complementation tests of ethyl methanesulfonate-induced Ste- mutants revealed three novel ste loci, designated ste11, ste12 and ste13. These genes were mapped on chromosome II. ste11 was allelic to stex; ste12 was tightly linked to ste2 (0.8 cM). Homozygous diploid strains harboring either of these ste alleles were defective in meiosis. Vegetative cells of the ste11 mutant showed the normal resting state in stationary phase. In contrast, the ste12 and ste13 mutant cells rapidly lost viability after nutrient deprivation and the latter were sensitive to heat shock in stationary phase. The transcript level of the mat1-Pi and mei2 genes was greatly reduced in these mutants. These results indicate that the ste12 and ste13 genes are responsible not only for entry into sexual reproduction but also for entry into the G0 phase.

Ypt7p, a fission yeast (Schizosaccharomyces pombe) homologue of Rab7 GTPase, mediates fusion of endosomes to vacuoles and homotypic vacuole fusion. Here, we report that Ypt7p plays important roles in sporulation. Most ypt7Delta asci produced less than four spores, which were apparently immature and germinated at low frequency. Furthermore, ypt7Delta cells were defective in development of the forespore membranes. Vacuoles in sporulating cells were found to undergo extensive homotypic vacuole fusion to form a few large compartments occupying the entire cytoplasm of asci. This extensive vacuole fusion depended on Ypt7p.

In the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, sporulation involves de novo synthesis of forespore membrane (FSM) within the cytoplasm of mother cells. The FSM ultimately becomes the plasma membrane of the developing ascospores. Several protein components of the FSM have been identified. Visualization of these proteins has demonstrated the dynamic nature of the genesis and development of the FSM. It begins to develop at the differentiated outer plaque of the spindle pole bodies (SPBs) and extends outwards, encapsulating each of the haploid nuclei produced by meiosis. Several coiled-coil proteins are specifically recruited to the SPBs and play indispensable roles in FSM assembly. Temporal and spatial coordination of meiotic nuclear divisions and membrane assembly is of special importance. Comparison of the processes of FSM assembly in these yeasts shows that the basic mechanism has been conserved, even though the individual proteins involved are often different. Understanding these dynamic aspects of yeast sporulation will help to elucidate a general mechanism for the cellularization of cytoplasm containing multiple nuclei.

It was almost 35 years ago that Bresch and coworkers (Bresch et al. 1968) first isolated and analyzed sporulation-deficient mutants of Schizosaccharomyces pombe, a few years before the isolation of similar mutants of Saccharomyces cerevisiae. They genetically defined 23 meiosis- and sporulation-specific genes, which were subsequently cloned one after another in the 1980 s (e.g. Shimoda and Uehira 1985; Shimoda et al. 1987). Investigations of meiosis-specific genes have unveiled a negative control mechanism for the initiation of meiotic differentiation. By contrast, studies of the molecular cell biology of sporulation only got under way in the past few years (Ikemoto et al. 2000; Nakase et al. 2001; Nakamura et al. 2001). With completion of the sequencing of the S. pombe genome (Chap. 2; Wood et al. 2002), genome-wide screenings of meiotic and sporulation genes have been conducted (Mata et al. 2002).

We have defined five sev genes by genetic analysis of Schizosaccharomyces pombe mutants, which are defective in both proliferation and sporulation. sev1+/cdt2+ was transcribed during the G1-S phase of the mitotic cell cycle, as well as during the premeiotic S phase. The mitotic expression of cdt2+ was regulated by the MCB-DSC1 system. A mutant of a component of DSC1 affected cdt2+ expression in vivo, and a cdt2+ promoter fragment containing MCB motifs bound DSC1 in vitro. Cdt2 protein also accumulated in S phase and localized to the nucleus. cdt2 null mutants grew slowly at 30° and were unable to grow at 19°. These cdt2 mutants were also medially sensitive to hydroxyurea, camptothecin, and 4-nitroquinoline-1-oxide and were synthetically lethal in combination with DNA replication checkpoint mutations. Flow cytometry analysis and pulsed-field gel electrophoresis revealed that S-phase progression was severely retarded in cdt2 mutants, especially at low temperatures. Under sporulation conditions, premeiotic DNA replication was impaired with meiosis I blocked. Furthermore, overexpression of suc22+, a ribonucleotide reductase gene, fully complemented the sporulation defect of cdt2 mutants and alleviated their growth defect at 19°. These observations suggest that cdt2+ plays an important role in DNA replication in both the mitotic and the meiotic life cycles of fission yeast.

The Schizosaccharomyces pombe spo14-B221 mutant was originally isolated as a sporulation-deficient mutant. However, thespo14+gene is essential for cell viability and growth. spo14+is identical to the previously characterizedstl1+gene encoding a putative homologue of Saccharomyces cerevisiae Sec12, which is essential for protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. In the spo14 mutant cells, ER-like membranes were accumulated beneath the plasma membrane and the ER/Golgi shuttling protein Rer1 remained in the ER. Sec12 is a guanine nucleotide exchange factor for the Sar1 GTPase. Overproduction ofpsr1+coding for an S. pombe Sar1 homologue suppressed both the sporulation defect ofspo14-B221 and cold-sensitive growth of newly isolatedspo14-6 and spo14-7 mutants. These results indicate that Spo14 is involved in early steps of the protein secretory pathway. The spo14-B221 allele carries a single nucleotide change in the branch point consensus of the fifth intron, which reduces the abundance of the spo14 mRNA. During meiosis II, the forespore membrane was initiated near spindle pole bodies; however, subsequent extension of the membrane was arrested before its closure into a sac. We conclude that Spo14 is responsible for the assembly of the forespore membrane by supplying membrane vesicles.

The fission yeast Schizosaccharomyces pombe has three putative ubiquitin-protein ligases of the Nedd4/Rsp5 family, named Pub1p, Pub2p and Pub3p. Pub1p has been reported to be involved in cell cycle regulation and proliferation under acidic pH conditions. Here we characterize Pub2p, which contains a conserved HECT domain and a WW domain but lacks a C2 domain. Transcription of the pub2(+) gene was constitutive and further enhanced by nitrogen starvation. A pub2-null mutation gave no remarkable phenotypes, but intensified temperature sensitivity in a pub1Delta background. Moderately overexpressed pub2(+) suppressed the temperature sensitivity of pub1Delta cells, which suggests that the function of Pub2p overlaps with that of Pub1p. Overexpression of pub2(+) by a strong nmt1 promoter in wild-type strains caused growth arrest and cell elongation, probably owing to defects in G2 progression or the G2/M transition. Unlike Pub1p, however, overexpression of Pub2p did not reduce the levels of Cdc25p. Pub2-GFP was found throughout the cell, especially at the cell surface in the polar regions. Pub2p contains a conserved cysteine residue (Cys639) in its putative catalytic HECT domain that can be thiol-ubiquitinated. Substitution of Cys639 by alanine (Pub2CA) caused a functional defect, because growth arrest and cell elongation were not induced by overexpression of Pub2CA. A chimeric Pub1 protein, in which the HECT domain was replaced by the Pub2 HECT domain, completely suppressed the temperature sensitivity of pub1Delta cells, suggesting that the HECT domain of Pub2p has the catalytic activity of a ubiquitin ligase. We conclude that Pub2p is a HECT-type ubiquitin-protein ligase that shares partially overlapping function with Pub1p.

The mes1+ gene of Schizosaccharomyces pombe is required for the second meiotic division. The single 75-nt intron in mes1 is spliced out only in meiotic cells. Here we report a cis-acting element which is responsible for meiosis-specific splicing. Both 5′ and 3′ splice sites of the mes1 intron deviate from the consensus sequence. Point mutations which altered these sites so that they conformed to the consensus, however, did not affect the splicing pattern of mes1. Neither replacement of the mes1 intron with the constitutively spliced intron of the nda3 gene, nor replacement of the 3′ exon with E. coli lacZ changed the splicing pattern. In contrast, deletion of the 5′ terminal 125 nt from the 5′ exon derepressed splicing in vegetative cells, implying that this 5′ terminal sequence, named SRE (mes1 splicing repression element), inhibits splicing of the downstream intron. A potential stem-loop structure in the SRE is predicted. Disruption of this stem structure by mutation abolished the repression of mes1 splicing in vegetative cells. Overexpression of the SRE sequence on a multicopy plasmid also relieved the repression of splicing of the authentic mes1 transcripts. These results suggest that as yet unknown trans-acting factors inhibit splicing of the mes1 transcript in vegetative cells by interacting with the cis-element SRE.

In Schizosaccharomyces pombe wild-type cells, the nucleus positions in the middle of the cell where the cortical actin ring is assembled prior to septum formation. ste12 mutants contain a few large vacuoles. In a considerable fraction of ste12 cells, the nuclei and septa were eccentrically positioned. Both extension of spindle microtubules in the anaphase and post-mitotic migration of sister nuclei to the cell poles were partially disrupted, probably due to enlarged vacuoles. In spite of the eccentric positioning of nuclei, the cortical actin ring overlays the displaced pre-mitotic nucleus. This observation supports the notion that the nucleus dictates the division site in fission yeast.

Spindle pole bodies in the fission yeast Schizosaccharomyces pombe are required during meiosis, not only for spindle formation but also for the assembly of forespore membranes. The spo15 mutant is defective in the formation of forespore membranes, which develop into spore envelopes. The spo15(+)gene encodes a protein with a predicted molecular mass of 223 kDa, containing potential coiled-coil regions. The spo15 gene disruptant was not lethal, but was defective in spore formation. Northern and western analyses indicated that spo15(+) was expressed not only in meiotic cells but also in vegetative cells. When the spo15-GFP fusion gene was expressed by the authentic spo15 promoter during vegetative growth and sporulation, the fusion protein colocalized with Sad1p, which is a component of spindle pole bodies. Meiotic divisions proceeded in spo15delta cells with kinetics similar to those in wild-type cells. In addition, the morphology of the mitotic and meiotic spindles and the nuclear segregation were normal in spo15delta. Intriguingly, transformation of spindle pole bodies from a punctate to a crescent form prior to forespore membrane formation was not observed in spo15delta cells. We conclude that Spo15p is associated with spindle pole bodies throughout the life cycle and plays an indispensable role in the initiation of spore membrane formation.

The mei4+ gene of the fission yeast Schizosaccharomyces pombe was cloned by functional complementation. The mei4 disruptant failed to complete meiosis-I but could proliferate normally. mei4+ was transcribed only in meiosis-proficient diploid cells after premeiotic DNA replication. The mei4+ open reading frame encodes a 57-kDa serine-rich protein comprised of 517 amino acids with a forkhead/HNF3 DNA-binding domain in the amino-terminal region. Transcription of spo6+, a gene required for sporulation, was dependent on the mei4+ function. Two copies of the GTAAAYA consensus sequence, proposed as the binding site for human forkhead proteins, were found in the promoter region of spo6+. A gel mobility shift assay demonstrated the sequence-dependent binding of the GST-Mei4 forkhead domain fusion protein to DNA fragments with one of the consensus elements. Deletion of this consensus element from the spo6 promoter abolished the transcription of spo6+ and resulted in a sporulation deficiency. One-hybrid assay of Mei4 which was fused to the Gal4 DNA-binding domain localized the transcriptional activation domain in the C-terminal 140 amino acids of Mei4. These results indicate that Mei4 functions as a meiosis-specific transcription factor of S. pombe.

To isolate Arabidopsis cDNAs that encode signal transducers and components involved in the regulation of meiosis, a trans-complementation analysis was performed using a Schizosaccharomyces pombe meiosis-defective mutant in which the genes for pheromone receptors were disabled. One cDNA obtained in this screening encodes a polypeptide, named AML1, that shows significant similarity to S. pombe Mei2 protein and has three putative RNA-recognition motifs like as Mei2. Mei2 is involved in the regulation of meiosis in fission yeast. Northern blot analysis showed that the AML1 gene is expressed in each organ. The possible functions of AML1 are discussed.

When the fission yeast Schizosaccharomyces pombe is starved for nitrogen, the cells are arrested in the G1 phase, enter the G0 phase and initiate sexual development. The ste13 mutant, however, fails to undergo a G1 arrest when starved for nitrogen and since this mutant phenotype is not suppressed by a mutation in adenylyl cyclase (cyr1), it would appear that ste13+ either acts independently of the decrease in the cellular cAMP level induced by starvation for nitrogen, or functions downstream of this controlling event. We have used functional complementation to clone the ste13+ gene from an S. pombe genomic library and show that its disruption is not lethal, indicating that, while the gene is required for sexual development, it is not essential for cell growth. Nucleotide sequencing predicts that ste13+ should encode a protein of 485 amino acids in which the consensus motifs of ATP-dependent RNA helicases of the DEAD box family are completely conserved. Point mutations introduced into these consensus motifs abolished the ste13+ functions. The predicted Ste13 protein is 72% identical to the Drosophila melanogaster Me31B protein over a stretch of 391 amino acids. ME31B is a developmentally regulated gene that is expressed preferentially in the female germline and may be required for oogenesis. Expression of ME31B cDNA in S. pombe suppresses the ste13 mutation. These two evolutionarily conserved genes encoding putative RNA helicases may play a pivotal role in sexual development.