Index INTRODUCTION MATERIALS AND METHODS Yeast strains, media and genetic techniques. Construction of cta4-HA. Northern blot analysis. Nucleotide sequence analysis of the sev4-L5 allele. Immunofluorescence microscopy. RESULTS AND DISCUSSION Isolation of a sporulation-deficient sev4-L5 mutant. Identification of the sev4+ gene. Expression and localization of Cta4. sev4-L5 was defective in development of the FSM. References

INTRODUCTION

Sporulation in yeasts provides a model for gametogenesis in higher eukaryotes. An important step leading to ascospore formation is de novo assembly of the plasma membrane of newborn spores (Neiman, 1998; Shimoda, 2004). The spore plasma membrane is formed in the cytoplasm of zygotes as a double-layered intracellular membrane, termed the forespore membrane (FSM) in the fission yeast Schizosaccharomyces pombe (Yoo et al., 1973; Hirata and Tanaka, 1982; Tanaka and Hirata, 1982). The nascent FSM extends by fusing with vesicles derived from the ER/Golgi and encapsulates each of the meiotic nuclei (Hirata and Tanaka, 1982; Tanaka and Hirata, 1982; Nakamura et al., 2001). The inner membrane of the FSM becomes the plasma membrane of the spores. Formation of the FSM initiates during meiosis II near the spindle pole body (SPB), a microtubule-organizing center in yeast that is equivalent to the centrosome. Before formation of the FSM, the SPB undergoes a morphological change from a dot to a crescent shape (Hagan and Yanagida, 1995), and this modification seems to be essential for FSM formation (Hirata and Shimoda, 1994; Ikemoto et al., 2000). These facts imply that the SPB plays an important role in membrane assembly, but the mechanistic details are still elusive.

Genetic and molecular analyses of many sporulation-specific mutants in S. pombe have dissected the molecular events that occur during meiosis and sporulation (Bresch et al., 1968; Kishida and Shimoda, 1986; Shimoda and Nakamura, 2004). In addition to specific genes termed spo, we have also screened for asporogenous mutants out of a collection of temperature-sensitive mutants (Yoshida et al., 2003). These mutants, referred to as “sev” (for sporulation genes essential for vegetative growth), have defined five genes. sev1⁺ is identical to cdt2⁺, which is responsible for mitotic and premeiotic DNA replication (Yoshida et al., 2003). In this article, we report the analysis of sev4⁺, which encodes Cta4, a putative Ca²⁺-ATPase.

Ca²⁺ is an essential component of cellular signal transduction. The intracellular concentration of Ca²⁺ is strictly regulated, and perturbation of Ca²⁺ homeostasis causes various disorders in cell growth and function. Analysis of the cta4-null mutant previously revealed that Ca²⁺ homeostasis is crucial for growth and cell polarity (Okorokova-Facanha et al., 2002). Influx of Ca²⁺ into cells is triggered during conjugation in budding yeast (Iida et. al., 1994). The Ca²⁺-binding protein calmodulin and its downstream protein phosphatase calcineurin are also involved in cell polarity and mating in yeast (Liu et al., 1990; Cyert et al., 1991; Ohya and Botstein, 1994; Yoshida et. al., 1994). To data, however, little information is available on the requirement of intracellular Ca²⁺ homeostasis for meiosis and sporulation. Here we report that mutation of sev4/cta4 causes severe disorders in sporulation.

MATERIALS AND METHODS

Yeast strains, media and genetic techniques.

The S. pombe strains used in this study are listed in Table 1. Complete medium YE and minimal medium EMM2+N were used for growth. Sporulation media MEA, SSA and EMM2–N were used for mating and sporulation (Egel and Egel-Mitani, 1974; Gutz et al., 1974; Moreno et al., 1991). The original sev4-L5 mutant was derived from C982-16C. The method for synchronous meiosis has been described in our previous report (Yoshida et al., 2003).

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Table 1. Strain list

To construct a cta4-null allele, a major part of the ORF (amino acids 64 to 993) was replaced by a ura4⁺ cassette (Grimm et. al., 1988). The DNA fragment containing the cta4::ura4⁺ allele was introduced into the diploid strain SYD1 and uracil prototrophs were isolated. Correct integration of the disrupted allele at the sev4⁺ locus was verified by Southern blot analysis, as described previously (Yoshida et al., 2003).

Construction of cta4-HA.

Triple hemagglutinin (HA) epitope-tagged cta4⁺ was constructed as follows. The cta4⁺ coding region was amplified by PCR using primers CCCGTCGAC(SalI)GGGGAGTAAGGCTTTA and GGG-GCGGCCGC(NotI)AATTACGAAGAACAAT. The amplified DNA fragment was digested with SalI and NotI and then inserted into pTN218 containing the HA (3x) and the nmt1 terminator (Nakamura et. al., 2002) to yield a cta4-HA integration plasmid pSY05004. pSY05004 was linearized at a single StuI site in the Cta4-coding region and transformed into TN29 to create SY70, in which a single copy of the Cta4-HA allele with a cta4 native promoter was integrated at the chromosomal cta4 locus.

Northern blot analysis.

Total RNA was prepared from the pat1-114 mutant strain (JZ670) at hourly intervals after induction of meiosis (Jensen et al. 1983). RNA was fractionated on a 1.0% agarose gel containing 3.7% formaldehyde (Thomas, 1980), blotted and then probed with the cta4⁺-specific probe. The probe DNA was labeled with [³²P]dCTP by the random primer method (Feinberg et al., 1983).

Nucleotide sequence analysis of the sev4-L5 allele.

The complete sev4-L5 ORF was amplified by PCR by using genomic DNA prepared from SY13 as a template. Amplified DNA fragments were directly sequenced with an ABI-PRISM 310 autosequencer (Applied Biosystems, CA, USA). The nucleotide sequence of the sev4-L5 allele was compared with the published sequence of cta4⁺ (Okorokova-Facanha et al., 2002).

Immunofluorescence microscopy.

For cell fixation, we followed the method of Hagan and Hyams (1988) using glutaraldehyde and paraformaldehyde. Cta4-HA was visualized by indirect immunofluorescence microscopy using rat anti-HA antibody 3F10 (Boehringer Mannheim, Mannheim, Germany) and Alexa 546-conjugated goat anti-rat IgG (Molecular Probes, Eugene, USA). The SPB was visualized by indirect immunofluorescence microscopy using rabbit anti-Sad1 antibody and Alexa 546-conjugated goat anti-rabbit IgG (Molecular Probes). For microtubule staining, TAT-1 anti-α-tubulin antibody (Woods et al., 1989) and Cy3-conjugated secondary antibody (Sigma Chemical Co., St. Luis, USA) were used. To visualize the nuclear chromatin region, cells were stained with 4’,6-diamidino-2-phenylindole (DAPI) at 1 μg/ml. Stained cells were observed under a fluorescence microscope (model BX51; Olympus, Tokyo) equipped with a charge-coupled device (CCD) camera (Cool-SNAP; Roper Scientific, San Diego, USA).

RESULTS AND DISCUSSION

Isolation of a sporulation-deficient sev4-L5 mutant.

In a genetic screen for sporulation-deficient mutants present in a collection of temperature-sensitive mutants of S. pombe, we previously isolated sev mutants and identified five sev loci (Yoshida et al. 2003). Genetic analysis of the sev4-L5 mutant indicated that its mutant phenotype was due to a single recessive mutation. sev4-L5 cells were unable to form colonies either at 37°C or at 19°C on YEA plates (Fig. 1A). The mutant cells grew very poorly in EMM2+N supplemented with 0.5 M CaCl₂ or in Ca²⁺-free EMM2+N containing 10 mM EGTA (data not shown). The growth in the latter medium was recovered by adding 20 mM CaCl₂ (data not shown). A homothallic haploid strain carrying sev4-L5 was incubated in nitrogen-limited medium MEA at the permissive temperature (28°C) for two days. Cells conjugated to form zygotes at a lower efficiency than did wild-type control cells, and notably produced no asci (Table 2 and Fig. 1B).

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Fig. 1. Phenotype of the sev4 mutant. (A) Colony formation at different incubation temperatures. Wild-type (C996-11D) and sev4-L5 (SY15) cells were incubated on YE plates at 28°C for 2 days, at 37°C for 2 days, or at 19°C for 3 days. (B) Mating and sporulation ability of the sev4-L5 mutant. Wild-type (C996-11D) and sev4-L5 (SY15) cells were incubated on MEA plates at 28°C for 2 days. After fixation with 70% ethanol, cells were stained with DAPI. Bar, 10 μm.

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Table 2. Efficiency of conjugation and sporulation in sev4 mutant

Identification of the sev4⁺ gene.

An S. pombe genomic library (pTN-L1) (Nakamura et al., 2001) was introduced into the sev4-L5 mutant. Plasmid DNA was prepared from a few Spo⁺ transformants, and both termini of the inserted fragment were sequenced to identify the cloned gene. Comparison with the S. pombe genome sequence database (http://www.sanger.ac.uk/Projects/S_pombe/) showed that the insert contained two ORFs. Subcloning experiments determined the ORF (SPACUNK4.07c, also known as cta4⁺) responsible for the complementation activity, and integration mapping verified that the cloned gene was indeed the defined sev4⁺/cta4⁺ gene.

cta4⁺ encodes a cation-transporting P-type ATPase that specifically localizes to the endoplasmic reticulum (ER) (Okorokova-Facanha et al., 2002). The cta4-null mutation markedly increases the cellular Ca²⁺ concentration, indicating that Cta4 is responsible for Ca²⁺ homeostasis in S. pombe (Okorokova-Facanha et al., 2002). Sequencing of the sev4-L5 allele revealed that it contained a single mutation of G to A at nucleotide 1844. This mutation predicts the substitution of Gly615, which is located in the conserved ATP-binding site, with a glutamate residue. As the phenotype of the sev4-L5 mutant closely resembled that of the cta4-null mutant, the ATP-binding activity must be essential for the function of Cta4.

Expression and localization of Cta4.

To examine the expression of cta4⁺, we estimated the mRNA abundance by Northern blot analysis. Synchronous meiosis was conducted in the pat1-114 mutant, JZ670 (Iino and Yamamoto, 1995). The cta4 hybridization signal was detected in the cell sample at 0 hr (vegetative cells) and intensified at around 6–7 hr after the induction of meiosis, corresponding to meiosis I (Fig. 2). cta4 mRNA was also present in exponentially growing cultures (data not shown). This result is consistent with DNA microarray data showing that transcription of cta4⁺ is transiently upregulated during meiosis (Mata et al., 2002). We thus conclude that cta4⁺ transcription is constitutive in vegetative cells and is upregulated significantly during meiosis.

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Fig. 2. Northern blot analysis of cta4+ mRNA. The pat1-114 homozygous diploid strain (JZ670) was incubated in EMM2–N for 15 hr at 25°C and then shifted to 34°C to induce synchronous meiosis. At hourly intervals, aliquots were removed for RNA preparation. (A) Autoradiogram of RNA blots with a cta4-specific probe. Ethidium bromide staining of ribosomal RNAs is presented as a loading control (bottom). (B) To monitor the meiotic nuclear divisions, the number of nuclei per cell was determined after DAPI staining.

A GFP-Cta4 fusion protein has been reported to localize to the ER (Okorokova-Facanha et al., 2002). To verify this result, we constructed a strain carrying a single copy of the cta4-HA allele that was expressed under the control of a cta4 native promoter. The integrant strain was then transformed with pREP82(13g6-GFP), which was a ura4⁺ -carrying version of pREP81(13g6-GFP) (Nakamura-Kubo et al. 2003), to visualize an ER marker protein 13g6 (Brazer et. al., 2000). Both signals were colocalized to the nuclear periphery and beneath the plasma membrane; this localization is typical of proteins associated with the ER (Fig. 3). These findings support the idea that Cta4 is a cation-transporting ATPase localized to the ER.

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Fig. 3. Localization of Cta4-HA to the ER. Vegetative cells of an integrant strain SY70 was fixed, treated with anti-HA antibody (3F10), and stained with Alexa 594-conjugated anti-rat IgG antibody and DAPI. A phase-contrast image is also presented. Bar 10 μm.

sev4-L5 was defective in development of the FSM.

The cta4-null mutant showed elevated levels of intracellular Ca²⁺ concentration (Okorokova-Facanha et al., 2002). We thus examined whether externally added CaCl₂ influenced conjugation and sporulation in wild-type cells. Homothallic wild-type strain (FS26) was cultured in nitrogen-free liquid medium, EMM²–N, containing different concentrations of CaCl₂. Cells were cultured with shaking at 28°C for 2 days. CaCl₂ at concentrations higher than 0.5 M markedly inhibited conjugation. At lower concentrations, 0.1–0.4 M, CaCl₂ moderately inhibited conjugation, but did not significantly affect sporulation (data not shown).

As external Ca²⁺ addition preferentially blocked the zygote formation, we studied the requirement of Ca²⁺ homeostasis for the later stages of sexual development, meiosis and sporulation, with the sev4-L5 mutant. First, the kinetics of meiosis in diploid strains was monitored by DAPI staining of the nuclear chromatin region. As shown in Fig. 4, timing of the meiotic nuclear divisions in the sev4-L5 homozygous diploid was similar to that in the wild-type strain with a slight increase in the remaining mononucleate cells. Thus, it seems unlikely that the sporulation deficiency in sev4-L5 is due to a failure in the progression of meiosis.

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Fig. 4. Kinetics of meiotic nuclear divisions in the sev4-L5 mutant. Diploid strains homozygous for sev4+ (SYD2) and sev4-L5 (SYD3) were incubated in EMM+N for 16 hr at 28°C and then shifted to EMM2–N to induce meiosis. To monitor the meiotic nuclear divisions, the number of nuclei per cell was determined after DAPI staining.

Formation of the FSM, as visualized by GFP-Psy1, commences during meiosis II (Nakamura et al., 2001). Wild-type and sev4-L5 cells expressing GFP-Psy1 were cultured at 28°C on SSA sporulation medium. Fixed cells were immunostained with an antibody against α-tubulin (TAT-1) (Woods et. al., 1989) or an anti-Sad1 antibody (Hagan and Yanagida, 1995) to monitor microtubules or the SPB, respectively. Progression of meiosis was also traced by staining cells with DAPI. Assembly of the FSM began at metaphase II near the spindle poles in both strains (Fig. 5). Cup-shaped structures typical of an early stage of FSM were observed in the sev4-L5 mutant (Fig. 5, metaphase II), but extension of the FSM appeared to be aberrant (Fig. 5, anaphase II). As a result, malformed and anucleated prespores were frequently produced. Finally, we analyzed quantitatively the terminal morphology of the FSM in post-meiotic, tetranucleate cells. In most wild-type cells, the FSM developed into closed membrane compartments termed prespores, each of which contained one haploid nucleus (Fig. 6A, Class I). By contrast, normal nucleated prespores were rarely found in sev4-L5; instead, this mutant exhibited abnormal morphology that could be classified into three main groups: zygotes that formed four prespores, but some of which were anucleated (Fig. 6A, Class II); zygotes that formed malformed and/or anucleated prespores (Fig. 6A, Class III); and zygotes that formed only amorphous aggregates of the FSM (Fig. 6A, Class IV). The distribution of these classes in wild-type and sev4-L5 zygotes is summarized in Fig. 6B. Normal zygotes containing four nucleated prespores were rarely found in sev4-L5.

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Fig. 5. Formation of the FSM in the sev4-L5 mutant. Homothallic strains of sev4+ (YN68) and sev4-L5 (SY60) were cultured on SSA plates for 24 hr and then fixed. GFP-Psy1 signals were observed under a fluorescence microscope. Immunostaining of microtubules (A) and SPBs (B) was performed with anti-α-tubulin and anti-Sad1 antibodies, respectively. Nuclear chromatin regions were stained with DAPI. Bar, 10 μm.

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Fig. 6. Aberrant assembly of the FSM in the sev4-L5 mutant. Homothallic strains of sev4+ (YN68) and sev4-L5 (SY60) were cultured on SSA plates for 2 days at 28°C. (A) Typical morphologies of the FSM and prespores visualized by GFP-Psy1. Cells were also stained with DAPI. Classification of zygotes: Class I, zygotes containing four normal nucleated prespores; Class II, zygotes containing anucleated prespores; Class III, zygotes containing malformed prespores; and Class IV, zygotes containing no prespores (only aggregates). (B) Relative frequency of cell types. Approximately 200 cells were scored for each sample.

Similar aberrant FSM morphology has been observed in several asporogenous mutants such as spo3, spo14 and spo20 (Nakase et. al., 2001; Nakamura et. al., 2001; Nakamura-Kubo et. al., 2003), in which the fusion of membrane vesicles is impaired. Cta4 might be required for the vesicle fusion process to extend the FSM. We also noted that the structural modification of the compact SPBs to a crescent shape occurred only incompletely (Fig. 5B). The S. pombe calmodulin homologue encoded by cam1⁺ localizes to the SPBs (Moser et. al., 1997). A missense cam1-F116 mutant is also defective in sporulation (Takeda and Yamamoto, 1987) and shows marked defects in FSM formation (our unpublished data). This finding that the structural alteration of the SPB that normally takes place prior to formation of the FSM occurs only partially in the sev4-L5 mutant suggests the possibility that disturbances in Ca²⁺ homeostasis may inhibit the function of the SPB in constructing the FSM.

We thank O. Niwa of Kazusa DNA Research Institute for affinity-purified antibodies against Sad1, K. Gull of the University of Manchester for the anti-α-tubulin antibody, TAT-1, and S. L. Forsburg of The Salk Institute for plasmids. We also thank M. Morita for her excellent technical assistance. The present study was supported by Grants-in-Aid for Scientific Research on Priority Area from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) to C. S.(No.16013242) and to T. N. (No. 14037263).