Index Introduction Materials and Methods Yeast strains, media and culture conditions Plasmid construction Gene disruption of sec9 In vitro mutagenesis of sec9 by PCR Immunofluorescence microscopy Results Identification of SNAP-25 homologue in fission yeast SpSec9 is essential for cell viability Transcriptional regulation of sec9+ Isolation of sporulation-deficient sec9 mutants The sec9-10 mutant is defective in forespore membrane formation sec9+ interacts with syntaxin1 homologue, psy1+ Discussion Acknowledgments References

Introduction

Sporulation is the process of gametogenesis in the fission yeast Schizosaccharomyces pombe, which involves a regulated program of cell development that includes two overlapping events, meiosis and spore formation. The latter event requires assembly of double-layered intracellular membranes, termed forespore membranes (FSMs), which is equivalent to the prospore membrane in Saccharomyces cerevisiae (Yoo et al., 1973). During meiosis II, FSM are assembled by the fusion of membrane vesicles. From metaphase II to anaphase II, the spindle pole body (SPB), which plays a crucial role in spindle microtubule formation, undergoes a morphological transformation from a single plaque into a multilayered structure. Membrane vesicles are then transported to the vicinity of the modified SPBs and subsequently fuse there to generate FSMs. As the nucleus divides in meiosis II, the FSM extends, and eventually encapsulates, each of the four nuclei. Assembly of FSMs provides a model system for studying the de novo biogenesis of membrane compartments within the cytoplasm (Yoo et al., 1973, Tanaka and Hirata, 1982; Hirata and Shimoda, 1992, 1994; Ikemoto et al., 2000; Nakase et al., 2001; Nakamura et al., 2001; Nakamura-Kubo et al., 2003; Takegawa et al., 2003b; Shimoda, 2004).

Recent studies suggest that a general protein secretion apparatus is involved in FSM assembly (Nakase et al., 2001, 2004; Nakamura et al., 2001; Nakamura-Kubo et al., 2003; Koga et al., 2004). The S. pombe Sec12 homologue, Spo14, is necessary for proper construction of the FSM (d’Enfert et al., 1992; Nakamura-Kubo et al., 2003). Sec12 is responsible for vesicle transport from the endoplasmic reticulum (ER) to the Golgi apparatus by activating the Sar1 GTPase in S. cerevisiae (Nakano et al., 1988). This protein is known as a GEF (GTP-GDP exchange factor) for a small GTPase, Sar1 (Nakano and Muramatsu, 1989; Barlowe and Schekman, 1993). S. pombe Spo20 protein (SpSpo20) is structurally and functionally related to the major S. cerevisiae phosphatidylinositol/phosphatidylcholine-transfer protein Sec14, which is required for vesicle formation from the Golgi apparatus (Bankaitis et al., 1989, 1990; Nakase et al., 2001). SpSpo20 regulates formation of the FSM, in addition to its known roles in post-Golgi vesicle trafficking (Nakase et al., 2001). The soluble NSF attachment protein receptor (SNARE) proteins play a central role both in providing specificity and in catalyzing the fusion of vesicles with the target membrane (Protopopov et al., 1993; Sollner et al., 1993; Rothman, 1994; Rothman and Warren, 1994; Pelham, 1999; Jahn et al., 2003). There are at least 20 different SNAREs in a higher eukaryote cell and 17 in an S. pombe cell, each associated with a particular membrane-enclosed organelle involved in the secretory pathway (Takegawa et al., 2003a). These transmembrane proteins exist as complementary sets of vesicle membrane SNAREs (v-SNAREs), and target membrane SNAREs (t-SNAREs). These SNARE proteins have characteristic helical domains, which are responsible for interactions between a specific pair of v-SNARE and t-SNARE. In the secretion of neurotransmitter, the SNAREs responsible for docking and fusion of synaptic vesicles with the plasma membrane consist of three proteins: synaptobrevin, syntaxin-1 and SNAP-25 (Rothman, 1994). The former two are transmembrame proteins each of which contributes one α-helix to the complex. SNAP-25 is a peripheral membrane protein contributing two α-helices to the four-helix bundle. We isolated the S. pombe psy1⁺ gene encoding a syntaxin 1-like protein as a dose-dependent suppressor of the sporulation-deficient mutant, spo3. Psy1 localizes to the plasma membrane during vegetative growth. psy1⁺ is essential for vegetative growth, and its transcription is further enhanced during sporulation. Interestingly, Psy1 disappears from the plasma membrane of the mother cell immediately after the first meiotic division and reappears at the nascent FSM. These results support the idea that the FSM is assembled by the fusion of membrane vesicles, mediated by the SNARE complex (Nakamura et al., 2001). In budding yeast, SEC9 and SPO20 encode SNAP-25 homologues (Brennwald et al., 1994; Neiman, 1998). S. cerevisiae Sec9 (ScSec9) and Spo20 (ScSpo20) interact with the syntaxin 1-like protein Sso1/Sso2 (Ssop) and the synaptobrevin-like protein Snc1/Snc2 (Sncp) (Gerst et al., 1992; Aalto et al., 1993; Protopopov et al., 1993). The ScSec9/Ssop/Sncp complex mediates the fusion of exocytic vesicles with the plasma membrane during vegetative growth, whereas ScSpo20/Ssop/Sncp mediates the fusion of the prospore membrane during sporulation (Neiman, 1998).

In this study, we identified a SNAP-25 homologue, SpSec9, whose existence was predicted from the S. pombe genome sequence. The sec9⁺ gene is essential for vegetative growth, and its transcription is further enhanced during sporulation. We also isolated the sporulation-deficient mutant, sec9-10, in which assembly of the FSM is severely impaired. Our analysis suggests that SpSec9 plays an essential role both in cytokinesis and in sporulation.

Materials and Methods

Yeast strains, media and culture conditions

S. pombe strains used in this study are listed in Table I. Strains were grown in complete medium YEA supplemented with 75 μg/ml adenine sulfate and 50 μg/ml uracil. Malt extract medium MEA and synthetic sporulation medium SSL–N and MM–N were used for mating and sporulation. These media were described by Egel and Egel-Mitani, 1974, Gutz et al., 1974, and Moreno et al., 1990. S. pombe cells were grown at 30°C and sporulated at 28°C except for the sec9-10 mutant, which was grown and sporulated at 25°C.

Plasmid construction

pREP1(ade6) (Tamai et al., unpublished) was digested with PstI, filled in, and then ligated with an ApaI linker, yielding pL-A. Plasmid pL-A(syb1) was constructed as follows. The two oligonucleotides 5'-CCCCTCGAG(XhoI)TGAACCTTTCGCAAGGGATTC-3' and 5'-CCCGCGGCCGC(NotI)AGGGGAGCAAATATACTAC-3' were used to amplify the syb1⁺ gene. The corresponding PCR product was digested with XhoI and NotI, and then subcloned into the corresponding sites in pAL-KS (Tanaka et al., 2000), yielding pAL(syb1). The ApaI-SacI fragment of pAL(syb1) was inserted into the corresponding sites in pL-A, yielding pL-A(syb1). Plasmid pL-A(psy1) was constructed as follows. The two oligonucleotides 5'-CCCGTCGAC(SalI)AATGAATAAAGCAAACGAT-3' and 5'-CCCGAGCTC(SacI)ATCTAACCGGCCATATCACT-3' were used to amplify the psy1⁺ gene. The corresponding PCR product was digested with SalI and SacI, and then subcloned into the corresponding sites in pAL-KS, yielding pAL(psy1). The ApaI-SacI fragment of pAL(psy1) was inserted into the corresponding sites in pL-A, yielding pL-A(psy1). Plasmid pL-A(sec9) was constructed as follows. The two oligonucleotides 5'-CCCCTCGAG(XhoI)TAGTCCATCGGAAAGACAGAA-3' and 5'-CCCGAGCTC(SacI)TAGCGGCATCAAAGCTGCTCA-3' were used to amplify the sec9⁺ gene. The corresponding PCR product was digested with XhoI and SacI, and then subcloned into the corresponding sites in pAL-KS, yielding pAL(sec9). The ApaI-SacI fragment of pAL(sec9) was inserted into the corresponding sites in pL-A, yielding pL-A(sec9).

Gene disruption of sec9

The two oligonucleotides 5'-CCCCTCGAG(XhoI)TAGTCCATCGGAAAGACAGAA-3' and 5'-AGTGTGCGGCCGC(NotI)CTCGTAGTTTTGGTTAGCTAT-3' were used to amplify the sec9⁺ gene by PCR. The PCR product was digested with XhoI and NotI, and then ligated into the same sites in pBluescript II (STRATAGENE, La Jolla, CA, USA), yielding pBS(sec9). A 1.8-kb ura4⁺ fragment (Grimm et al., 1988) was then inserted into the internal PstI site, yielding pBS(sec9::ura4⁺). A 4.8-Kb NspV-Cfr10I fragment containing the interrupted sec9 allele (sec9::ura4⁺) was used to transform strain TN75. The disruption was confirmed by Southern hybridization (data not shown).

In vitro mutagenesis of sec9 by PCR

PCR was used to introduce a random point mutation into the sec9⁺ gene (one cycle at 95°C for 3 min followed by 30 cycles at 95°C for 30 sec, 50°C for 30 sec, and 72°C for 3 min with Taq polymerase). The forward and reverse primers were 5'-ACACACACAGTCGAC(SalI)TAGTCCATCGGAAAGACAGAAAAATG-3' and 5'-CCACCAACAGAGCTC(SacI)CTCGTAGTTTTGGTTAGCTATGAG-3', respectively. The amplified DNA fragment contained the promoter and terminator regions, in addition to the sec9⁺ ORF. The S. pombe genomic library pTN-L1 (Nakamura et al., 2001) was included in the reaction mixture as a template. The amplified fragment was digested with SalI and SacI and cloned into pBR(leu1) (Nakamura-Kubo et al., 2003). The resulting library was digested at the SnaBI site within the leu1⁺ gene and then integrated at the leu1 locus of a heterozygous diploid carrying a sec9::ura4⁺ allele (TN363). Colonies of transformants on SSA plates were treated with ethanol to kill non-sporulating vegetative cells and were then spread again on SSA sporulation plates, which were then exposed to iodine vapor (Gutz et al., 1974). Iodine-negative (white) colonies were selected and inspected for zygotic ascus formation.

Immunofluorescence microscopy

Cells were fixed according to the procedure of Hagan and Hyams (1988) using glutaraldehyde and paraformaldehyde. For microtubule staining, the anti-α-tubulin antibody TAT-1 (Woods et al., 1989) was used with Cy3-conjugated anti-mouse IgG (Sigma, St Louis, MO, USA) at a 1:1000 dilution. The nuclear chromatin region was stained with DAPI (4',6-diamidino-2-phenylindole) at 1 μg/ml. Stained cells were observed under a fluorescence microscope (model BX51; Olympus, Tokyo) and images were captured using a Cool SNAP CCD camera (Roper Scientific, San Diego, CA, USA).

Results

Identification of SNAP-25 homologue in fission yeast

The SNAP-25-like protein (SPBC26H8.02C), designated sec9⁺, was found by means of a conventional BLAST search of an S. pombe genome database (The Sanger Institute, UK) for S. cerevisiae Sec9. sec9⁺ encodes a 46.2-kDa protein containing 419 amino acids. Budding yeast has duplicated genes, SEC9 and SPO20. ScSpo20 is dispensable for growth but essential for formation of prospore membranes. In contrast, ScSec9 is essential for vegetative growth (Brennwald et al., 1994; Neiman, 1998). SpSec9 is the sole SNAP-25-like protein in S. pombe. The N-terminal region of SpSec9 does not share similarity to homologues from other organisms. However, over the C-terminal 228 residues, the SpSec9 protein shares 41, 28, 26, and 23% identity and 61, 48, 41, and 41% similarity with the budding yeast ScSec9, ScSpo20, zebrafish SNAP-25A, and human SNAP-25 proteins, respectively (Fig. 1). In neurons, the SNARE complex is composed of a bundle of four helices with synaptobrevin and syntaxin 1 contributing one helix each and SNAP-25 contributing two. SpSec9 is predicted to have two α-helical regions, which are highly conserved among SNAP-25 proteins. These structural features strongly suggest that SpSec9 functions as a plasma membrane t-SNARE component.

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Fig. 1. Primary structure of the fission yeast SpSec9 protein. Comparison of the amino acid sequence of the conserved domains among SpSec9, SNAP-25 homologues from S. cerevisiae (ScSec9 and ScSpo20), Danio rerio (DrSNAP25), and Homo sapiens (HsSNAP25). Identical amino acids are shown in white against black. Similar amino acids are shaded.

SpSec9 is essential for cell viability

To explore the consequences of complete loss of sec9⁺ function, a plasmid was constructed in which the ura4⁺ cassette was inserted at the PstI site within the sec9⁺ ORF. After transformation of the S. pombe diploid TN75 with a linear DNA fragment containing the sec9 disruption allele, Ura⁺ transformants were obtained. Tetrad analysis indicated that every ascus consisted of two viable and two inviable spores, and that all viable spores were phenotypically Ura^–. Microscopic observation of nonviable meiotic progeny showed that these spores could not germinate (data not shown). Therefore, sec9⁺ is essential for vegetative cell growth and spore germination.

Transcriptional regulation of sec9⁺

Log-phase cells of a homothallic haploid strain (MKW5) were incubated in the sporulation medium MM–N, and the sec9 mRNA abundance was monitored by Northern analysis. mRNA was detected in vegetative cells and was found to increase during sporulation (data not shown). To determine exactly when the rise in sec9 mRNA level occurred, a similar Northern analysis was carried out using a pat1-114 mutant. Meiosis was found to proceed in a synchronous fashion (Iino et al., 1995). The level of sec9 mRNA began to increase about 6 hr after induction and peaked at about 9 hr, when cells were in early meiosis II (Fig. 2A, 2B).

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Fig. 2. Expression of sec9+ during meiosis. (A) Transcription of sec9+ in pat1-driven meiosis. Meiosis in the diploid strains JZ670 (mei4+) and AB4 (mei4Δ), homozygous for pat1-114, was synchronously induced by a temperature shift-up (Iino et al., 1995). At intervals, total RNA was prepared from S. pombe cultures (Jensen et al., 1983) and fractionated on a 1.0% gel containing 3.7% formaldehyde as previously described (Thomas, 1980). The approximate quantity of RNA was assessed by staining with ethidium bromide. (B) Meiotic nuclear division of JZ670 was monitored by DAPI staining. Open circles, mononucleate; closed circles, binucleate; squares, tetranucleate cells. (C) Transcription of sec9+ is induced by ectopic overproduction of Mei4 in vegetative cells. TN4 cells harboring plasmid pREP1(mei4+) were grown in MM medium without thiamine (Mei4 OP, +) or MM with thiamine (Mei4OP, –) (Maundrell., 1993). After an incubation of 13, 15 and 17 hr, total RNA was subjected to Northern analysis. A Mei4-dependent gene spo6+ is included as a positive control.

The mei4⁺ gene encodes a forkhead transcription factor that regulates an array of genes required for meiosis and sporulation (Horie et al., 1998; Abe and Shimoda, 2000; Nakamura et al., 2001; Watanabe et al., 2001; Nakamura et al., 2002). To determine whether the elevation in sec9 mRNA during sporulation is dependent on Mei4, the sec9 mRNA level was measured in a mei4Δ mutant. As shown in Fig. 2A, accumulation of sec9 mRNA was completely abolished in the mei4Δ mutant. Furthermore, ectopic overexpression of mei4⁺ was found to induce sec9⁺ mRNA in vegetative cells (Fig. 2C). sec9⁺ has a consensus recognition sequence for Mei4, GTAAAYA (Horie et al., 1998) in the 5' upstream region. We conclude that transcription of sec9⁺ during meiosis is strictly regulated by Mei4.

Isolation of sporulation-deficient sec9 mutants

To determine whether SpSec9 is involved in sporulation, an attempt was made to isolate sporulation-deficient mutant by random PCR mutagenesis (Materials and Methods). A single sporulation-deficient mutant, sec9-10 was identified. sec9-10 cells failed to develop spores but completed normal meiotic nuclear divisions (data not shown).

In addition to causing a defect in ascospore formation, the sec9-10 mutation compromised vegetative growth. As shown in Fig. 3B, the sec9-10 mutant grew well at 25°C but was unable to form colonies at 37°C. Thus, the sec9-10 mutation confers temperature sensitivity for growth. It is known that mutations in several genes involved in membrane trafficking cause defects in cytokinesis (Nakase et al., 2001; Poloni and Simanis, 2002; Wang et al., 2002; Cheng et al., 2002; Edamatsu and Toyoshima, 2003). For example, the sec8⁺ gene, which encodes a component of the exocyst complex, is essential for cell separation (Wang et al., 2002). Therefore, the cell morphology of sec9-10 mutants incubated at permissive and restrictive temperatures was examined. sec9-10 mutants were indistinguishable from wild type cells with respect to septum formation when incubated at 25°C (Fig. 3C). In marked contrast, sec9-10 cells exhibited a rather uniform arrest morphology at the restrictive temperature (Fig. 3C). At 12 hr after the shift to 34°C, approximately 43% of the sec9-10 cells had a single septum, and 4% exhibited multiple septa (Table II). In wild-type cells, only 13% of the cells had a single septum (Table II). These results indicate that SpSec9 plays an important role in cytokinesis, especially in cell separation.

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Fig. 3. Phenotypes of the sec9-10 mutant. (A) MM59-4D (wild type) and TN9SP10 (sec9-10) were sporulated on SSA medium for 2 days at 25°C. Bar, 10 μm. (B) Strains MM59-4D (wild type) and TN9SP10 (sec9-10) were streaked on complete medium (YEA) and incubated at 25°C or 37°C for 3 days. (C) MM59-4D (wild type) and TN9SP10 (sec9-10) were incubated in liquid complete medium (YEL) for 12 hr at 25°C or 34°C. Cells were fixed and stained with calcofluor and DAPI. Bars, 10 μm.

We next determined the mutation point of the sec9-10 allele. The mutant gene was isolated from genomic DNA by PCR. Nucleotide sequencing demonstrated that sec9-10 is the result of a single nucleotide change (T to C) that results in replacement of leucine 228 with proline in the conserved N-terminal α-helical region.

The sec9-10 mutant is defective in forespore membrane formation

To examine in detail how the sec9-10 mutation impairs sporulation, the assembly of FSMs in the sec9-10 mutant was analyzed, using GFP-tagged Psy1, a syntaxin 1-like protein (Nakamura et al., 2001). Overexpression of GFP-Psy1 did not overcome the sporulation defect in sec9-10 cells. Progression of meiosis was monitored by observing the construction and elongation of spindle microtubules. In wild type cells, most haploid nuclei produced by meiotic second divisions were encapsulated by the FSM (Fig. 4A). In sec9-10 mutant cells, FSMs initiated normally at both poles of the meiosis II spindles (Fig. 4A), but extension of the FSMs was soon blocked, resulting in anucleated small prespores (Fig. 4B). These results indicated that the FSM initiated normally, but its subsequent development was abnormal. In conclusion, SpSec9 appears to be required for the normal construction of the FSM.

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Fig. 4. Aberrant assembly of FSMs in sec9-10. Assembly of the FSM during metaphase II and anaphase II (A), and in post-meiosis (B). TN402 (wild type) and TN396 (sec9-10) were cultured on SSA medium for 1 day. Fixed cells were doubly stained with the anti-α-tubulin and DAPI. Bars, 10 μm.

sec9⁺ interacts with syntaxin1 homologue, psy1⁺

In higher eukaryotes, SNAP-25 forms a complex with syntaxin-1 and synaptobrevin, and this interaction is important for specific membrane fusion. S. pombe has a synaptobrevin homologue, Syb1, which is involved in cytokinesis and cell elongation (Edamatsu and Toyoshima, 2003). To examine whether overproduction of Syb1 and Psy1 suppresses the sec9-10 mutation in S. pombe, a multicopy plasmid harboring either syb1⁺ or psy1⁺ was introduced into sec9-10 strains. Psy1 was found to suppress sec9-10 well at 34°C but poorly at 37°C. In contrast, Syb1 could not suppress sec9-10 even at 34°C (Fig. 5). These data indicate that sec9⁺ interacts genetically with the syntaxin1 homologue, psy1⁺. With respect to sporulation, overexpression of both genes was tested and found not to suppress the sporulation defect of the sec9-10 mutant.

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Fig. 5. Suppression of temperature sensitivity of sec9-10 by psy1+. TN9SP10 (sec9-10) cells transformed with empty vector (pL-A), pL-A(syb1), pL-A(psy1), or pL-A(sec9) were serially diluted and plated onto YEA medium. Photographs were taken after 2 days of incubation at 25, 34, and 37°C.

Discussion

This is the first report indicating that the S. pombe SNAP-25 homologue, SpSec9, plays essential roles in cell separation, a final step of cytokinesis, and in assembly of the FSM, an important step in sporulation. While SpSec9 is the sole SNAP-25 homologue in S. pombe, two SNAP-25 homologues exist in S. cerevisiae, ScSec9 and ScSpo20. Interestingly, formation of the prospore membrane in budding yeast does not require one of the SNAP-25 paralogs, ScSec9, while its sporulation-specific counterpart, ScSpo20, is indispensable. These findings imply that precursor vesicles for spore membranes are provided through a general secretory pathway and that sporulation-specific components are substituted in the case of the SNAP-25 homologue (Neiman, 1998). Recent studies have shown that ScSpo20 is subject to both positive and negative regulation by its amino terminal domain. A short, amphipathic helix in the N-terminal region, which binds to acidic phospholipids such as phosphatidic acids, is essential for proper localization of ScSpo20 to the prospore membrane. Thus, the N-terminal region appears to be an important determinant for meiosis-specific function of Spo20 (Neiman et al., 2000; Nakanishi et al., 2004). However, an analogous amphipathic helix was not found in the N-terminal region of S. pombe Sec9. The mechanism by which the SNAP-25 homologue regulates FSM formation might be quite different between the two yeasts.

The sec9-10 mutant is temperature-sensitive for growth yet exhibits a sporulation-defective phenotype at temperatures permissive for proliferation. Perhaps SpSec9 plays a functional role in sporulating cells but not in vegetative cells at these permissive temperatures. Alternatively, the threshold activity level of SpSec9 might be required for sporulation is higher than that required for vegetative growth. We presently favor the latter model because the sec9 mRNA level increased dramatically during sporulation. Other sporulation mutants such as spo20-KC104 and spo20-H6 also show a sporulation-defective phenotype at temperatures permissive for proliferation, supporting this notion. sec9-10 was also defective in cell separation at a restrictive temperature. In cell poles and the medial septation sites, membranes are actively remodeled by both biosynthetic and degradative mechanisms. These regions therefore may require the robust recruitment of membrane vesicles carrying secretory and membrane-associated proteins for cell surface growth and septation. Indeed, proteins required for septum formation and cell separation localize in the medial region of the cell as ring-like structures. Additionally, recent studies have revealed that a mutation in several genes involved in the membrane trafficking (Sec8, Sec10, Sec13, SpSpo20, Syb1, and Ypt3) causes a cytokinesis defect (Nakase et al., 2001; Poloni and Simanis, 2002; Wang et al., 2002; Cheng et al., 2002; Edamatsu and Toyoshima. 2003). Therefore, it is possible that SpSec9 might play a role in the transport of secretory proteins to these growing sites.

In S. cerevisiae, ScSec9 localizes to the plasma membrane during vegetative growth and both ScSec9 and ScSpo20 localize to the prospore membrane during sporulation (Brennwald et al., 1994; Neiman et al., 2000). We tried to observe the localization of SpSec9 using GFP-tagged SpSec9. However, GFP-SpSec9 was not fully functional and showed no characteristic localization.

The sec9-10 mutation site is positioned in one of the two conserved α-helices that are essential for formation of the SNARE complex. Therefore this mutation may reduce the affinity for the other t-SNARE, Psy1. Overexpression of Psy1 suppressed the temperature sensitivity, but not sporulation defect of sec9-10. These data suggest that the phenotype of sec9-10 could be due to in vivo instability of the SpSec9-Psy1 complex. The process of sporulation might require a more stable SpSec9-Psy1 complex than vegetative growth. Alternatively, the Sec9-10 mutant protein might be more labile than wild type Sec9. Indeed, cam1-F116 mutation, which reduces the abundance of the Cam1 protein level both in vegetative growth and in sporulation, exhibits sporulation-specific phenotype (Takeda et al., 1989).

The fluorescence microscopic analysis showed that, in sec9-10 mutants, FSM formation initiated normally near the SPB during meiosis II, but subsequent development into closed membrane compartments containing a nucleus, called prespores, was severely impaired. The sporulation-specific mutants, spo3 and spo14, exhibit similar phenotypes. Compared to these mutants, the FSM in the sec9-10 mutant arrested at an earlier stage of membrane formation. These data suggest that SpSec9 functions at a very early stage of FSM extension. This is consistent with the fact that the SNARE complex plays a central role in membrane vesicle fusion.

In summary, we have shown that a fission yeast SNAP-25 homologue, SpSec9, functions in completion of cell septation in vegetative cells and also plays a crucial role in sporulation, especially in biogenesis of the FSM. The present study together with our previous work demonstrate that spore formation accompanies a dynamic membrane fusion process which occurs under the strict control of SNARE proteins. Further work will be needed to understand how these SNARE proteins function at the molecular level during sporulation.

Acknowledgments

We thank Dr. K. Gull of the University of Manchester for the anti-α-tubulin antibody, TAT-1, Dr. M. Yamamoto of the University of Tokyo for strains, and Ms. Y. Maeda of Osaka City University for plasmids. The present study was supported in part by Grants-in-Aid for Scientific Research “B” (14380338) and Priority Areas “Genome Biology” (15013249) from the Ministry of Education, Science, Sports and Culture of Japan to C. S. and Grants-in-Aid for Scientific Research on Priority Area ‘Cell Cycle Control’ (16026240) and ‘Life of Proteins’ (14037263) from the Ministry of Education, Science, Sports and Culture of Japan to T. N. A few strains used in this study were provided by the Yeast Genetic Resource Center Japan (http://bio3.tokyo.jst.go.jp/jst/).