INTRODUCTION

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Sporulation in the yeast Saccharomyces cerevisiae involves a regulated program of cell development that includes premeiotic DNA replication, the two meiotic divisions, and the encapsulation of haploid nuclei within spore walls (15). Sporulation is initiated when MATa/MAT cells are starved of nitrogen in the presence of a nonfermentable carbon source, such as acetate, and is under the control of master regulatory genes that include IME1, IME2, and RME1 (31, 36). In yeast cells, the nuclear envelope remains intact throughout meiosis and the intranuclear segregation of chromosomes generates four bulges in the nucleus, each next to the respective spindle pole body (for a review, see reference 8).

Progression through the sporulation program depends on the sequential expression of at least four temporally distinct groups of sporulation-specific genes, classified as early, middle, mid-late, and late (31, 36). Sporulation in S. cerevisiae thus provides a model system to study the temporal control of gene expression during development. The mid-late and late genes are activated at the time of the meiotic divisions and spore formation, and this activation is accompanied and followed by morphological changes that result in spore formation. Such changes include (i) the formation of a flattened membrane sac (the "prospore wall") closely apposed to the cytoplasmic faces of the spindle pole bodies, (ii) the extension of the prospore walls along the outer surface of the nuclear envelope, (iii) the separation of the prospore walls from the spindle pole bodies and the nuclear envelope and the movement of cytoplasm and organelles into the intervening space, (iv) the final engulfment of the nuclear lobes (containing the haploid chromosome sets) and associated cytoplasm, and (v) the deposition of spore wall components between the two membranes of the prospore wall (2, 8, 20, 37, 38, 40).

The final differentiated spore wall offers increased protection to stress conditions, compared to the wall of the vegetative cell, and consists of four layers (35). The two inner layers are formed by closely juxtaposed glucans and mannans and often appear as a single layer strongly resembling the vegetative cell wall when examined by electron microscopy (29). The third and fourth layers are spore-specific structures and are composed primarily of chitosan and dityrosine, respectively. They are thought to confer upon the spore wall its protective nature (4, 6, 7). Chitosan, a -(1,4)-D-glucosamine homopolymer, is produced by deacetylation of nascent chains of chitin, a -(1,4)-N-acetyl-D-glucosamine homopolymer produced by the action of chitin synthases (14). The deacetylation reaction is catalyzed by the enzyme chitin deacetylase (28), which is encoded by two recently identified sporulation-specific genes (10). The outermost layer of the spore wall is the dityrosine coat, which is composed of an insoluble macromolecule containing a high number of cross-linked tyrosine residues that is synthesized in a two-step reaction catalyzed by the products of the sporulation-specific genes DIT1 and DIT2 (5).

In this report we describe the characterization of a novel yeast gene named SWM1 (spore wall maturation) that is transcribed during vegetative growth but whose expression is strongly induced during the sporulation process with kinetics similar to those of middle sporulation-specific genes. SWM1 is required for normal activation of mid-late and late genes during sporulation and, in consequence, swm1 mutants fail to assemble the spore wall, a phenotype similar to that described for sps1 and smk1 mutants (18, 30).

	MATERIALS AND METHODS

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Plasmids used. Plasmid pSU4, used to generate the swm1::hisG deletion allele, was constructed by PCR amplification of the 5'-end (from 291 to 1) and the 3'-end (from +524 to +876) flanking regions with specific oligonucleotide primers and cloning the PCR fragments in Bluescript SK(+) vector (Stratagene), yielding plasmid pSU3. The two fragments were separated by a synthetic BamHI site that was used to clone a 3.8-kb BamHI-BglII fragment carrying the URA3::hisG cassette (1) to generate plasmid pSU4.

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Physical and functional map of EXG2 and SWM1 (YDR260c). At the top is the map of the EXG2 and SWM1 chromosomal region, with arrows indicating the coding sequences. Immediately below is a representation of the region replaced in the construction of the exg2::LEU2 allele (YPA69 strain). Constructs used to test for complementation of the sporulation defect of YPA69 are indicated (pJC5, pRN30, and pPS47), with arrows designating the regions of each ORF present in the plasmids. The ability of the strains to sporulate is indicated at the right. The lower line represents the extent of the deletion in the swm1 allele, in which the complete coding region was replaced with the URA3 gene flanked by hisG repeats. Restriction sites: B, BamHI; P, PstI; S, SmaI; Sa, Sau3A; X, XhoI.

Yeast strains and growth conditions. Table 1 lists the yeast strains used in this study. The wild-type diploid strain YPA24 was constructed by mating strains W303-1A and 131-20 (45). The diploid strain YPA207, homozygous for the swm1::hisG allele, was constructed by transforming strains W303-1A and 131-20 with plasmid pSU4 digested with XhoI and NotI to release a linear fragment containing the deletion cassette. Several independent transformants from each haploid strain were grown on plates containing 5-FOA (3) to select for colonies that had lost the URA3 marker (which is flanked by direct repeats of hisG). Replacement of the SWM1 coding sequence by the hisG fragment was confirmed by Southern blot analysis of genomic DNA obtained from Ura colonies. Finally, a MATa swm1::hisG strain (YPA203) and a MAT swm1::hisG strain (YPA202) were mated to construct diploid strain YPA207, which is isogenic to the wild-type strain YPA24. Heterozygous diploids YPA205 and YPA206 were constructed by crossing, respectively, YPA202 and YPA203 with the wild-type strains from the opposite mating type.

RNA isolation and Northern analysis. Cells (1.3 × 10⁹) were collected at different time intervals after transfer to sporulation medium, and total RNA was prepared by the method described by Percival-Smith and Segall (44). For Northern blot analysis, 5 µg of RNA was denatured and transferred to Hybond membranes (Amersham) according to the manufacturer's instructions. The DNA probes used to detect the different transcripts were as follows: SWM1, a 360-bp internal fragment (from +38 to +398) obtained by PCR; SPS1, the 1.02-kb EcoRV-BglII fragment of plasmid pSU9; SMK1, the 1.8-kb SpeI-XhoI fragment of plasmid pSU10 (which includes the coding sequence as well as 5'- and 3'-flanking regions); HOP1, the 1.3-kb BamHI-HindIII fragment of plasmid pNH33-2 (24); SPO12, the 0.45-kb BamHI-EcoRI fragment of plasmid pRE129 (34); SSG1, the 1.2-kb SacI-SalI fragment of plasmid pPS23 (45); DIT1, a 0.7-kb fragment obtained by PCR amplification of the region from 41 to +657; SPS100, a 0.75-kb fragment obtained by PCR amplification of the region from 14 to +745; and ACT1, the 1.7-kb BamHI-HindIII fragment. The probes were radioactively labeled by the random priming method (16). The specificity of the mRNA detected by the SWM1 probe used was confirmed by using a swm1 mutant.

Resumption of mitotic growth and resistance to stress. Resumption of growth was assessed by using cells grown in YEPA to a concentration of 1 × 10⁷ to 2 × 10⁷ cells/ml and then transferred to sporulation medium. At various times during sporulation, samples of wild-type and mutant cells were withdrawn, diluted, plated on YEPD, and then incubated 2 days at 30°C before the numbers of viable colonies were counted. To determine the efficiency of haploidization, we used the appearance of the recessive cycloheximide resistance allele cyh2 by replica plating the germination plates on YEPD plates containing cycloheximide (10 µg/ml). The sensitivity of sporulating cells to heat shock treatment (55°C) or to enzymatic digestion (glusulase) was determined as described by Briza and coworkers (4).

Fluorescence-activated cell sorter (FACS) analysis. Cells grown on YEPD to the early log phase were fixed and stained with propidium iodide according to the protocol previously described by Hutter and Eipel (27). Flow cytometry analysis was performed on a Becton Dickinson FACSort analyzer.

Microscopy. For light microscopy, cells were fixed in ethanol and stained with DAPI (4',6-diamidino-2-phenylindole) as previously described (46). Samples were viewed and photographed as a wet mount with a Zeiss Axiophot microscope equipped for Nomarski optics and epifluorescence. For visualization of the GFP-Swm1 fusion protein, cells that had been in sporulation medium for 10 h were fixed with 3.7% formaldehyde for 1 h, washed briefly with phosphate-buffered saline (PBS), resuspended in PBS buffer containing DAPI, and observed with a Nikon E800 microscope with an FITC-HYQ (Chroma) filter set. Pictures were taken with a Photometrics Sensys CCD camera.

	RESULTS

Top Abstract Introduction Materials and methods Results Discussion References

Identification of the SWM1 gene. In our studies with EXG2, a gene encoding a 1,3--glucanase expressed during the S. cerevisiae vegetative cycle, we observed that a diploid strain carrying a homozygous deletion that replaces the whole EXG2 coding region, as well as 466 nt of the 3' region by the LEU2 gene (exg2::LEU2 in Fig. 1) renders diploid strains unable to sporulate (12). However, this deletion also eliminates the first 58 amino acids of a small ORF located downstream from EXG2, named YDR260c in the yeast genome sequencing project. In order to test whether the sporulation defect was due to the absence of the Exg2 protein or the YDR260c gene product, strain YPA69 (exg2::LEU2) was transformed with plasmids carrying different DNA fragments containing the EXG2-YDR260c region (Fig. 1), and the ability of the cells to sporulate was tested. Our results indicated that plasmid pJC5, carrying the full-length EXG2 gene, was unable to complement the sporulation defect, while plasmids pRN30 (carrying YDR260c and part of the 3' end of the EXG2 coding sequence) and pPS47 (carrying only YDR260c) restored the ability to form spores in the homozygous diploid mutant strain (Fig. 1). These results thus suggested that the lack of the protein encoded by YDR260c was responsible for the sporulation defect initially attributed by us to the exg2 mutation.

SWM1 is required for ascospore development. To confirm that SWM1 is required for sporulation, a gene replacement that deleted the SWM1 coding region (swm1::URA3::hisG) was performed (Fig. 1). Sporulation and dissection of spores from an swm1/SWM1 heterozygous diploid revealed no differences in growth between swm1 and wild-type haploid segregants. SWM1 is therefore not required for vegetative growth or germination.

Expression of SWM1 is induced during the sporulation process. To pinpoint the exact time at which SWM1 serves its role in sporulation, the meiotic time course of SWM1 expression was examined by Northern analysis of wild-type cells at various stages in the differentiation process (Fig. 2A). An RNA molecule of 0.9 kb was detected by using a radiolabeled SWM1 probe, a finding consistent with the predicted size of the gene. SWM1 transcripts were present at the time when the cells were shifted to the nitrogen-deficient medium (0 h), but a sharp rise in the amount of SWM1 RNAs occurred in the middle period of the sporulation process (6 h), with maximal accumulation being observed between 9 and 12 h. SWM1 RNA levels remained high during the time the first mature asci were observed (12 to 15 h) but thereafter slowly declined. This observation indicates that the SWM1 induction profile is similar to that of genes expressed midway through the sporulation process (36), although SWM1 expression is not only restricted to the meiotic process.

View larger version (41K): [in this window] [in a new window]   FIG. 2.   Expression of the SWM1 gene during the sporulation process. (A) Meiotic time course of SWM1 expression. RNA purified from wild-type cells (strain AP1a/) at the indicated times after transfer to sporulation medium was hybridized with a radioactively labeled SWM1 probe. The top panel represents the percentage of mature asci at each time point. (B) Expression of SWM1 in MAT/MAT cells. RNA purified from strain AP1a/ at 0 or 10 h (lanes 1 and 2, respectively) after transfer to sporulation medium or from strain AP1/ at 0 or 10 h (lanes 3 and 4, respectively) was hybridized with the SWM1 probe. In both experiments, the ACT1 probe was used to test for equal loading of RNA in all lanes.

SWM1 is not required for meiotic nuclear divisions. To study the sporulation defect in more detail, the homozygous swm1 diploid YPA207 and the isogenic parental strain YPA24 were transferred to liquid sporulation medium, and the cells were examined by phase-contrast microscopy to monitor asci formation and by fluorescence microscopy with the DNA-specific fluorophore DAPI to follow the meiotic divisions. DAPI staining revealed that both meiosis I and meiosis II occurred at approximately the same time in mutant and wild-type cells, the majority of bi- and tetranucleate cells appearing between 8 and 16 h (Fig. 3). However, we also observed that mutant cells consistently displayed a slight reduction in their ability to complete the second meiotic division (69% in the wild-type cells versus 59% in swm1 cells). Despite this, most of the cells that had completed meiosis I went on to complete meiosis II but failed to form spores, as assessed by phase-contrast microscopy.

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Time course of meiosis in swm1 cells. Samples of cells from sporulating cultures of the wild-type strain YPA24 (A) or the swm1 mutant YPA207 (B) were fixed, stained with DAPI, and examined by fluorescence microscopy to determine the percentage of cells that had completed meiosis I () or meiosis II (). Cells that appeared to be binucleate, trinucleate, or tetranucleate by DAPI staining were considered to have completed meiosis I. Cells that appeared to be trinucleate or tetranucleate were considered to have completed meiosis II. Samples were also examined by phase-contrast microscopy to monitor ascus formation ().

View larger version (106K): [in this window] [in a new window]   FIG. 4.   Microscopic appearance of wild-type and swm1 mutant asci during the sporulation process. Rows 1 and 3, differential interference contrast microscopy (DIC) photomicrographs; rows 2 and 4, corresponding photomicrographs of DAPI-stained cells. Wild-type cells (strain YPA24) at 8, 12, and 24 h after transfer to sporulation medium present a characteristic tetranuclear organization and birefringent spore walls. swm1 cells (strain YPA207) at 8, 12, and 24 h after transfer to sporulation medium do not display discernible spore walls by DIC microscopy. These mutant cells are clearly tetranucleate after 12 h in sporulation medium.

SWM1 is required for assembly of functional spore walls. In wild-type cells, birefringent spore walls were first observed after 12 to 14 h in sporulation medium. By contrast, the swm1 mutant strain failed to assemble spore walls, even after prolonged incubation in sporulation medium. Microscopic examinations suggested that swm1 mutants are blocked in development before the assembly of the spore wall, the structure that confers a high degree of resistance to stress conditions. The viability of the swm1 mutant incubated in sporulation medium for 24 h was 56% that of wild-type sporulating cells and 30% that of wild-type sporulating cells when plated 4 days after transfer to the sporulation medium (Fig. 5A). This indicates that swm1 mutant cells are able to resume mitotic growth after incubation in sporulation medium, although their viability decreases after a prolonged incubation in it.

View larger version (15K): [in this window] [in a new window]   FIG. 5.   Resistance of wild-type and swm1 asci to prolonged incubations in sporulation medium, exposure to heat shock, or enzymatic (glusulase) digestion. (A) Wild-type cells from the strain YPA24 () or the isogenic swm1 strain YPA207 () were incubated in sporulation medium for the indicated times (at 30°C) before the plating efficiencies were assayed. (B and C) Wild-type (strain YPA24 []) or swm1 cells (strain YPA207 []) were incubated in sporulation medium for 24 h and then assayed for plating efficiency after exposure to 55°C (B) or to glusulase (C) for the indicated times (see Materials and Methods).

Haploid progeny can be recovered from swm1-arrested cells. Compartmentalization of the haploid genome is thought to be dependent on the formation of prospore walls around the four nuclear lobes that arise after meiosis. The prospore envelope traps some cytoplasmic material and then serves as the scaffold for the deposition of the spore-wall-specific material (37, 38, 40). Upon germination, the four spores present within the ascal sac are released, and growth of the individual spores gives rise to haploid progeny.

View larger version (26K): [in this window] [in a new window]   FIG. 6.   FACS analysis shows that swm1/swm1 cells are able to generate haploid progeny upon germination. Cells were grown in YEPD, fixed, and stained with propidium iodide prior to analysis in a Becton Dickinson FACSort. (A) Scan of haploid SWM1 cells (W303-1A). The peaks of cells marked 1N and 2N represent cells in the G1 and the G2 phases of the cell cycle, respectively. (B) Scan of haploid swm1 cells (YPA203). (C) Scan of diploid wild-type strain YPA24. The peaks of cells marked 2N and 4N represent cells in the G1 and the G2 phases of the cell cycle, respectively. (D) Scan of diploid mutant strain YPA207. (E and F) Scan of two of the cycloheximide-resistant clones obtained after sporulation and germination of the swm1/swm1 diploid strain YPA207. The graphs depict relative DNA content (x axis) versus cell number (y axis).

Spore walls are aberrant in swm1 cells. To assess the nature of the sporulation defect in swm1 cells at high resolution, we used electron microscopy to examine cells that had been under sporulation conditions for 24 h. The ultrastructure of a typical wild-type ascus is shown in Fig. 7A and B. The innermost layers of the spore wall, which often appear as a single electron-transparent layer, are similar in composition to the vegetative cell wall, which contains mainly glucans and mannans (7, 29). The outermost surface of the spore wall consists of a cross-linked insoluble macromolecule containing a large amount of dityrosine (4, 6) and often appears as a very thin, osmiophilic layer. This layer is responsible for the resistance of spores to degradative enzymes and organic solvents (4) and is closely linked, perhaps by covalent linkages, to an underlying chitin and chitosan layer (7), which appears as a more diffuse osmiophilic layer.

View larger version (112K): [in this window] [in a new window]   FIG. 7.   Electron microscopy of wild-type and swm1 mutant cells incubated for 24 h in sporulation medium. (A and B) wild-type strain YPA24. (C to H) swm1 mutant strain YPA207. Single asci at low magnification are depicted in panels A, C, E, G, and H, while higher magnifications of spore walls are shown in panels B, D, and F. In panel A, a typical ascus of the wild-type strain YPA24 showing three of the four spores clearly presents a well-defined spore wall surrounding all of them. In panel B, one portion of the wall of the three spores present in the ascus at higher magnification clearly shows the inner, electron-transparent components, which appear as a single layer followed by the chitin-chitosan layer closely juxtaposed next to the dityrosine coat. In panels C to H, a variety of spore wall defects are apparent in swm1 mutant asci. Mutant spores are surrounded by an electron-dense region with a heterogeneous structure (see the text for a detailed description of the aberrant morphology in the mutant). Scale bars, 1 µm (A, C, E, G, and H) and 0.4 µm (B, D, and F).

SWM1 is required for normal expression of late sporulation-specific genes. Several classes of temporally distinct sporulation-specific genes, referred to as early, middle, mid-late, and late, are sequentially expressed as the sporulation program proceeds (for a review, see references 31 and 36). To determine whether deletion of the SWM1 gene affects the pattern of sporulation-specific gene expression, we used Northern blot analysis to measure the transcript levels of various genes in isogenic wild-type and swm1 diploid cells incubated in sporulation medium for different periods of time. The timing and relative expression levels of the early sporulation-specific gene HOP1 (24) and the middle sporulation-specific gene SPO12 (34) were indistinguishable between mutant and wild-type cells (Fig. 8). These results are consistent with the microscopic observations indicating that sporulation initiation and the execution of meiotic landmark events are unaffected in swm1 cells. However, the timing of induction of several of the mid-late and late genes tested was different between wild-type and mutant cells. Thus, the expression of the mid-late SSG1 gene (also known as SPR1) (39, 45) was slightly delayed in swm1 cells, with maximal expression being detected between 12 and 14 h after transfer to nitrogen-deficient medium (in contrast to 10 h in wild-type cells). More-dramatic effects were found in another two genes that are required for spore wall maturation: the expression of DIT1 (5) and SPS100 (32) was delayed and the level of transcript accumulation was greatly reduced in the swm1 mutant (Fig. 8). These results therefore suggest that SWM1 is required for the normal expression of mid-late and late sporulation-specific genes, which are involved in spore cell wall formation and maturation, and are consistent with the morphological defects observed by electron microscopy.

View larger version (75K): [in this window] [in a new window]   FIG. 8.   Expression of sporulation-specific genes in wild-type and swm1 mutant cells. RNA was purified from wild-type (strain YPA24) and swm1 cells (strain YPA207) at the indicated times after transfer to sporulation medium. RNA blots were sequentially hybridized with the following radioactively labeled gene-specific probes: SWM1, HOP1, SPO12, SSG1, DIT1, and SPS100. The ACT1 gene was used to test for equal loading of RNA in all lanes.

SPS1 and SMK1 expression is not regulated by Swm1p. Because deletion of the SPS1 or SMK1 genes, two components of a sporulation-specific mitogen-activated protein (MAP) kinase cascade, has similar effects on the expression of SPS100 to those described here (18, 30), it seemed possible that SWM1 might be required to induce the expression of these two genes during the sporulation process. To test this possibility, we used the same set of RNA blots to compare the pattern of expression of SPS1 and SMK1 between isogenic wild-type and swm1 diploid strains. We found that the timing and relative expression levels of both protein kinase-encoding genes were indistinguishable between mutant and wild-type cells (Fig. 9A), indicating that the SWM1 is not required for the correct expression of SPS1 and SMK1. Interestingly, the induction of SWM1 expression during the sporulation process coincided in time with the induction of SPS1 and SMK1 transcripts.

View larger version (76K): [in this window] [in a new window]   FIG. 9.   (A) Expression of SMK1 and SPS1 in wild-type and swm1 mutant cells. The same RNA blots used in Fig. 8 were stripped and sequentially hybridized with radioactively labeled probes for SPS1 and SMK1. For a better comparison, the panels corresponding to SWM1 and ACT1 expression are also shown. (B) Expression of SWM1 in wild-type and mutant smk1 cells. RNA purified from wild-type (strain NKY278) and smk1 (strain LAKY30) at the indicated times after transfer to sporulation medium was hybridized with an SWM1 radioactively labeled probe, and the ACT1 gene was used to test for equal loading of RNA in all lanes.

Genetic analysis of the relationship between Swm1p and Sps1p-Smk1p. The similarity of the phenotypes observed in swm1 mutant cells to those reported for the deletion of SMK1 and SPS1 (18, 30) suggested that the corresponding products might operate in the same pathway. To test this hypothesis, we constructed a set of isogenic strains carrying all possible combinations of deletions in the SWM1, SPS1, and SMK1 genes. If Swm1p is a downstream component of the Sps1p-Smk1p signaling pathway and is involved in functions controlled by this pathway, then deletion of SWM1 would be expected to have a similar effect to the deletion of SMK1 or SPS1; however, if Swm1p operates in a pathway parallel to the sporulation-specific MAP kinase pathway, then smk1 or sps1 would be expected to enhance the swm1 mutant defects. These possibilities were tested by examining the expression of the late gene SPS100 by Northern blot analysis in wild-type and single- and double-mutant homozygous diploid strains (Fig. 10A). Expression of SPS100 in wild-type and swm1 mutant cells was similar to the pattern described in Fig. 8. Accumulation of SPS100 transcripts in the smk1 or sps1 strains was both delayed and reduced, as described previously (18, 30) (Fig. 10A). This analysis clearly shows that SPS100 expression is more severely affected in swm1 cells than in isogenic smk1 or sps1 mutant cells, suggesting that both proteins do not act in the same linear pathway. The timing of induction of SPS100 transcription in the swm1 smk1 double mutant was similar to the pattern observed in the swm1 mutant, although the level of induction was reduced. Similarly, expression in the double sps1 swm1 mutant was also reduced compared with any of the single mutants. Interestingly, no additive effect was observed when the sps1 and smk1 mutations were combined. These results indicate that Swm1p does not lie in the same linear pathway as Sps1p-Smk1p. Additional support for this idea was obtained when the resistance of the different diploid strains to incubation in sporulation medium was analyzed (Fig. 10B). The viability of the double mutants swm1 smk1 and swm1 sps1 (20 and 18%, respectively) after 72 h of incubation in sporulation medium was more reduced than that of any of the single mutant strains swm1, smk1, or sps1 (30, 29, and 43%, respectively). By contrast, the double-mutant smk1 sps1 showed a viability similar to that of the single smk1 mutant (30 versus 29%) after 3 days of incubation in sporulation medium. Together, these results indicate that although Swm1p is required for normal expression of the late sporulation-specific genes, it is not a component of the Smk1p signaling pathway necessary for the late events of the sporulation program.

View larger version (45K): [in this window] [in a new window]   FIG. 10.   (A) Expression of SPS100 in wild type (WT) and in single- and double-deletion mutants. RNA from strains YPA24 (WT), YPA207 (swm1), LS35 (smk1), LS36 (sps1), LS34 (smk1 swm1), LS37 (swm1 sps1), and LS45 (smk1 sps1) that had been incubated in sporulation medium for the indicated time periods was hybridized with a radioactively labeled probe specific for the SPS100 gene. After the probe stripping, the ACT1 gene was applied to same filters to test for equal loading of RNA in all lanes. (B) Resistance of wild-type and single- and double-mutant cells to prolonged incubations in sporulation medium. Wild-type cells from the strain YPA24 (*) or the isogenic strains YPA207 (swm1 []), LS35 (smk1 []), LS36 (sps1 []), LS34 (swm1 smk1 []), LS37 (sps1 swm1 []) and LS45 (sps1 smk1 []) were incubated in sporulation medium for the indicated times before direct assay of the plating efficiencies.

Localization of Swm1p in sporulating cells. To determine the intracellular localization of Swm1p, we constructed a GFP-SWM1 fusion expressed under the control of the SWM1 promoter and present in a centromeric plasmid. The GFP-Swm1 fusion protein is fully functional and it completely restores sporulation ability when introduced into an swm1 mutant (not shown). The localization of Swm1p was examined in diploid cells containing the plasmid that had been in sporulation medium for 10 h. At this time, a fraction of the cells had already undergone meiosis I and II. As shown in Fig. 11, GFP-Swm1p appears to be a nuclear protein, whose GFP fluorescence coincides with the DAPI staining in bi- and tetranucleate cells. The signal was completely absent from cells carrying the untagged wild-type SWM1 gene (not shown). This result indicates that a large fraction of Swm1p appears to be concentrated in the nucleus.

View larger version (66K): [in this window] [in a new window]   FIG. 11.   Localization of GFP-Swm1p in sporulating cells. Cells from strain YPA207 carrying a GFP-SWM1 fusion in the centromeric vector Ycplac33 were incubated for 10 h in sporulation medium, fixed, stained with DAPI, and photographed.

	DISCUSSION

Top Abstract Introduction Materials and methods Results Discussion References

We have characterized a novel gene, SWM1, that is required for the final steps of the sporulation process to be completed. When diploid yeast cells are deprived of nitrogen in the presence of a nonfermentable carbon source, such as acetate, they enter the meiotic program, in which a reductional division (meiosis I) is rapidly followed by a mitosis-like division (meiosis II) to generate four haploid nuclei. The final steps involve encapsulation of the newly formed meiotic products in an extremely resistant spore wall, which consists of four layers: the two inner ones are closely juxtaposed and are similar in composition to the vegetative cell wall, while the two outer ones are spore-specific structures, composed of chitin and chitosan (the third) and dityrosine (the fourth). The genes required to execute these landmark meiotic events exhibit a tight temporal expression pattern and have been classified as early, middle, mid-late, and late (36). Our results indicate that Swm1p functions as a positive effector of the machinery responsible for spore wall component assembly.

SWM1 (YDR260c) codes for a 170-amino-acid protein with a predicted molecular size of 19,356 Da. Similarity searches against sequences present in the databases revealed no homology with any known protein, either from S. cerevisiae or from any other organism. Also, after several databases were searched, no relevant motifs or patterns hinting to the function of the protein were found in the sequence. Analysis of the expression pattern of SWM1 revealed that it is expressed at a low basal level during the vegetative cycle but strongly activated during the sporulation process, the expression peak coinciding with other middle sporulation-specific genes, such as SPO12 or SMK1. Similar expression kinetics have been described for the SPR3 gene, which encodes a sporulation-specific homolog of the yeast Cdc3/10/11/12 family of septins (42). Detailed examination of the promoter region of the SPR3 gene showed that its pattern of expression is regulated by at least two essential elements: a palindromic sequence containing a sporulation-specific element, termed MSE (midsporulation element) and an ABFI element (42, 43). The MSE element alone has been shown to confer sporulation-specific regulation, suggesting that it is directly involved in regulating the timing of expression of the SPR3 gene, and it is also present in other middle sporulation-specific genes, such as SPS4, SPR2, or SMK1 (43). Recently, it has been shown that direct binding of Ndt80p to the MSE is necessary for activating transcription of the middle sporulation genes (11, 21). The ABFI element [consensus sequence CGTNNNN(G/A)(T/C) GA(TC)] is normally found upstream from the MSE element and is also essential for normal regulation of SPR3 expression (42). The promoter region of SWM1 also contains a perfect match to the MSE consensus sequence [gNCRCAAA(A/T)] in an inverted orientation, between positions 77 and 85, which is preceded by a sequence that shows an almost perfect match (7 of 8 nt) with the ABFI consensus sequence (147 to 135). These two elements, as described for SPR3, may be responsible of the induction of expression of SWM1 that we observed during the sporulation process.

Several lines of evidence indicate that Swm1p is required for completion of the final steps of spore wall maturation. First, although no mature spores are formed after incubation in sporulation medium, swm1 cells were able to complete meiosis I and meiosis II with efficiencies similar to that of wild-type cells, as assayed by DAPI staining and microscopic observation. Second, after incubation in sporulation medium, the mutant cells were able to resume mitotic growth upon transfer to rich medium, generating viable haploid progeny, as has been shown for other mutants arrested at different stages of the sporulation process, such as spo14 (25, 26) or sps1 (18). The recovery of haploid progeny from swm1 mutants was an indication that postmeiotic compartmentalization had occurred, at least to some degree, in some of the mutant cells. Third, cells lacking the SWM1 gene are more sensitive to heat shock or enzymatic digestion than wild-type cells, indicating that they are unable to assemble a functional spore cell wall, the cellular structure responsible for the extreme resistance to stress conditions characteristic of mature spores. Finally, examination of the morphology of swm1 defective asci by electron microscopy revealed the formation of four distinct compartments inside each cell that were generally smaller than wild-type spores. The appearance of the mutant spores was variable, and they were surrounded by a rudimentary cell wall with a fibrillar aspect. Taken together, all of these results indicate that Swm1p functions late in sporulation, at the time when maturation and assembly of the spore cell wall occurs.

Relation of Swm1p and the Sps1p-Smk1p MAP kinase pathway. Two yeast genes, SPS1 and SMK1, encode protein kinases that play a regulatory role in spore packaging: Sps1p is a homolog of the protein kinase STE20 (44% identity in the catalytic domain) (18), while Smk1p is a homolog of MAP kinases, sharing 40% identity with FUS3 (30). Both sps1 and smk1 null mutations cause defects in spore wall formation. On the basis of their common functions and structural features, it has been proposed that the two proteins may act in a sporulation-specific MAP kinase pathway. Indeed, by analogy with the pheromone response pathway, Sps1p might govern the activity of a MEKK-MEK-MAP kinase module containing Smk1p as its MAP kinase, although no epistasis analysis has been done to confirm this proposal (18, 22, 30, 31). From these analyses, it was suggested that although Sps1p and Smk1p are required for activation of late sporulation-specific genes, they have opposite roles in the expression of the mid-late gene DIT1 because, whereas a sps1 mutant overexpresses DIT1, an smk1 mutant underexpresses it. This difference was interpreted assuming that Sps1p may have a second function in addition to the presumed stimulation of Smk1p activity (31).