The Fission Yeast RNA-Binding Protein Meu5 Is Involved in Outer Forespore Membrane Breakdown during Spore Formation

In Schizosaccharomyces pombe, the spore wall confers strong resistance against external stress. During meiosis II, the double-layered intracellular forespore membrane (FSM) forms de novo and encapsulates the nucleus. Eventually, the inner FSM layer becomes the plasma membrane of the spore, while the outer layer breaks down. However, the molecular mechanism and biological significance of this membrane breakdown remain unknown. Here, by genetic investigation of an S. pombe mutant (E22) with normal prespore formation but abnormal spores, we showed that Meu5, an RNA-binding protein known to bind to and stabilize more than 80 transcripts, is involved in this process. We confirmed that the E22 mutant does not produce Meu5 protein, while overexpression of meu5+ in E22 restores the sporulation defect. Furthermore, electron microscopy revealed that the outer membrane of the FSM persisted in meu5∆ spores. Investigation of the target genes of meu5+ showed that a mutant of cyc1+ encoding cytochrome c also showed a severe defect in outer FSM breakdown. Lastly, we determined that outer FSM breakdown occurs coincident with or after formation of the outermost Isp3 layer of the spore wall. Collectively, our data provide novel insights into the molecular mechanism of spore formation.


Introduction
Sporulation in the fission yeast Schizosaccharomyces pombe is a common cellular process that occurs in response to nutrition starvation, especially nitrogen starvation [1,2]. Sporulation consists of two coordinated processes: meiosis and spore morphogenesis. During meiosis II, a double-layered intracellular membrane called the forespore membrane (FSM), which subsequently becomes the spore plasma membrane, is formed de novo on the cytoplasmic side of the spindle pole body [1]. The FSM then expands by the fusion of membrane vesicles and eventually encapsulates a haploid nucleus generated by meiotic nuclear division, producing the "prespore", namely the membrane-bound precursor of the spore [1,[3][4][5]. Spore wall formation proceeds by the deposition of spore wall materials 2.5. Isolation of meu5 + The E22 mutant was transformed with the S. pombe genomic library pTN-L1 [4], containing Sau3AI fragments constructed in the multicopy plasmid pAL-KS [25]. Transformants were incubated on SD medium at 28 • C for 4 days and then transferred onto SSA medium. Colonies were exposed to iodine vapor [26]. Iodine staining-positive colonies were removed and inspected for recovery of sporulation after confirmation by fluorescence microscopy. Plasmids were isolated from these candidates and their nucleotide sequences were determined.

Reverse Transcription PCR
Total RNA from sporulating cells was extracted by the glass bead method using ISOGEN reagent (Nippon Gene, Toyama, Japan) and DNase I (Stratagene, La Jolla, CA, USA) treatment. A ReverTra Ace-α-kit (Toyobo, Osaka, Japan) was used for cDNA synthesis. Reverse transcription-PCR (RT-PCR) was performed using two sets of forward and reverse primers (itr1f/itr1r and itr2f/itr2r) (Table S2).

Western Blotting
BW46 and BW47 cells carrying a single chromosomal copy of meu5 + or the meu5-E22 gene tagged with three tandem copies of the HA epitope, respectively, were precultured to mid-log phase in MM+N medium and then transferred into MM-N medium to induce sporulation. Samples of the culture were collected at specific intervals, and crude cell extracts were prepared as previously described [27]. Proteins were resolved by SDS-PAGE on a 12% Bis-Tris gel and then transferred onto a polyvinylidene difluoride membrane (Immobilon-P, Millipore, Darmstadt, Germany). Blots were probed with rat anti-HA 3F10 antibody (Roche Diagnostics, Indianapolis, IN, USA) and mouse anti-α-tubulin TAT-1 antibody [28] at a 1:10,000 dilution. Immunoreactive bands were detected by using ECL Select chemiluminescence (GE Healthcare, Little Chalfont, UK) with horseradish peroxidase-conjugated goat anti-rat immunoglobulin G (BioSource International, Camarillo, CA, USA) and sheep anti-mouse IgG (GE Healthcare) at a 1:100,000 dilution.

Construction of the Plasmid for meu5 + Overexpression
Plasmid pBW1 was constructed as follows. A meu5 + fragment including the promoter and terminator regions was amplified by PCR using the primers meu5pc.1 and meu5pc.2 (Table S2). The resulting fragment was digested with SacI and ApaI restriction enzymes, and inserted into the corresponding site of pDblet [29], yielding pBW1.

The E22 Mutant Shows a Defect in Spore Maturation
In a previous study, we isolated a number of novel sporulation-deficient mutants from mutagenized S. pombe cells, in which the FSM was visualized by GFP-tagged Psy1 [30]. To investigate aspects of the sporulation mechanism, here we focused on the E22 mutant, whose asci produced four spores, but the spores were apparently abnormal ( Figure 1A). Like other sporulation-deficient mutants, the E22 mutant formed an iodine staining-negative colony on sporulation medium ( Figure 1B). Under the differential interference contrast (DIC) microscope, spores from the E22 cells were dull in appearance as compared with spores from wild-type cells ( Figure 1A). Mating frequency and progression of meiotic nuclear division were normal in the E22 mutant as judged by the observation of nuclear staining. These data indicate that a late step of spore formation is defective in the E22 mutant.

The E22 Mutant Shows a Defect in Spore Maturation
In a previous study, we isolated a number of novel sporulation-deficient mutants from mutagenized S. pombe cells, in which the FSM was visualized by GFP-tagged Psy1 [30]. To investigate aspects of the sporulation mechanism, here we focused on the E22 mutant, whose asci produced four spores, but the spores were apparently abnormal ( Figure 1A). Like other sporulation-deficient mutants, the E22 mutant formed an iodine staining-negative colony on sporulation medium ( Figure  1B). Under the differential interference contrast (DIC) microscope, spores from the E22 cells were dull in appearance as compared with spores from wild-type cells ( Figure 1A). Mating frequency and progression of meiotic nuclear division were normal in the E22 mutant as judged by the observation of nuclear staining. These data indicate that a late step of spore formation is defective in the E22 mutant.  In S. pombe sporulation, spore wall formation initiates after closure of the FSM [1]. Because the FSM is a double unit membrane, FSMs labelled by GFP-Psy1 are transiently observed as a double ring by fluorescence microscopy, and then the outer ring disappears. Coincidently, the outline of spores becomes clearly visible under a DIC microscope. Hereafter, we refer to spores whose outlines are clearly visible under a DIC microscope as mature spores.
We evaluated the efficiency of spore maturation in the E22 mutant. At 48 h after the induction of sporulation, most wild-type asci had mature spores. By contrast, only 12% of E22 asci had mature spores ( Figure 1A,C). Fluorescence microscopy revealed that most E22 asci included four prespores surrounded by GFP-Psy1, similar to wild-type asci, suggesting that prespore formation proceeds normally in E22 cells ( Figure 1D). However, most wild-type spores lost the outer FSM, while the majority of E22 mutant spores retained the outer FSM. To evaluate the efficiency of outer FSM breakdown, we roughly classified the asci into two types: those in which the outer FSM of any of the four spores was observed (type I); and those in which the outer FSM of all four spores disappeared (type II). We then determined the frequency of type II asci ( Figure 1E). At 48 h after the induction of sporulation, more than 80% of wild-type asci had completed outer FSM breakdown. In the E22 mutant, by contrast, the abundance of type II asci was less than 20% ( Figure 1A,E). Taken together, these data indicate that the E22 mutant is defective in breakdown of the outer FSM.

The E22 Mutation Is Located in the meu5 + Gene Encoding the RNA-Binding Protein Meu5
The gene responsible for the E22 mutation was identified by transforming the E22 mutant with a genomic library and isolating clones that showed restoration of the sporulation defect (see Materials and Methods). The resulting gene was identical to the previously characterized meu5 + / crp79 + gene [31,32], which encodes a protein of 710 amino acids with three RNA recognition motifs. A literature search revealed that meu5 + is upregulated during meiosis [31], while Meu5 binds to and stabilizes the transcripts of more than 80 genes [33]. Hereafter, we refer to the E22 mutant as meu5-E22.
Nucleotide sequence analysis demonstrated that the meu5-E22 mutant harbored a single nucleotide change (from T to G) in the second intron of the meu5 + gene (Figure 2A). The consensus sequence GTANG at the 5 end of the intron was mutated to GGANG in intron 2 of meu5-E22. To detect whether the splicing efficiency of intron 2 was reduced in meu5-E22, we conducted reverse transcription PCR analysis with primers encompassing intron 1 and intron 2 ( Figure 2A). Total RNA was prepared from sporulating cells of wild type and the meu5-E22 mutant. As expected, the splicing efficiency of intron 2 but not intron 1 was strikingly reduced in the meu5-E22 mutant ( Figure 2B). Thus, this single nucleotide mutation inhibits pre-mRNA splicing of intron 2 in the meu5-E22 mutant.
To examine whether the meu5-E22 mutant synthesizes Meu5 protein correctly, we constructed strains carrying a single chromosomal copy of either meu5 or the meu5-E22 gene tagged with three tandem copies of the HA epitope. In western blot analysis, no Meu5-E22-HA protein band was detected in the meu5-E22 mutant, whereas a Meu5-HA was detected in wild type ( Figure 2D). This result further suggested that the abnormal phenotype of meu5-E22 was caused by the absence of Meu5 protein.
In fact, the meu5∆ deletion strain exhibited essentially the same phenotype as meu5-E22, and the outer FSM breakdown defect of both mutants was restored by transformation with a plasmid overexpressing meu5 + ( Figure 2E). Considering these results, we concluded that the defect in outer FSM breakdown in the meu5-E22 mutant is indeed caused by a defect in Meu5 protein synthesis.

Meu5 Is Dispensable for Initiation of FSM Formation and Expansion of the FSM
As described above, prespores seemed to form normally in the meu5-E22 mutant ( Figure 1A). To confirm this, we observed the initiation of FSM formation and expansion of the FSM in wild-type and meu5∆ strains expressing GFP-Psy1 in more detail (Figure 3). Progression of meiosis was monitored by observing the formation and elongation of spindle microtubules. At metaphase II, four semicircle signals of GFP-Psy1 emerged from the vicinity of a nucleus and then elongated to encapsulate the nucleus. Most haploid nuclei produced by meiotic second divisions were encapsulated by the FSM in both wild-type ( Figure 3A) and meu5∆ mutant cells ( Figure 3B), and the processes were indistinguishable. Similar to meu5-E22, meu5∆ asci had prespores with a double ring of GFP-Psy1. Interestingly, in some of the meu5 mutant asci, the two outer FSMs of two spores were fused ( Figure S1). By contrast, this phenotype was not observed in wild-type asci. Together with the results of a previous study in which meu5∆ cells were able to mate and proceed through the meiotic divisions with normal kinetics [33], these data indicate that Meu5 is dispensable for the progression of meiosis and FSM expansion.

Meu5 Is Dispensable for Initiation of FSM Formation and Expansion of the FSM
As described above, prespores seemed to form normally in the meu5-E22 mutant ( Figure 1A). To confirm this, we observed the initiation of FSM formation and expansion of the FSM in wild-type and meu5∆ strains expressing GFP-Psy1 in more detail (Figure 3). Progression of meiosis was monitored by observing the formation and elongation of spindle microtubules. At metaphase II, four semicircle signals of GFP-Psy1 emerged from the vicinity of a nucleus and then elongated to encapsulate the nucleus. Most haploid nuclei produced by meiotic second divisions were encapsulated by the FSM in both wild-type ( Figure 3A) and meu5∆ mutant cells ( Figure 3B), and the processes were indistinguishable. Similar to meu5-E22, meu5∆ asci had prespores with a double ring of GFP-Psy1. Interestingly, in some of the meu5 mutant asci, the two outer FSMs of two spores were fused ( Figure  S1). By contrast, this phenotype was not observed in wild-type asci. Together with the results of a previous study in which meu5∆ cells were able to mate and proceed through the meiotic divisions with normal kinetics [33], these data indicate that Meu5 is dispensable for the progression of meiosis and FSM expansion.

Meu5 Is Involved in Breakdown of the Outer FSM
We used electron microscopy to investigate the spores in meu5∆ in more detail. First, we conducted quick-freeze deep-etch replica electron microscopy [34,35]. In this method, the specimen is frozen in less than a millisecond, and then fractured and exposed to etching and shadowing by platinum, enabling high-contrast images to be obtained [36,37]. As previously reported [38], wild-type spores were observed as a characteristic surface structure from which many protrusions projected outward ( Figure 4A,C). By contrast, meu5∆ spores exhibited a rather smooth surface ( Figure 4B,D).

Meu5 Is Involved in Breakdown of the Outer FSM
We used electron microscopy to investigate the spores in meu5∆ in more detail. First, we conducted quick-freeze deep-etch replica electron microscopy [34,35]. In this method, the specimen is frozen in less than a millisecond, and then fractured and exposed to etching and shadowing by platinum, enabling high-contrast images to be obtained [36,37]. As previously reported [38], wildtype spores were observed as a characteristic surface structure from which many protrusions projected outward ( Figure 4A,C). By contrast, meu5∆ spores exhibited a rather smooth surface ( Figure  4B,D). We also conducted thin-section electron microscopy using the freeze-substitution technique. As shown in Figure 5, both wild-type and meu5∆ asci had four spores, but the spore wall in meu5∆ was thicker than that in wild type. Consistent with the results of fluorescence microscopy and quickfreeze deep-etch replica electron microscopy, the outer membrane of the FSM persisted in meu5∆ spores ( Figure 5D,F). By contrast, wild-type spores were surrounded by the electron-dense structure of the Isp3 layer ( Figure 5C,E). It should be noted that, in meu5∆, the amount of Isp3 is severely reduced [33] and Isp3-GFP did not localize on the surface of spores by fluorescence microscopy (our unpublished data). Taken together, these data further support the notion that Meu5 is involved in breakdown of the outer membrane.  We also conducted thin-section electron microscopy using the freeze-substitution technique. As shown in Figure 5, both wild-type and meu5∆ asci had four spores, but the spore wall in meu5∆ was thicker than that in wild type. Consistent with the results of fluorescence microscopy and quick-freeze deep-etch replica electron microscopy, the outer membrane of the FSM persisted in meu5∆ spores ( Figure 5D,F). By contrast, wild-type spores were surrounded by the electron-dense structure of the Isp3 layer ( Figure 5C,E). It should be noted that, in meu5∆, the amount of Isp3 is severely reduced [33] and Isp3-GFP did not localize on the surface of spores by fluorescence microscopy (our unpublished data). Taken together, these data further support the notion that Meu5 is involved in breakdown of the outer membrane.

Cytochrome c Is Involved in Breakdown of the Outer FSM
Meu5 has many targets known to be involved in the progression of meiotic nuclear division and sporulation, including FSM expansion [33,[39][40][41]. Nevertheless, meu5∆ did not show any defects in these processes. To determine additional genes involved in breakdown of the outer layer, we observed the FSM in several S. pombe clones harboring a mutant of the target genes of Meu5. Of the 80+ targets of meu5 + including sporulation-related genes [33], we tested 60 mutants. Notably, cyc1∆ cells exhibited the most severe phenotype with regard to breakdown of the outer layer of the FSM (Table 1 and Figure 6). Notably, the role of cytochrome c, a highly conserved mitochondrial protein

Cytochrome c Is Involved in Breakdown of the Outer FSM
Meu5 has many targets known to be involved in the progression of meiotic nuclear division and sporulation, including FSM expansion [33,[39][40][41]. Nevertheless, meu5∆ did not show any defects in these processes. To determine additional genes involved in breakdown of the outer layer, we observed the FSM in several S. pombe clones harboring a mutant of the target genes of Meu5. Of the 80+ targets of meu5 + including sporulation-related genes [33], we tested 60 mutants. Notably, cyc1∆ cells exhibited the most severe phenotype with regard to breakdown of the outer layer of the FSM (Table 1 and Figure 6). Notably, the role of cytochrome c, a highly conserved mitochondrial protein in eukaryotes and most prokaryotes, in sporulation remains unknown. Interestingly, a comparison of DIC images with fluorescence images revealed that spores were clearly visible in most cyc1∆ asci even though the outer FSM remained present (Figure 6), suggesting that outer FSM breakdown is independent of the formation of "visible spores" under a DIC microscope.  To examine whether meu5 + and cyc1 + are necessary for the formation of viable spores, we measured the colony-forming ability of their mutant asci ( Figure S2). Both meu5∆ and cyc1∆ asci formed less colonies than wild type, suggesting that these genes are related to forming viable spores.

Localization of Isp3 to the Spore Periphery Precedes Outer Membrane Breakdown
In S. cerevisiae, breakdown of the outer membrane occurs after completion of the glucan layer [20]. Lastly, we investigated the timing of outer membrane breakdown in S. pombe. Interestingly, fluorescence microscopy revealed that the double ring of the mCherry-Psy1 signal persisted even when the Isp3-GFP ring had formed in S. pombe ( Figure 7A), suggesting the possibility that breakdown of the outer FSM layer occurs after formation of the Isp3 layer. As described above, the amount of Isp3 is severely reduced in meu5∆ [33], therefore we used cyc1∆ cells simultaneously expressing mCherry-Psy1 and Isp3-GFP to observe this process. The Isp3-GFP signal almost completely overlapped with the outer ring of FSM in cyc1∆ spores ( Figure 7B), supporting this possibility.

Localization of Isp3 to the Spore Periphery Precedes Outer Membrane Breakdown
In S. cerevisiae, breakdown of the outer membrane occurs after completion of the glucan layer [20]. Lastly, we investigated the timing of outer membrane breakdown in S. pombe. Interestingly, fluorescence microscopy revealed that the double ring of the mCherry-Psy1 signal persisted even when the Isp3-GFP ring had formed in S. pombe ( Figure 7A), suggesting the possibility that breakdown of the outer FSM layer occurs after formation of the Isp3 layer. As described above, the amount of Isp3 is severely reduced in meu5∆ [33], therefore we used cyc1∆ cells simultaneously expressing mCherry-Psy1 and Isp3-GFP to observe this process. The Isp3-GFP signal almost completely overlapped with the outer ring of FSM in cyc1∆ spores ( Figure 7B), supporting this possibility.

Discussion
Similar to nuclear and mitochondrial membranes, the precursor of the spore membrane (the FSM in S. pombe and the prospore membrane in S. cerevisiae) comprises two units, and the spore wall is formed between these two units. It has been shown that, during or after formation of the glucan layer in S. cerevisiae, the outer prospore membrane breaks down and the chitosan layer is subsequently synthesized [20]. Electron microscopy has also revealed that, in S. pombe, the outer FSM disappears during spore wall formation [1]. Therefore, it is of interest to know how breakdown of the outer membrane occurs and how it is achieved without damaging the forming spore. In this study, we have provided evidence that, in contrast to S. cerevisiae, the outer FSM breaks down after or coincident with the formation of the outermost Isp3 layer in S. pombe. Why is the timing of the outer membrane breakdown different between the two yeasts? The glucan layer of the S. cerevisiae spore wall is dominated by β-glucans, whereas the S. pombe spore wall contains both αand β-glucans. Furthermore, previous studies have indicated that the chitin/chitosan content is lower in S. pombe than in S. cerevisiae spore walls, although chitin synthases and chitin deacetylase are important for proper sporulation [15][16][17]. The most significant difference between the spore wall of the two yeasts is the composition of the outermost layer: in S. cerevisiae, the major constituent of this "dityrosine layer" is the modified, cross-linked diamino acid N-N-bisformyl-dityrosine [42,43], whereas S. pombe spores are coated by a proteinaceous surface layer comprising mainly Isp3 [18,19]. Thus, we presume that these differences may determine the timing of outer membrane breakdown.
We also identified two genes, meu5 + and cyc1 + , involved in breakdown of the outer membrane. A previous RNA-binding protein immunoprecipitation assay revealed that Meu5 binds to and stabilizes more than 80 target transcripts including the cyc1. [33]. Meu5 was originally isolated as an auxiliary mRNA export factor [32], but Meu5 is not directly involved in the export of the cyc1 mRNA [33]. Therefore, it is more likely that Meu5 is involved in the stabilization rather than the export of the cyc1 mRNA.
The meu5∆ mutant did not show a defect in the progression of meiosis or FSM expansion, but was deficient in spore maturation (Figure 3; [33]). Because Meu5 is an RNA-binding protein, we presumed that targets of Meu5 might be directly involved in outer membrane breakdown. Unexpectedly, however, only cyc1∆ cells showed a significant defect in outer membrane breakdown among 60 strains harboring a disruption of a target gene of Meu5 (Table 1). Cytochrome c is a small hemeprotein that is loosely associated with the inner membrane of the mitochondrion and is essential for the electron transport system. In S. pombe, cyc1 + is not essential for cell viability in normal growth; however, cyc1∆ cells show a deficiency in respiration and loss of viability in the stationary phase [44,45]. As a mitochondrial protein involved in the electron transfer system, cytochrome c does not seem to be directly involved in membrane breakdown. Indeed, how the mitochondrion is involved in sporulation is little known. In fact, most cyc1∆ cells appeared to proceed through normal meiosis and expansion of the FSM. In addition, cyc1∆ cells formed visible spores with a clear spore boundary (mature spores), which also suggests that spore maturation is independent of breakdown of the outer FSM (Figures 6 and 7). In S. cerevisiae, efficient segregation of mitochondria into spores requires a component of a protein coat found at the leading edge of the prospore membrane [46]; however, it remains to be determined how mitochondrial function is involved in spore formation.
What is the physiological significance of the breakdown of the outer membrane? The release of spores from the ascus was defective or significantly delayed in meu5∆ cells [33], suggesting the possibility that outer membrane breakdown is important for spore release. Effective spore release may be important for the spread of spores in the natural environment. Interestingly, mutual adherence between two neighboring spores was occasionally observed in the meu5 mutant ( Figure S1), supporting the possibility.
We also directly measured the viability of meu5∆ and cyc1∆ asci. Many of them were able to germinate, although to some extent they were less viable than those of wild type ( Figure S2), suggesting the possibility that membrane breakdown is important for proper formation of the spore wall and spore germination. Notably, however, mutation of neither isp3 + nor mde10 + , both targets of Meu5 and responsible for spore wall formation, affected spore viability [19,38]. Moreover, breakdown of the membrane occurred in these mutants (Table 1 and Figure 6). Given that both Meu5 and cytochrome c are involved in various cellular processes, it is unlikely that the reduced spore viability in these mutants directly reflects the ability to form spore walls.
In the present study, we did not identify genes directly involved in breakdown of the outer membrane. In the late stage of sporulation, vacuoles are known to fuse extensively to form a few large membranous compartments that occupy the whole cytoplasm [47]. It is possible that the vacuolar membrane may directly contact the outer FSM and degrade it. Isolation of factors directly involved in breakdown of the outer membrane will be a future challenge.