CRR1, a gene encoding a putative transglycosidase, is required for proper spore wall assembly in Saccharomyces cerevisiae

Francisco Gómez-Esquer1,{dagger}, Jose M. Rodríguez-Peña1, Gema Díaz1,{dagger}, Estefanía Rodriguez1, Peter Briza2, César Nombela1 and Javier Arroyo1

1 Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain
2 Department of Molecular Biology, University of Salzburg, A-5020 Salzburg, Austria

Correspondence
Javier Arroyo
jarroyo{at}farm.ucm.es


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In Saccharomyces cerevisiae, sporulation is a developmental process that converts a single cell into four haploid spores. The four haploid nuclei are encapsulated within multilayered spore walls that protect them against stressful conditions. The formation of the spore-specific cell wall is a highly coordinated process that requires the participation of enzymic activities for biosynthesis, degradation, and cross-linking between components. Here the sporulation-specific gene CRR1, encoding a putative transglycosidase that is required for proper spore wall assembly, is described. Both the transcription of CRR1 and the synthesis of Crr1p were induced biphasically under sporulating conditions, with a first expression peak displaying kinetics similar to those of the middle to middle-late sporulation-specific genes, and a second late peak after 24 h under these conditions. Localization studies revealed that Crr1p localized to the spore wall that surrounds each of the four ascospores within the mature asci. Mutation of this gene had no effect on the efficiency of spore formation. However, crr1 mutant spores were sensitive to hydrolytic enzymes such as glusulase and to heat-shock treatments, underscoring the importance of this gene in the proper formation and assembly of the ascospore wall. Moreover, the deletion of CRR1 had additive effects with respect to the sensitivity of cda1 cda2 mutants to these treatments. Interestingly, overexpression of CRR1 not only complemented the phenotype of the crr1 strain but also rendered spores more resistant to the stress conditions than the wild-type. Like other mutants impaired in the formation of the spore outer layer, crr1 mutants were permeable to Calcofluor White. Finally, detailed analysis by electron microscopy of the spore walls in the crr1 mutants revealed a defect in the assembly of the spore wall components, suggesting a role for Crr1p in the cross-linking between the inner (glucan/mannoprotein) and the outer (chitosan/dityrosine) spore layers.


Abbreviations: GFP, green fluorescent protein; SFH, short flanking homology

A video image of the three-dimensional localization of Crr1–GFP in the spore, showing the rotation around the z axis, is available as supplementary data with the online version of this paper (at http://mic.sgmjournals.org).

{dagger}Present address: Facultad de Ciencias de la Salud, Universidad Rey Juan Carlos, 28922 Madrid, Spain.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In Saccharomyces cerevisiae, meiotic development (sporulation) is induced when diploid a/{alpha} cells are starved of essential nutrients in the presence of a non-fermentable carbon source (Kupiec et al., 1997). This process is controlled by the master regulatory genes RME1, IME1 and IME2 (Mitchell, 1994), and involves a coordinated series of genetic and morphological processes, including all the events necessary for meiosis, spore wall formation and packaging of the four meiotic products into an ascus (for a review, see Kupiec et al., 1997). All these events depend on the sequential activation of temporally distinct classes of sporulation-specific genes, classified as early, middle, mid-late and late (Kupiec et al., 1997; Mitchell, 1994). This transcriptional programme has been now fully characterized at the genomic level by the use of DNA microarrays (Chu et al., 1998; Primig et al., 2000).

The sporulation programme begins when cells exit the mitotic cycle and enter the meiotic prophase. The meiotic prophase is followed by the meiosis I reductional and meiosis II equational divisions. Spore wall morphogenesis initiates with the outgrowth of the prospore wall, a double-membrane structure formed from the outer plaques of each meiosis II spindle pole body (Deng et al., 1993; Knop & Strasser, 2000). As meiosis progresses, the prospore membrane extends along the outer surface of the nuclear envelope, under the control of specific septins (Fares et al., 1996), and engulfment of the four haploid meiotic products, together with some portions of the mother-cell cytoplasm and organelles, occurs. Deposition of the spore wall components in the luminal space between the two layers of the prospore wall leads to the formation of mature spore walls (Guth et al., 1972; Lynn & Magee, 1970; Moens & Rapport, 1971; Neiman, 1998).

Analysis of mature spore walls by electron microscopy reveals the presence of four layers (Kreger-van Rij & Veenhuis, 1970). The two inner layers, consisting mainly of glucans and mannoproteins, are similar in morphology and chemical composition to that of the vegetative cell wall (Briza et al., 1988; Katohda et al., 1984). The two outer layers are thought to contribute to the mechanical rigidity and the resistance to chemical and enzymic attack of the spore wall (Briza et al., 1988, 1990b), and hence confer the protective nature of this structure to adverse environmental conditions. The main component of the third layer is chitosan (Briza et al., 1988), although chitin is also present. Chitosan, a 1,4-{beta}-D-glucosamine homopolymer, is produced by deacetylation of chitin chains (Kafetzopoulos et al., 1993a), this reaction being catalysed by two chitin deacetylase isozymes encoded by the CDA1 and CDA2 genes, respectively (Christodoulidou et al., 1996; Kafetzopoulos et al., 1993b), whose deletion affects ascospore wall assembly (Christodoulidou et al., 1999). The outermost layer of the spore wall is mainly composed of dityrosine. Dityrosine forms an insoluble macromolecule containing a high number of cross-linked tyrosine residues in its LL-, DL, and DD configurations (Briza et al., 1986, 1990b). This polymer is synthesized in a two-step process catalysed by the products of the genes DIT1 and DIT2 (Briza et al., 1994, 1996), with the formation of N-formyl tyrosine and N,N'-bis-formyl dityrosine in the first and second steps, respectively. Dtr1p, a sporulation-specific member of the major facilitator superfamily involved in multidrug resistance, has recently been shown to be involved in the transport of bis-formyl dityrosine from the cytoplasm of the prospore to the spore wall (Felder et al., 2002).

Events of spore wall assembly are controlled by regulatory proteins. Swm1p, a protein localized to the nucleus during the sporulation process (Ufano et al., 1999), together with Sps1p and Smk1p, members of the Smk1p-MAP kinase signalling pathway (Neigeborn & Mitchell, 1991), are required for full expression of the mid-late and late genes during the sporulation programme. The timing of synthesis of the different layers is controlled by these regulatory proteins. The execution of the distinct sporulation events, like the formation of the glucan, chitosan and dityrosine layers, requires distinct Smk1p activity thresholds (Wagner et al., 1999).

With knowledge of the S. cerivisiae genome sequence, many new genes potentially involved in spore morphogenesis have been pinpointed. As well as the classic genetic screenings used to identify contributors to the meiotic pathway in S. cerevisiae, genomic-based screenings (Briza et al., 2002; Enyenihi & Saunders, 2003; Rabitsch et al., 2001) have been performed, leading to the identification of many genes involved in this process. However, many sporulation genes still remain to be characterized.

Here, we describe the characterization of CRR1. This gene belongs to a family of cell-wall-related genes, recently characterized in our laboratory, that includes CRH1, CRH2 and CRR1 (Rodriguez-Peña et al., 2000, 2002). The products encoded by CRH1 and CRH2 are GPI cell wall proteins involved in the cross-linking between cell wall polymers, probably glucan and chitin, at different stages of the S. cerevisiae vegetative cell cycle (Rodriguez-Peña et al., 2000). CRR1 is poorly expressed under vegetative growth, but highly expressed under sporulation conditions (Rodriguez-Peña et al., 2000). The existence of a catalytic domain conserved between CRH1, CRH2 and CRR1 suggests a role for this gene in spore wall construction. As deduced from the results presented here, CRR1 is required for proper spore wall assembly during spore formation.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains and media.
The Saccharomyces cerevisiae strains used in this study are listed in Table 1. For routine experiments, yeast cells were grown in YEPD (1 % yeast extract, 2 % peptone, 2 % glucose). To induce sporulation, diploid cells were grown in YEPD for 12 h, transferred to YEPA medium (0·5 % yeast extract, 0·6 % yeast nitrogen base, 0·5 % peptone, 1 % potassium acetate, 1·02 % potassium biphthalate, pH 5·5), and adjusted to an optical density of 0·1 at 600 nm. The cells were grown for at least three generations and harvested by centrifugation when the cellular concentration had reached 1–2x107 cells ml–1, washed twice with sporulation medium (1 % potassium acetate), and resuspended at 1·5x107 cells ml–1 in the same sporulation medium supplemented with the appropriate auxotrophic requirements. After 48 h of incubation at 30 °C, ascus formation was monitored by phase-contrast microscopy. Sporulation rates (expressed as the number of asci divided by the total number of cells) of 55 and 85 % were found in the FY1679 and YPA24 strains, respectively. The Escherichia coli strain used as the plasmid host was DH5{alpha} [supE44 {Delta}lacU169 ({pi}80 lacZ{Delta}M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1]. For selective growth, bacteria were grown on Luria–Bertani (LB) medium containing 100 mg ampicillin l–1.


View this table:
[in this window]
[in a new window]
 
Table 1. Yeast strains used in this study

 
Yeast genetics and phenotypic analyses.
Tetrad analyses were performed by standard micromanipulation procedures. Markers of the segregants were verified on SD (2 % glucose, 0·16 % yeast nitrogen base without amino acids, 0·5 % ammonium sulfate, plus the appropriate amount of amino acids) plates lacking a particular amino acid. The sensitivity or resistance of the segregants to geneticin (encoded by the KanMX4 module) or hygromycin B (encoded by the hphMX4 module) was monitored on YEPD plates containing 200 mg geneticin l–1 (Gibco-BRL) or 300 mg hygromycin B l–1 (Roche Diagnostics), respectively. The sensitivity of asci to heat-shock treatment (55 °C) or to enzymic digestion (glusulase) was determined as described by Briza et al. (1990a). Briefly, in the case of glusulase treatment, about 2x107 cells were collected from the sporulation medium after 72 h of growth and transferred to 100 µl undiluted glusulase (type H-2; Sigma). At several times during incubation at room temperature, 50 µl aliquots were taken from the cell suspension and plated onto YEPD plates. To determine heat tolerance, cell cultures were kept at 4 °C for 12 h and diluted to a density of 3x106 cells ml–1 in distilled water. Then, 1 ml of the cell suspension was transferred to an Eppendorf tube, which was immersed in a water bath at 55 °C. Aliquots (100 µl) were taken at different times during heat treatment and plated onto YEPD plates. For both phenotypic studies, the plates were incubated for 2 days at 30 °C before the number of colonies was counted. In parallel, cell survival to heat shock at 55 °C was measured at different times by spotting aliquots of cells onto YEPD plates.

Plasmid and deletant strain construction.
Standard molecular biology techniques for DNA manipulations and bacterial transformations were used as described elsewhere (Sambrook et al., 1989). In the construction of the haploid CRR1 deletant strains (FG01 and FG02), the CRR1 complete ORF was deleted, except for the start and stop codons, by using the short flanking homology (SFH) PCR technique (Wach et al., 1997), which allowed the replacement of the target ORF by the hphMX4 selection marker from plasmid pAG32 (Goldstein & McCusker, 1999). The CR1 and CR2 primers devised for this purpose are listed in Table 2. The SFH deletion cassette was obtained using the Expand High Fidelity PCR System (Roche Diagnostics). ORF replacement was identified by PCR with the help of diagnostic primers that bind either outside the target ORF (V2 and V3) or within the selection marker (HIG1 and HIG2). Finally, both crr1 strains were mated to generate the homozygous crr1{Delta} diploid strain FG10 (see Table 1).


View this table:
[in this window]
[in a new window]
 
Table 2. Oligonucleotides used in this study

 
In order to generate strains lacking CDA1 and CDA2 ORFs (FG03 and FG04, respectively), we took advantage of the adjacent localization of both ORFs (CDA1 and CDA2) in the yeast chromosome XII to delete them simultaneously using a unique selection marker. The deletion cassette, containing the KanMX4 marker that confers resistance to geneticin, was obtained by means of the SFH technique using the primers CDASFW and CDASRV (see Table 2) and plasmid pFA6a-KanMX4 (Wach et al., 1994) as a template. ORF replacement was identified by PCR with the help of the diagnostic primers CDASver1 and CDASver2, which bind outside the target ORFs, and K2 and K3 primers, which bind within the selection marker. Eventually, haploid strains were mated to generate the homozygous cda1 cda2 diploid strain FG20. The same strategy was used to generate the haploid crr1 cda1 cda2 MATa and MAT{alpha} strains (FG05 and FG06, respectively) and the homozygous crr1 cda1 cda2 diploid strain FG30.

Cloning of the CRR1 gene.
The complete CRR1 ORF plus 598 bp and 479 bp of upstream and downstream sequences, respectively, was amplified from genomic DNA (FY1679 strain) by PCR using the primers YLRup and YLRdw (see Table 2). The amplification parameters were 10 min at 97 °C and then 1 min at 94 °C, 1 min at 53 °C, 2 min at 72 °C, for 30 cycles. The PCR product of 2389 bp was cloned into the pGEM-T vector (Promega) (pJP1 plasmid). Sequence verification was carried out by sequencing both DNA strands using the walking-primer strategy on an ABI 377 automated DNA sequencer (Applied Biosystems). Plasmid pJV21C was constructed by ligating the SacII/SacI insert from pJP1 into the SacII/SacI-cleaved pRS416 vector (centromeric vector; URA3; Ampr) (Sikorski & Hieter, 1989). The restriction enzymes used in this work were provided by Roche Diagnostics.

Construction of GFP and MYC fusions.
In order to create a fusion protein of Crr1p with the green fluorescent protein (GFP) from Aequorea victoria, we took advantage of a GFP cassette flanked by two SpeI restriction sites generated previously (Rodriguez-Peña et al., 2000, 2002). We performed site-directed mutagenesis of CRR1 to create an artificial SpeI restriction site. Plasmid pJV21C containing the CRR1 ORF was used as a template for PCR. Primers were designed to bring about the change from ACTCAA to ACTAGT (SpeI recognition sequence) at the 3' end of the CRR1 ORF (24 bases from the stop codon). Briefly, two PCRs were run in parallel using the primer pairs YLR1/YLR2 and YLR3/YLR4 (see Table 2). Both the YLR1–YLR2 (496 bp) and YLR3–YLR4 (388 bp) PCR products were used as overlapping templates in a second PCR with YLR1 and YLR4 as external primers. Thus, a final product of 864 bp was generated, corresponding to a mutated internal fragment of the ORF. This PCR product was verified by sequencing and subcloned into KpnI-cleaved pJV21C, producing plasmid pJV21D, hence replacing the wild-type sequence with the mutant sequence. The GFP cassette was SpeI-digested and introduced in-frame into pJV21D to give pJV21F. Finally, the multi-copy plasmid pJV21G was constructed by ligating the XbaI/EcoRI insert from pJV21F into the XbaI/EcoRI-cleaved Yep352 vector.

To obtain a C-terminal 6Myc-tagged version of Crr1p, plasmid pRS306CRR1m was constructed. The complete CRR1 ORF, except for a triplet encoding the stop codon, was amplified with the BamHI-containing primers CRR1MYCFV and CRR1MYCRV (Table 2). The PCR product was digested with BamHI, and the resulting fragment was introduced into plasmid pRS306m (6-Myc epitope in the integrative URA3 vector pRS305; Sikorski et al., 1989) to generate pRS306CRR1m. To integrate CRR1-6myc into the CRR1 locus, pRS306CRR1m was linearized by AspI digestion and transformed in a W303-1A strain. The heterozygous diploid strain (FG40) was constructed by crossing W303-1A 6Myc-Crr1 (FG35) with the wild-type strain from the opposite mating type ({alpha}131-20).

Microscopy techniques.
For phase-contrast, fluorescence and indirect immunofluorescence microscopy, cells were examined with an Eclipse TE2000U microscope (Nikon). Digital images were acquired with an Orca C4742-95-12ER charge-coupled device camera (Hamamatsu) and Aquacosmos Imaging Systems software. For Calcofluor staining, cells were incubated for 1 min in the presence of 10 µg Calcofluor White ml–1.

For visualization of the GFP–Crr1 fusion protein, cells cultured in sporulation medium for 24 h were harvested by gentle centrifugation, washed twice with PBS, and finally resuspended in PBS. Samples were observed with an Eclipse TE-300 microscope (Nikon) attached to a Bio-Rad MRC1024 confocal system.

For electron microscopy, samples were prepared according to the protocol described by Miret et al. (1992). Briefly, about 2x107 cells were collected after 24 h of incubation in sporulation medium and fixed with 500 µl fixative solution (sodium cacodylate buffer, pH 7·2, supplemented with 1·5 % glutaraldehyde and 2 % paraformaldehyde), and then incubated at 4 °C for 24 h. Samples were then washed, and treated with 1 % potassium permanganate for 90 min. Fixed cells were dehydrated through a graded series of ethanol and embedded in Embed 812 resin (Electron Microscopy Science). Thin sections were stained and examined with a Zeiss EM902 electron microscope.

Quantification of mRNAs using real-time quantitative RT-PCR.
Total RNA was isolated from cells (1·3x109), collected at different time intervals after transfer to sporulation medium, by the acidic phenol method, as described previously (Ausubel et al., 1993). First-strand cDNAs were synthesized from 2 µg total RNA, using the Reverse Transcription System (Promega), following the recommendations of the manufacturer. As a control for genomic contamination, the same reactions were performed in the absence of reverse transcriptase. Real-time PCR was performed using an ABI 7700 instrument (Applied Biosystems) in a final volume of 25 µl containing 5 µl of a 25-fold dilution of the reverse transcription reaction and 12·5 µl of the 2x TaqMan Universal PCR Master Mix (Applied Biosystems), together with the specific forward and reverse primers (Sigma) and the corresponding gene-specific TaqMan probe (FAM and VIC labelled for CRR1 and ACT1, respectively), designed using Primer Express Software 2.0 (Applied Biosystems). Real-time PCR conditions were selected according to the universal conditions (default conditions) recommended by the manufacturer of the instrument. Each cDNA was assayed in at least duplicate PCR reactions. Basic analysis was performed using the SDS 1.9.1 software (Applied Biosystems). For relative quantification, the abundance of each gene (CRR1 and ACT1) was determined by preparing standard curves (four fivefold serial dilutions) for each gene from a common cDNA sample. For all experimental samples, the target quantity was determined from the standard curve and was divided by the target quantity of the calibrator (reference sample). Thus, the calibrator (t=0) became the 1x sample and all other quantities were expressed as an n-fold difference relative to the calibrator. In addition, amplification of ACT1 (endogenous control) was used to standardize the amount of sample added to each reaction mixture. The following probes and forward/reverse primers were used: ACT1, 5'-VIC-CATGAAGGTCAAGATCATTGCTCCTCCAG-TAMRA-3', 5'-ATCACCGCTTTGGCTCCAT-3'/5'-CCAATCCAGACGGAGTACTTTCTT-3'; and CRR1, 5'-FAM-TCGATTTCACACATAGCGGCTACACATCA-TAMRA-3', 5'-GAGGCAGAGAAAATGTTGGAAGA-3'/5'-TTCCCGCTACTCGCTTCAA-3'.

Preparation of yeast extracts and immunoblot analysis.
Cell collection (20 ml at different time intervals after transfer to sporulation medium), lysis, collection of proteins, fractionation by SDS-PAGE and transfer to nitrocellulose membranes were performed as described by Martin et al. (2000). Detection of actin and Myc-tagged proteins was carried out using mouse anti-actin monoclonal antibody C4 (ICN Biomedicals) and mouse anti-c-myc monoclonal antibody 9E10 (Covance Richmond), respectively.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Sporulation-specific expression of YLR213c (CRR1)
We had previously identified CRR1 as a member of the putative family of transglycosidases which includes CRH1, CRH2 and CRR1 (Rodriguez-Peña et al., 2000). In that work, we characterized the role of CRH1 and CRH2 in the cell wall construction process under vegetative growth conditions, and we showed that CRR1 was mainly expressed under the meiosis developmental programme, suggesting a role for this gene in the construction of the spore wall. Here, to characterize the expression of CRR1 under sporulation conditions more precisely, wild-type YPA24 cells were grown in pre-sporulation media, transferred to sporulation media, and incubated for 24 h at 30 °C. The expression of CRR1 was monitored at different times by means of real-time PCR, as described in Methods. As shown in Fig. 1(a), CRR1 was activated 6 h after transfer of the cells to the sporulation medium, with a peak of expression at about 9 h: at this time CRR1 was induced about threefold with respect to t=0. The induction subsequently declined (10–14 h), disappearing after 15 h. This pattern of expression, characteristic of genes expressed midway through the sporulation process (mid to mid-late genes), almost coincides with the pattern of expression reported for this gene in the two meiotic transcriptome analyses already carried out (Chu et al., 1998; Primig et al., 2000). These authors monitored expression until 12 h. However, here we extended the analysis until 24 h, detecting a second peak of expression between 18 and 24 h, with a maximum sixfold induction, indicating the biphasic expression of CRR1.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 1. (a) Time-course of CRR1 transcription. Levels of CRR1 mRNA were measured by quantitative RT-PCR at the times indicated after transfer of the cells to the sporulation medium. Relative CRR1 mRNA levels are indicated with respect to t=0 and normalized by ACT1 mRNA levels measured at the same intervals. (b) Time-course of Crr1p induction during sporulation. Samples of strain YPA24 carrying Crr1p–Myc were removed from the sporulation medium at the times indicated, and extracts were made up and analysed by Western blotting using anti-Myc antibodies. The protein load was monitored using anti-actin antibodies. The relative levels of Crr1p–Myc with respect to actin protein levels are represented in the graphics. MM, molecular mass.

 
To examine the CRR1 gene product, an epitope-tagged allele was constructed by fusing a sequence encoding six copies of the c-myc epitope to the 3' end of the coding region. The original CRR1 allele in the YPA24 strain was replaced by this epitope-tag allele (see Methods), and the levels of Crr1p–Myc were followed through the sporulation process by Western blotting of cell extracts using anti-c-myc antibodies. As shown in Fig. 1(b), the protein was detected 10 h after transfer to the sporulation medium, with a first peak of expression after 12 h. The levels of the protein decreased to undetectable levels after 17 h, but then increased again after 20 h, with a second peak of maximum expression 24 h after transfer to sporulating conditions, in accordance with the transcriptional activation of CRR1 at this time. Therefore, the levels of Crr1p during the sporulation process are in agreement with the transcriptional activation described above, suggesting that the induction of Crr1p is basically regulated at the transcriptional level.

Crr1p localizes to the spore wall envelope
To elucidate the possible role of Crr1p during sporulation, we performed localization studies using a fusion protein in which a GFP had been inserted at the C-terminus of Crr1p. Cells expressing Crr1–GFP were obtained by transformation of the FY1679 strain with plasmid pJV21G. This is a multi-copy plasmid in which the transcription of CRR1–GFP is controlled by the CRR1 promoter. Forty-eight hours after transfer to sporulation conditions, in mature spores, the Crr1–GFP protein localized to the spore wall, surrounding the four haploid asci (Fig. 2a). This pattern was not observed in cells transformed with the empty vector (data not shown). Confocal analysis of the spores expressing Crr1–GFP revealed the three-dimensional aspect of this localization (Fig. 2b). This localization pattern suggests that Crr1p may be required for spore wall synthesis during sporulation.



View larger version (63K):
[in this window]
[in a new window]
 
Fig. 2. Localization of Crr1p in mature spores. A homozygous crr1{Delta} strain, transformed with the plasmid pJV21G expressing the Crr1p–GFP fusion protein, was sporulated. After 48 h growing under sporulating conditions, cells were analysed by confocal microscopy (a). (b) Confocal images obtained with a distance between stacks of 0·2 µm were used to reconstruct the three-dimensional localization of Crr1–GFP in the spore. A video image showing the rotation around the z axis of the three-dimensional reconstructed image is available as supplementary data with the online version of this paper at http://mic.sgmjournals.org. Four slides corresponding to different grades of rotation of the reconstructed three-dimensional confocal image of spores expressing Crr1–GFP are shown in (b).

 
Mutation of CRR1 leads to spores sensitive both to lytic enzymes and to stress conditions
To determine whether CRR1 plays a role in spore morphogenesis, diploid homozygous mutants in this gene were tested for their ability to form spores. A crr1{Delta} homozygous diploid strain and its isogenic wild-type strain (FY1679) were transferred to sporulation medium, and their ability to form ascospores was monitored by phase-contrast microscopy. The efficiency of spore formation was not affected by the mutation, since mutant strains in CRR1 sporulated as efficiently as wild-type cells, with a percentage of cells with normal ascospores of about 55 % after 72 h.

The resistance of spores to environmental stress mainly depends on the presence of an intact cell wall (Briza et al., 1990a; Pammer et al., 1992). On the basis of the possible participation of CRR1 in spore wall construction, we examined the spore thermotolerance of the wild-type and mutant crr1{Delta} after exposure to 55 °C for different periods of time, as well as the sensitivity of the spores to the lytic enzyme glusulase. As shown in Figs 3(a) and 3(c), mutant spores were more sensitive to heat shock than wild-type spores. Thirty minutes of exposure to heat shock reduced viability by two orders of magnitude in the mutant strain compared to the wild-type, while no viable spores were detected in the mutant strain after 60 min of temperature treatment (Fig. 3c).



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 3. Phenotypic characterization of the ascospores from the crr1/crr1 mutant strain. Wild-type cells ({circ}), the isogenic crr1{Delta} strain ({square}) and the crr1{Delta} strain transformed with the plasmid pRS416-CRR1 ({blacklozenge}) from FY1679 background (a, c and d) and YPA24 background (b) were incubated in sporulation medium for 24 h at 28 °C and then assayed for plating efficiencies after exposure to 55 °C (a, b and c) or to glusulase (d) at the indicated times (see Methods). Data in (a), (b) and (d) are indicated as a percentage of the initial survival rate (t=0). Tolerance of the spores to 55 °C was also measured in the YPA24-derived strains indicated (c) by spotting cells in YEPD after heat shock at the different times indicated. For each strain, serial 1 : 10 dilutions of untreated cells were spotted as a control for cell growth. WT, wild-type.

 
A single-copy plasmid containing the complete ORF of YLR213c (pRS416-CRR1) restored the temperature-sensitive phenotype of the mutant strain to the wild-type (Fig. 3a, c), indicating that the absence of CRR1 was indeed responsible for the phenotype. Furthermore, a multi-copy plasmid containing the CRR1 gene (Yep352-CRR1) not only restored the phenotype of the mutant strain but even rendered these spores clearly more resistant to heat shock than the wild-type ones (Fig. 3c).

The germination efficiency after different times of treatment with the lytic enzyme glusulase was also determined. As shown in Fig. 3(d), this efficiency was reduced from nearly 100 % in the wild-type to 65 % in the crr1{Delta} spores after 45 min of treatment, and from 90 % to 50 % after 2 h in the presence of the enzyme. This phenotype was complemented by the expression of CRR1 from a multi-copy plasmid (Fig. 3d). To rule out any strain-specific effect, we carried out the deletion of CRR1 in the YPA24 strain (San Segundo et al., 1993). In this strain, the deletion led to phenotypes identical to those observed in the original strain background, both for the heat-shock (Fig. 3b) and glusulase treatments (data not shown).

Taken together, these results support the idea that ascospore wall maturation does not proceed properly in the crr1/crr1 mutant. Based on these results, we looked for genetic interactions between CRR1 and other genes involved in spore wall construction. For this purpose, we constructed triple-deleted strains in CRR1, CDA1 and CDA2. CDA1 and CDA2 code for the two chitin deacetylases involved in the synthesis of chitosan, and strains mutated in both genes are defective in the synthesis and assembly of the outermost spore wall chitosan and dityrosine layers (Christodoulidou et al., 1999). The sensitivity of these mutants to temperature shock (55 °C) was tested. As shown in Fig. 4, deletion of CDA1 and CDA2 slightly increased the sensitivity of the crr1 mutant strain at 55 °C, again suggesting a role for Crr1p in spore wall assembly.



View larger version (37K):
[in this window]
[in a new window]
 
Fig. 4. Genetic interactions of CRR1 with CDA1 and CDA2. YPA24 wild-type and the isogenic crr1{Delta} (FG10), cda1{Delta} cda2{Delta} (FG20) and crr1{Delta} cda1{Delta} cda2{Delta} (FG30) strains were grown under sporulating conditions for 24 h. Then, cell survival after exposure to 55 °C at the indicated times was measured by spotting cells in YEPD (Methods). Serial 1 : 10 dilutions of untreated cells of each strain were spotted as a control for cell growth.

 
crr1{Delta} spores have cell wall defects
To detect the chitosan layer it is possible to stain cells with Calcofluor White, which specifically stains chitin. Calcofluor stains sporulating cells before spore wall maturation. However, this staining is not observed in mature spores because the dye is unable to permeate through the mature outer spore wall unless the spore wall is damaged or missing. This is because the dityrosine layer on the spore surface prevents the dye from entering the spore wall and binding to the chitosan layer. Thus, staining of wild-type sporulating cells with Calcofluor resulted in the staining of less than 3 % of the mature spores (Fig. 5a). However, more than 40 % of the asci of the crr1 mutant strain contained spores whose chitosan layer became accessible for the dye and therefore displayed fluorescence after Calcofluor staining, indicating an alteration of their surface layer (Fig. 5b).



View larger version (60K):
[in this window]
[in a new window]
 
Fig. 5. Calcofluor staining of wild-type and crr1/crr1 diploid cells under sporulating conditions. Wild-type YPA24 cells (a) or mutant crr1/crr1 isogenic strain (FG10) (b) were collected 24 h after transfer to sporulation conditions, stained with Calcofluor White as described in Methods, and visualized by fluorescence microscopy.

 
To explore the spore wall defect in greater depth, the structure of the mutant spore walls in cells grown under sporulation conditions for 48 h was compared by electron microscopy with that of wild-type spore walls. Electron micrographs representative of wild-type asci are shown in Fig. 6(a). The innermost layer, appearing as an electron-transparent layer, is similar to the vegetative cell wall and contains glucans and mannoproteins (Briza et al., 1988; Katohda et al., 1984). The outermost layer consists of dityrosine, which forms highly cross-linked insoluble macromolecules, and appears as a thin and highly osmiophilic layer (Briza et al., 1990b). Underlying this layer, and closely linked to it, is the chitosan layer, which appears as a diffuse electron-dense layer (Briza et al., 1988). The electron-transparent layer and the two outer layers, the chitin/chitosan and dityrosine layers, were also present in the mutant spores (Fig. 6b). This was confirmed by chemical analysis of the spore walls, which revealed that chitosan and dityrosine levels were similar in the wild-type and mutant strains (data not shown). However, the images of the mutant spores clearly indicated the existence of an aberrant spore wall assembly process. A glance at the mutant spores indicated that the deposition of the two outer layers (chitosan and dityrosine layers) was not normal (Fig. 6b). Both electron-dense layers appeared as ruffles, instead of exhibiting the smooth aspect of wild-type spores. Instead of forming well-structured layers like the wild-type strain (Fig. 6a), both chitosan and dityrosine were deposited in an irregular fashion over the glucan layer. More detailed scrutiny of the images revealed that the ruffled appearance of the outermost layers in the mutants was the consequence of a problem in the linking between the glucan and chitosan layers. In many spores, the electron-dense (chitosan) layer was detached from the glucan/mannoprotein layer, and electron-transparent zones corresponding to the space between the two detached layers appeared (Fig. 6b, bottom panels). In some of the spores, some discontinuities in the chitosan layer were even detected. These results are in perfect agreement with the Calcofluor staining of mutant spores, and suggest that the probable function of Crr1p in the biogenesis of the spore wall is the cross-linking between glucan and chitosan.



View larger version (177K):
[in this window]
[in a new window]
 
Fig. 6. Electron micrographs of wild-type and crr1{Delta} mutants. (a) Single asci of wild-type strain FY1679 at low (left) and high (right) magnifications, showing a perfectly organized spore wall: the inner electron-transparent glucan layer (denoted with the arrow labelled ‘g’), followed by the chitin/chitosan layer (denoted with the arrow labelled ‘ch’) and the dityrosine outermost layer (denoted with the arrow labelled ‘d’); both of the latter appear as electron-dense layers. Old vegetative cell walls surround these structures. (b) Low (first two panels) and higher (remaining panels) magnifications of mutant crr1{Delta} spores. The outermost chitosan and dityrosine layers are aberrantly assembled over the glucan/mannoprotein layer, appearing as ruffled layers (marked with arrow heads). In most of the spores the dark electron-dense layer (the chitosan/dityrosine layer) is completely detached from the inner layer (denoted with arrows in the pictures). In some cases, discontinuities in the chitosan layer are even found (marked with an asterisk).

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Our knowledge of spore wall structure and biogenesis has increased considerably over the past few years, especially with respect to events related to the formation of the outer two layers, the chitin/chitosan and dityrosine layers. The enzymic activities responsible for the synthesis of chitosan have been characterized with precision. Chitin biosynthesis depends on CSIII activity (Pammer et al., 1992; Valdivieso et al., 1991; Sanz et al., 2002). The following step – the chitin deacetylation process – depends on the enzymic activities of Cda1p and Cda2p (Briza et al., 1988; Christodoulidou et al., 1996, 1999), and leads to the formation of the 1,4-{beta}-D-glucosamine polymer. There is a temporal order in the appearance of spore wall components, which begins after the closure of the prospore membrane (Lynn & Magee, 1970). Thus, mannoproteins are deposited as the prospore membrane forms, and after prospore membrane closure the glucan layers form, followed by the chitosan layer and finally the dityrosine layer (Tachikawa et al., 2001). The layers are deposited from innermost to outermost with respect to the spore cytoplasm. Although not yet characterized, enzymic activities must be necessary for the formation of cross-links between these spore wall polymers. As in vegetative cell wall biogenesis, little is known about the genes encoding these cross-linking activities.

Many gene families related to the vegetative cell wall construction process have members that are specifically induced under sporulation conditions. This is the case for SPS2, YCL048, GAS2 and GAS4, SSG1/SPR1, YPL130, CWP1, TIR2, CRR1 and others (Chu et al., 1998). However, no characterization of the role of these genes in the formation of the spore wall has been described, with the exception of SSG1/SPR1, which encodes a sporulation-specific exo-1,3-{beta}-glucanase (Muthukumar et al., 1993; San Segundo et al., 1993). Here, we have characterized the role of Crr1p in spore wall assembly. CRR1 encodes a protein of 422 aa with a predicted molecular size of 47·5 kDa, although the true molecular size deduced from Crr1p–Myc suggests some post-translational modification for the mature protein. Crr1p is encoded by the gene CRR1 (YLR213c), and shows significant similarity to Crh1p and Crh2p; the latter two proteins play an important role in the vegetative cell wall construction process by cross-linking cell wall polymers (Rodriguez-Peña et al., 2000). The three proteins belong to the 16th family of glycoside hydrolases (Henrissat & Bairoch, 1996), and include an N-terminal secretion signal for incorporation into the secretion pathway and a catalytic domain (DE-I/E-DXE) related to that of prokaryotic (1,3-1,4)-{beta}-glucanases and plant transglycosidases. In contrast to CRH1 and CRH2, CRR1 does not have the C-terminal domain for GPI anchor attachment.

The pattern of expression of CRR1 is characteristic of genes expressed midway through the sporulation process (mid to mid-late genes), being induced after 6–9 h under sporulating conditions in the strain background assayed here. As in many other genes with a similar expression pattern, detailed analysis of the CRR1 promoter revealed the existence of an MSE consensus box (–149/GTCACAAAAA/–141). This site is recognized by Ndt80, a transcription factor involved in the regulation of middle-sporulation-specific genes (Hepworth et al., 1998). Small variations in the time of induction have been reported for CRR1, depending on the strain background (this work; Chu et al., 1998; Primig et al., 2000), but the transcriptional kinetics are conserved. Similar temporal expression patterns have been described for those genes necessary for the formation of the spore walls (Briza et al., 1990a), suggesting a role for CRR1 in this process. Cluster analysis carried out by Primig et al. (2000), taking into account not only the time but also the levels of induction and the persistence of the induction, grouped CRR1 in a different cluster from that of DIT1, which is still highly induced after 12 h, or CDA1, which is also highly induced, but earlier. CRR1 grouped together with other genes involved in cell wall biogenesis, such as CWP1 and ECM37. Interestingly, we found that CRR1 was induced biphasically. In our hands, a second peak induction was found after 24 h under sporulating conditions. Only genes that are expressed very late in the sporulation process have been shown to be induced at these times. These genes are thought to play a role in spore maturation (Law & Segall, 1988), and therefore the second expression peak suggests that Crr1p is also necessary at late times for spore maturation.

Consistent with a role in spore wall biogenesis, Crr1p localizes to the surface of ascospores. Although the efficiency of spore formation was not affected by the absence of CRR1, mutants in this gene had clear defects in spore wall assembly, as deduced from the phenotypes observed. First, cells lacking CRR1 were more sensitive to heat shock and to enzymic digestion with glusulase than wild-type cells. Since the spore wall is the structure responsible for the resistance of mature spores to stress conditions, this observation clearly indicates that mutant cells in CRR1 are unable to assemble the spore walls properly. Second, crr1 cells were permeable to Calcofluor White, a dye that stains chitin and chitosan but is unable to permeate properly assembled spore walls. Third, the deletion of CRR1 in a double mutant in CDA1 and CDA2, the two genes responsible for the synthesis of the chitosan layer, led to an additive phenotype of resistance to temperature shock in the triple mutant. Taken together, all these data suggest a role for Crr1p in spore wall biogenesis.

In exactly which step of spore wall assembly is Crr1p involved? Our previous findings suggest that CRH1 and CRH2, the two members of the yeast transglycosydase family expressed during vegetative growth, are involved in the cross-linking between cell wall polymers, particularly at the sites of polarized growth. Based on differences in the alkali-soluble and -insoluble fractions in strains deleted in CRH1 and CRH2, and experiments to demonstrate co-localization of these proteins with chitin, it can be speculated that these proteins are involved in the cross-linking between glucan and chitin, possibly by transglycosylation reactions between these two polymers (Rodriguez-Peña et al., 2000). Since the putative catalytic domain in Crr1p (DEIDFE) is conserved with respect to that present in Crh1p (DEIDIE) and Crh2 (DELDYE), it is very likely that Crr1p would have a similar function in spore wall assembly. The function of Crr1p in the biogenesis of the spore wall would be the cross-linking between glucan and chitin/chitosan layers. Electron microscopy experiments comparing the structure of the spore wall in wild-type and mutant cells deleted in CRR1 support this hypothesis. In agreement with this, it was observed that overexpression of CRR1 rendered the spores more resistant to temperature shock. It could be envisaged that overexpression of a putative transglycosidase would lead to the construction of a more compact spore wall. Interestingly, this phenotype has also been observed, following overexpression of the homologous gene of the family, CRH2, in the case of vegetative cells (Rodriguez-Peña et al., 2000).

It is difficult to speculate about the nature of this cross-link and whether Crr1p is responsible for the cross-linking of chitin, chitosan, or both polymers to the glucan layer. Additional experiments will be needed to characterize the enzymic activity of Crr1p and to characterize further the precise link catalysed by this protein. However, to our knowledge, this is the first protein to be identified that is involved in the cross-linking between spore wall polymers.


   ACKNOWLEDGEMENTS
 
We thank all members of Research Unit 4 at the Department of Microbiology, C. V. Rodriguez de Aldana and Victor J. Cid for helpful and fruitful discussions. Raul García is acknowledged for bioinformatic analysis and graphic design. We also thank C. V. Rodriguez de Aldana for strains, and Rosa Pérez and María Isabel García Sáez at the Centro de Genómica y Proteómica-UCM for their help in Q-PCR and DNA sequencing. This work was supported by grants BIO2001-1345-C02-01 and GEN2001-4707-C08-04 from the Comision Internacional de Ciencia y Tecnología (CICYT) to Javier Arroyo, and by grant P14735-P09 from the Austrian Fonds zur Förderung der Wissenschaftlichen Forschung to Peter Briza. F. G. E. and E. R. were holders of a fellowship from the Universidad Complutense de Madrid.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Ausubel, F. M., Kingston, R. E., Brent, R., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (1993). Current Protocols in Molecular Biology. New York: Greene Publishing Associates & Wiley Interscience.

Briza, P., Winkler, G., Kalchhauser, H. & Breitenbach, M. (1986). Dityrosine is a prominent component of the yeast ascospore wall. A proof of its structure. J Biol Chem 261, 4288–4294.[Abstract/Free Full Text]

Briza, P., Ellinger, A., Winkler, G. & Breitenbach, M. (1988). Chemical composition of the yeast ascospore wall. The second outer layer consists of chitosan. J Biol Chem 263, 11569–11574.[Abstract/Free Full Text]

Briza, P., Breitenbach, M., Ellinger, A. & Segall, J. (1990a). Isolation of two developmentally regulated genes involved in spore wall maturation in Saccharomyces cerevisiae. Genes Dev 4, 1775–1789.[Abstract]

Briza, P., Ellinger, A., Winkler, G. & Breitenbach, M. (1990b). Characterization of a DL-dityrosine-containing macromolecule from yeast ascospore walls. J Biol Chem 265, 15118–15123.[Abstract/Free Full Text]

Briza, P., Eckerstorfer, M. & Breitenbach, M. (1994). The sporulation-specific enzymes encoded by the DIT1 and DIT2 genes catalyze a two-step reaction leading to a soluble LL-dityrosine-containing precursor of the yeast spore wall. Proc Natl Acad Sci U S A 91, 4524–4528.[Abstract]

Briza, P., Kalchhauser, H., Pittenauer, E., Allmaier, G. & Breitenbach, M. (1996). N,N'-Bisformyl dityrosine is an in vivo precursor of the yeast ascospore wall. Eur J Biochem 239, 124–131.[Abstract]

Briza, P., Bogengruber, E., Thur, A., Rutzler, M., Munsterkotter, M., Dawes, I. W. & Breitenbach, M. (2002). Systematic analysis of sporulation phenotypes in 624 non-lethal homozygous deletion strains of Saccharomyces cerevisiae. Yeast 19, 403–422.[CrossRef][Medline]

Christodoulidou, A., Bouriotis, V. & Thireos, G. (1996). Two sporulation-specific chitin deacetylase-encoding genes are required for the ascospore wall rigidity of Saccharomyces cerevisiae. J Biol Chem 271, 31420–31425.[Abstract/Free Full Text]

Christodoulidou, A., Briza, P., Ellinger, A. & Bouriotis, V. (1999). Yeast ascospore wall assembly requires two chitin deacetylase isozymes. FEBS Lett 460, 275–279.[CrossRef][Medline]

Chu, S., DeRisi, J., Eisen, M., Mulholland, J., Botstein, D., Brown, P. O. & Herskowitz, I. (1998). The transcriptional program of sporulation in budding yeast. Science 282, 699–705.[Abstract/Free Full Text]

Deng, C., Thomas, K. R. & Capecchi, M. R. (1993). Location of crossovers during gene targeting with insertion and replacement vectors. Mol Cell Biol 13, 2134–2140.[Abstract]

Enyenihi, A. H. & Saunders, W. S. (2003). Large-scale functional genomic analysis of sporulation and meiosis in Saccharomyces cerevisiae. Genetics 163, 47–54.[Abstract/Free Full Text]

Fares, H., Goetsch, L. & Pringle, J. R. (1996). Identification of a developmentally regulated septin and involvement of the septins in spore formation in Saccharomyces cerevisiae. J Cell Biol 132, 399–411.[Abstract]

Felder, T., Bogengruber, E., Tenreiro, S., Ellinger, A., Sa-Correia, I. & Briza, P. (2002). Dtrlp, a multidrug resistance transporter of the major facilitator superfamily, plays an essential role in spore wall maturation in Saccharomyces cerevisiae. Eukaryot Cell 1, 799–810.[Abstract/Free Full Text]

Goldstein, A. L. & McCusker, J. H. (1999). Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15, 1541–1553.[CrossRef][Medline]

Guth, E., Hashimoto, T. & Conti, S. F. (1972). Morphogenesis of ascospores in Saccharomyces cerevisiae. J Bacteriol 109, 869–880.[Medline]

Henrissat, B. & Bairoch, A. (1996). Updating the sequence-based classification of glycosyl hydrolases. Biochem J 316, 695–696.[Medline]

Hepworth, S. R., Friesen, H. & Segall, J. (1998). NDT80 and the meiotic recombination checkpoint regulate expression of middle sporulation-specific genes in Saccharomyces cerevisiae. Mol Cell Biol 18, 5750–5761.[Abstract/Free Full Text]

Kafetzopoulos, D., Martinou, A. & Bouriotis, V. (1993a). Bioconversion of chitin to chitosan: purification and characterization of chitin deacetylase from Mucor rouxii. Proc Natl Acad Sci U S A 90, 2564–2568.[Abstract]

Kafetzopoulos, D., Thireos, G., Vournakis, J. N. & Bouriotis, V. (1993b). The primary structure of a fungal chitin deacetylase reveals the function for two bacterial gene products. Proc Natl Acad Sci U S A 90, 8005–8008.[Abstract/Free Full Text]

Katohda, S., Konno, K., Sasaki, Y., Sazuki, K. & Sakamoto, S. (1984). Isolation and composition of the spore Saccharomyces cerevisiae. Agric Biol Chem 48, 895–901.

Knop, M. & Strasser, K. (2000). Role of the spindle pole body of yeast in mediating assembly of the prospore membrane during meiosis. EMBO J 19, 3657–3667.[Abstract/Free Full Text]

Kreger-van Rij, N. J. & Veenhuis, M. (1970). An electron microscope study of the yeast Pityrosporum ovale. Arch Mikrobiol 71, 123–131.[Medline]

Kupiec, M., Byers, B., Esposito, R. E. & Mitchell, A. P. (1997). Meiosis and sporulation in Saccharomyces cerevisiae. In The Molecular and Cellular Biology of the Yeast Saccharomyces cerevisiae: Cell Cycle and Cell Biology. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Law, D. T. & Segall, J. (1988). The SPS100 gene of Saccharomyces cerevisiae is activated late in the sporulation process and contributes to spore wall maturation. Mol Cell Biol 8, 912–922.[Medline]

Lynn, R. R. & Magee, P. T. (1970). Development of the spore wall during ascospore formation in Saccharomyces cerevisiae. J Cell Biol 44, 688–692.[Free Full Text]

Martin, H., Rodriguez-Pachón, J. M., Ruiz, C., Nombela, C. & Molina, M. (2000). Regulatory mechanisms for modulation of signaling through the cell integrity Slt2-mediated pathway in Saccharomyces cerevisiae. J Biol Chem 275, 1511–1519.[Abstract/Free Full Text]

Miret, J. J., Solari, A. J., Barderi, P. A. & Goldemberg, S. H. (1992). Polyamines and cell wall organization in Saccharomyces cerevisiae. Yeast 8, 1033–1041.[Medline]

Mitchell, A. P. (1994). Control of meiotic gene expression in Saccharomyces cerevisiae. Microbiol Rev 58, 56–70.[Medline]

Moens, P. B. & Rapport, E. (1971). Spindles, spindle plaques, and meiosis in the yeast Saccharomyces cerevisiae. J Cell Biol 50, 344–361.[Abstract/Free Full Text]

Muthukumar, G., Suhng, S. H., Magee, P. T., Jewell, R. D. & Primerano, D. A. (1993). The Saccharomyces cerevisiae SPR1 gene encodes a sporulation-specific exo-1,3-{beta}-glucanase which contributes to ascospore thermoresistance. J Bacteriol 175, 386–394.[Abstract]

Neigeborn, L. & Mitchell, A. P. (1991). The yeast MCK1 gene encodes a protein kinase homolog that activates early meiotic gene expression. Genes Dev 5, 533–548.[Abstract]

Neiman, A. M. (1998). Prospore membrane formation defines a developmentally regulated branch of the secretory pathway in yeast. J Cell Biol 140, 29–37.[Abstract/Free Full Text]

Pammer, M., Briza, P., Ellinger, A., Schuster, T., Stucka, R., Feldmann, H. & Breitenbach, M. (1992). DIT101 (CSD2, CAL1), a cell cycle-regulated yeast gene required for synthesis of chitin in cell walls and chitosan in spore walls. Yeast 8, 1089–1099.[Medline]

Primig, M., Williams, R. M., Winzeler, E. A., Tevzadze, G. G., Conway, A. R., Hwang, S. Y., Davis, R. W. & Esposito, R. E. (2000). The core meiotic transcriptome in budding yeasts. Nat Genet 26, 415–423.[CrossRef][Medline]

Rabitsch, K. P., Toth, A., Galova, M. & 11 other authors (2001). A screen for genes required for meiosis and spore formation based on whole-genome expression. Curr Biol 11, 1001–1009.[CrossRef][Medline]

Rodriguez-Peña, J. M., Cid, V. J., Arroyo, J. & Nombela, C. (2000). A novel family of cell wall-related proteins regulated differently during the yeast life cycle. Mol Cell Biol 20, 3245–3255.[Abstract/Free Full Text]

Rodriguez-Peña, J. M., Rodriguez, C., Alvarez, A., Nombela, C. & Arroyo, J. (2002). Mechanisms for targeting of the Saccharomyces cerevisiae GPI-anchored cell wall protein Crh2p to polarised growth sites. J Cell Sci 115, 2549–2558.[Abstract/Free Full Text]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Laboratory.

San Segundo, P., Correa, J., Vazquez de Aldana, C. R. & del Rey, F. (1993). SSG1, a gene encoding a sporulation-specific 1,3-{beta}-glucanase in Saccharomyces cerevisiae. J Bacteriol 175, 3823–3837.[Abstract]

Sanz, M., Trilla, J. A., Durán, A. & Roncero, C. (2002). Control of chitin synthesis through Shc1p, a functional homologue of Chs4p specifically induced during sporulation. Mol Microbiol 43, 1183–1195.[CrossRef][Medline]

Sikorski, R. S. & Hieter, P. (1989). A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19–27.[Abstract/Free Full Text]

Tachikawa, H., Bloecher, A., Tatchell, K. & Neiman, A. M. (2001). A Gip1p–Glc7p phosphatase complex regulates septin organization and spore wall formation. J Cell Biol 155, 797–808.[Abstract/Free Full Text]

Ufano, S., San Segundo, P., del Rey, F. & Vazquez de Aldana, C. R. (1999). SWM1, a developmentally regulated gene, is required for spore wall assembly in Saccharomyces cerevisiae. Mol Cell Biol 19, 2118–2129.[Abstract/Free Full Text]

Valdivieso, M. H., Mol, P. C., Shaw, J. A., Cabib, E. & Durán, A. (1991). CAL1, a gene required for activity of chitin synthase 3 in Saccharomyces cerevisiae. J Cell Biol 114, 101–109.[Abstract]

Wach, A., Brachat, A., Pohlmann, R. & Philippsen, P. (1994). New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10, 1793–1808.[Medline]

Wach, A., Brachat, A., Alberti-Segui, C., Rebischung, C. & Philippsen, P. (1997). Heterologous HIS3 marker and GFP reporter modules for PCR-targeting in Saccharomyces cerevisiae. Yeast 13, 1065–1075.[CrossRef][Medline]

Wagner, M., Briza, P., Pierce, M. & Winter, E. (1999). Distinct steps in yeast spore morphogenesis require distinct SMK1 MAP kinase thresholds. Genetics 151, 1327–1340.[Abstract/Free Full Text]

Received 8 May 2004; accepted 14 July 2004.



This Article
Abstract
Full Text (PDF)
Supplementary video file
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Gómez-Esquer, F.
Articles by Arroyo, J.
Articles citing this Article
PubMed
PubMed Citation
Articles by Gómez-Esquer, F.
Articles by Arroyo, J.
Agricola
Articles by Gómez-Esquer, F.
Articles by Arroyo, J.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS
Copyright © 2004 Society for General Microbiology.