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
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ABSTRACT |
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A video image of the three-dimensional localization of Crr1GFP 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).
Present address: Facultad de Ciencias de la Salud, Universidad Rey Juan Carlos, 28922 Madrid, Spain.
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INTRODUCTION |
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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-
-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.
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METHODS |
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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
diploid strain FG10 (see Table 1
).
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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 YLR1YLR2 (496 bp) and YLR3YLR4 (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 (
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 ml1.
For visualization of the GFPCrr1 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.
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RESULTS |
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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 Crr1GFP were obtained by transformation of the FY1679 strain with plasmid pJV21G. This is a multi-copy plasmid in which the transcription of CRR1GFP is controlled by the CRR1 promoter. Forty-eight hours after transfer to sporulation conditions, in mature spores, the Crr1GFP 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 Crr1GFP 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.
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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
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. 3
c).
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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
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.
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DISCUSSION |
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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-
-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 Crr1pMyc 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)-
-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 69 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.
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ACKNOWLEDGEMENTS |
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Received 8 May 2004;
accepted 14 July 2004.
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