YEAST VOL. 12: 983-990 (1996) Yeast Sequencing Reports Characterization of cwZl+, a Gene from Schizosaccharomyces pombe Whose Overexpression causes Cell Lysis CARLOS GODOY, MANUEL ARELLANO, MARGARITA DIAZ, ANGEL DURAN AND PILAR PEREZ* Znstituto de Microbiologia Bioquimira, Consejo Superior de Investigaciones Cientijicas and Universidad de Salamanca, 37007 Salamanca, Spain3 Received 6 February 1996; accepted 26 April 1996 From a Schizosaccharornyces pombe genomic library we have isolated the gene cwllf that causes cell lysis when it is overexpressed in the absence of an osmotic stabilizer. Southern hybridization showed that cwlI+ exists as a single copy in the S. pombe genome. The cwll+ gene nucleotide sequence revealed a putative open reading frame of 924 bp encoding a polypeptide of 308 amino acids with a calculated M, of 27 000. The cwll+ DNA hybridizes to a major RNA transcript of 1.5 kb whose 5' end maps at a position 452 bp upstream from the predicted translation start. Comparison of the amino acid sequence with those included in the current databases, showed no significant similarity to any known sequences. Cells overexpressing the cwll' gene under the control of the S. pombe nmt inducible promoter displayed a reduced cell wall content, were unable to separate after division and lysed drastically in the absence of osmotic stabilizer. Disruption of the cw11+ gene caused no noticeable phenotype, The sequence has been deposited in the EMBL data library under Accession Number X9445. KEY WORDS ~ fission yeast; dominant genetics; cell wall regulation INTRODUCTION The major structural polymer of budding and fission yeast cell wall is a linear 1,3-P-glucan branched with some 1,6-P-glucan (Fleet, 1991). In Saccharomyces cerevisiae, several genes involved in 1,3-P-glucan biosynthesis have been isolated by different approaches. Based on the sequence of internal peptides derived from a protein band enriched in the process of 1,3-p-glucan synthase purification, two 1,3-P-glucan synthase-related genes, GSCl and GSC2, have been isolated (Inoue et al., 1995). Genes defined by complementing mutations of resistance to 1,3-P-glucan synthesis inhibitors in S. cerevisiae such as echinocandin *Corresponding author. CCC 0749-503)3/96/100983-08 0 1996 by John Wiley & Sons Ltd analogs (Douglas et al., 1994; El-Sherbeini and Clemas, 1995), papulacandin B (Castro et al., 1995) or Hansenula mrakii killer toxin (Yamamoto et al., 1986; Hong et al., 1994; Kusuhara et al., 1994) have also been described. A third genetic strategy has been to isolate mutants hypersensitive to different agents known to affect the cell wall assembly, such as calcofluor white (Ram et al., 1994). With such techniques, several genes have been described. FKS1IETG1ICWH53ICND1I GSCIIPBRI (Douglas et al., 1994; Eng et al., 1994; Ram et al., 1995; Inoue et al., 1995; Castro et al., 1995) is a gene encoding a large polypeptide with 16 potential transmembrane domains that might be a structural component of 1,3-P-glucan synthase (Inoue et al., 1995). A second gene, FKS2I GSC2, highly homologous to FKSIIGSCI, has 984 been described (Inoue et al., 1995; Mazur et al., 1995) as a subunit of another yeast 1,3-p-glucan synthase. GNSl encodes a 40 kDa protein that is also predicted to be a membrane protein. It has no significant homology with any known protein and might constitute either a subunit or a regulator of 1,3-P-glucan synthase activity (El-Sherbeini and Clemas, 1995). H K R l is an essential gene that encodes a presumptive calcium-binding type I membrane protein. Its overexpression promotes resistance to H. mrakii killer toxin and increases the a-glucan content of the cell wall (Kasahara et al., 1994). Finally, KNR4ISMZl codes for a nuclear protein that might regulate cell wall synthesis (Hong et al., 1994). A different genetic approach to identify genes involved in the maintenance of cell wall integrity and cell viability has been the isolation of mutants displaying a thermosensitive lytic phenotype (Venkov et al., 1974; Cabib and Duran, 1975; Ribas et al., 1991). Mutants of this type have allowed the identification of the SLT2 gene from S. cerevisiae, encoding a serinehhreonine protein kinase from the family of MAP kinases (Torres et al., 1991), or the cwg2' gene from Schizosaccharomyces pombe, coding for the P-subunit of a type I geranylgeranyltransferase (Diaz et al., 1993). The plethora of mutants that can be isolated by these methods reflects the complexity of functions involved in cell wall integrity and cell viability. As a new approach in the search for genes involved in cell wall biosynthesis, we searched for genes that caused overexpression-mediated cell lysis in the absence of an osmotic stabilizer. Dominant genetics has been used for the identification of regulatory genes in different physiological events (Liu et al., 1992; Ramer et al., 1992). In this paper we describe the characterization of the S. pombe cwll+ gene whose overexpression causes osmotic lysis. MATERIALS AND METHODS Strains, growth conditions and genetic methods The S. pombe auxotrophic strains leul-32 (Kohli et al., 1977) and ura4-dl8 (Grimm et al., 1988) were used in this study. They were grown in YED medium (1% yeast extract, 1% glucose) or minimal medium, (1% glucose, 0.7% yeast nitrogen base without amino acids, 0.9 g/l KCl, 1 mg/l citric acid, 10 pg/l biotin, 1 mg/l calcium pantothenate, C . GODOY ET AL. 10 mg/l nicotine acid and 10 mg/l m-inositol) supplemented with the necessary requirements. Incubations were carried out at 28°C. Growth was monitored either by A600measurements or by cell number counting. Escherichia coli JM101, JM109 or DH5a were used. These were grown in LB medium (1% bactotryptone, 0.5% yeast extract, 1% NaC1) supplemented with 50 pg/ml ampicillin or 15 pg/ml tetracycline when appropriate. Solid medium plates contained 2% agar. Yeast extract, yeast nitrogen base, bactotryptone and agar were from Difco Laboratories, Detroit, Michigan, U.S.A. Plasmids and recombinant D N A methods The S. pombe genomic library was constructed in pDB262 (Wright et al., 1986). This vector contains the S. cerevisiae LEU2 gene which complements S. pombe leul-32, and part of the 2 pm circle, which allows high frequency transformation. The S. pombe expression plasmid pREP4 (Maundrell, 1993) was used for the overexpression of the cwll' gene under the control of the nmt inducible promoter. S. pombe was transformed by the protoplast method (Beach and Nurse, 1981) or the lithium chloride method (It0 et al., 1983). Other DNA manipulations were carried out by standard techniques (Sambrook et al., 1989). Southern analysis was performed using a radioactively labelled probe prepared as described (Rigby et al., 1982), or by the random primed method (Feinberg and Vogelstein, 1984), using a non-radioactive digoxigenin-dUTP labelled probe. Northern analysis was performed using total RNA extracted as described (Percivall-Smith and Segal, 1984). The electrophoresed RNA was transferred to nitrocellulose membranes and hybridized with a probe radioactively labelled by nick translation. The nucleotide sequence was determined by subcloning the 4 kb fragment bearing the cwll+ gene in to both KS - and KS' Bluescript vectors (Stratagene Inc., La Jolla, CA). A series of overlapping deletions in both orientations were created by unidirectional exonuclease I11 digestion. Singlestranded DNA was produced using the M13K07 helper phage and both strands were sequenced using the dideoxy chain-termination method (Sanger et al., 1977). The DNA sequence was translated to the predicted amino acid sequence using the DNAsis program. The amino acid sequence was thereafter compared with the 985 ISOLATION OF cwll', A LYTIC GENE A A C 8kb - - Figure 1. Micrographs of S. pombe led-32 cells grown in minimal medium at 28°C. (A) Wild type; (B) pCGl transformants: (C and D) pCG4 transformants grown in the absence of thiamine for (C) 14 h and (D) 18 h. The bar corresponds to 5 pm. sequences available in the EMBL data bases using the FASTA program. Analysis of mRNA 5' termini was carried out by primer extension essentially as described (Sambrook et al., 1989). Briefly, a synthetic oligonucleotide (sequence 5'-GATTTTTGGATTTT GCTGTAGG-3') complementary to nucleotide positions -262 to -284 of the cwll sequence was 5' end-labelled with [ Y - ~ ~ P ] A T(5000 P Ci/mmol, Amersham) and polynucleotide kinase. Poly (A) RNA from strain 972h- was denatured and hybridized with an excess of the labelled complementary oligonucleotide. Reverse transcriptase RAV-2 (Amersham) was then used to extend the primer, producing a labelled cDNA complementary to the RNA template. The cDNA was analysed by electrophoresis on a sequencing gel adjacent to the sequencing reactions of the c w l l c gene primed with the same oligonucleotide. Other methods S. pombe chromosomes were separated by alternating electric field electrophoresis with a Bio-Rad CHEF DR-I1 system (Carle and Olson, 1985) at 50V, switching the electric field every 30 min for 72 h. The separated chromosomes were transferred to a nitrocellulose membrane and hybridized with the cwll+ probe. Cell wall quantitation and fractionation was done by measuring [U-'4C]glucose IIII11 2kb - Figure 2. (A) Restriction map and sequencing strategy used in the determination of the nucleotlde sequence of the mpCGl insert. The position of the cwlZ+ gene is indicated with an arrow showing the direction of transcription. Thin arrows indicate the direction and the extent to which the sequence was determined. (B) Hybridization of the radioactively labeled pCGDl insert with genomic S. pombe DNA cut with: (1) X b d ; (2) PstI; (3) HindsII1. (C) Chromosomal localization of the cwll+ gene. The migration of the three S. pombe chromosomes is indicated. Southern blot was done using the same probe as in Figure 2B. (240 mCi/mmol, Amersham) uptake as described (Ribas et al., 1991). RESULTS Isolation of a plasmid containing the cwll+ gene S. pombe leul-32 h - was transformed with a S. pombe genomic DNA library constructed in the plasmid pDB262 (Wright et al., 1986). Assessment of cell lysis in the presence or absence of 1.2 Msorbitol was carried out by detection of red colonies in the presence of Phloxin B (Ribas et al., 1991). When autolysis occurred the colonies progressively stained red whereas they remained white when the cells did not lyse. Five leucine - z- - -5- --5 - 2 986 987 ISOLATION OF cu,lZ+. A LYTIC GENE region responsible for the lytic activity, the insert was shortened using the HindIII restriction sites present in the fragment (Figure 2A). The two plasmids obtained were unable to cause lysis in the absence of osmotic stabilizer and therefore the whole insert was sequenced on both DNA strands according to the strategy outlined in Figure 2A. A single open reading frame (ORF) was found between bases 2391-3315 of the insert and this putative gene was called cwll+ for ‘cell wall lysis’. Southern blot analysis of S. pombe genomic DNA cut with XbaI, Pstl, or HindIII using the whole insert as probe indicated no rearrangement of the DNA structure during cloning and that the S. pombe genome contains a single copy of the cloned gene (Figure 2B). The cwIl+ gene was located on chromosome 11, as shown in Figure 2C. cwII nucleotide sequence and transcription Two kilobases from the pCGDl insert nucleotide sequence containing the cwll+ gene are shown in Figure 3A. There is a single O R F 924 bp long and no consensus signals for intron splicing were observed. The predicted encoded protein has 308 amino acids with a Mr of 27000. The protein is relatively hydrophilic with a similar number of acidic and basic amino acids and a calculated isoelectric point of 6.4. The Kyte and Doolittle hydropathy plot of the product (Kyte and Doolittle, 1982) revealed no regions of marked hydrophobicity characteristic of N-terminal signal peptide sequences (Figure 3B). There is a potential membrane-spanning domain around amino acid 250, four potential N-glycosylation sites at amino acids 65, 113, 135 and 303, five potential phosphorylation sites for protein kinase C and six for casein kinase 11. A search of the GenBank and EMBL data banks did not reveal any significant resemblance of the cwll+ encoded protein to any described protein. Expression of the O R F was assessed by Northern blot analysis of S. pombe total mRNA using the HindIII-Hind111 fragment containing cwll+ as a probe. A single 1.5 kb transcript was detected in both the wild type and the pCG-transformed S. pombe (Figure 4A). The 5‘ flanking sequence of the coding region has a TATA element of 480 bp upstream from the translation start. In order to determine whether this could be part of the promoter, we identified the transcription initiation site by primer extension of an oligonucleotide complementary to the -262 + A B 1 2 ACGT - 2.3 Kb -1.3 Kb Figure 4. (A) Northern analysis of the w l I + transcript. Total RNA from S. ponzhe wild type (1) and transformed with pCGl (2) was resolved by agarose gel electrophoresis, transferred to a nylon membrane and hybridized to the radioactively labeled pCGDl insert. (B) Determination of the 5’ end of the c w l l f mRNA transcript by primer extension. The DNA sequence shown corresponds to the minus strand of the genomic DNA and was obtained using as primer the same oligonucleotide used to extend the mRNA with reverse transcriptase. The asterisk indicates the nucleotide matching the RNA 5’ end. to -284 nucleotides upstream from the ATG of the O R F in the sequence. One major transcript (Figure 4B) was detected at 452 bp upstream from the ATG. It is therefore likely that the mentioned ATATAA sequence at -480 bp would be part of the cwllf promoter. The 3‘ non-coding region contains two of the proposed (Miyake and Yamamoto, 1990) polyadenylation consensus sequences, AATATA and ATAAATA, 118 bp and 259 bp downstream from the O R F stop codon respectively. Sequences matching the S. cerevisiue termination motif (TAG . . . TATGT . . . TTT) might also be active in S. pombe (Hayles and Nurse, 1992) and are found before the second polyadenylation sequence. cwll+ gene overexpression und disruption On the basis of the observations with the plasmid pCGl, we cloned the cw11+ gene in the plasmid pREP4 under the control of the nmtl thiamine repressible promoter (Maundrell, 1993). When S. pombe was transformed with this plasmid (pCG4) and grown in minimal medium without thiamine, the cells did not separate after dividing (Figure 1C) but formed chains of cells that subsequently lysed (Figure 1D). The lysis first affected the mother cell and was prevented by sorbitol. In the presence of thiamine, pCG4 transformants grew as the wild type. We analysed the cell wall 988 C. GODOY ET AL. A probe b probea 1 B w t 1 2 3 4 5 w t 1 2 3 4 5 - 3 Kb - 3 Kb - 2 Kb - 2 Kb probe a probe b Figure 5. (A) Scheme of the cwll+ gene disruption. (B) Hybridiration of the HindIII-cut genomic DNA from the S. pomhe wild type and five selected disruptant clones to the probes indiated in (A). The probes were labeled with digoxigenin-dUTP by the random primed method. composition of pCG4 transformants to ascertain whether the (I-3)P-glucan content or any other major cell wall polymer might be affected by the cwll+ overexpression. No changes in cell wall composition were observed (data not shown) but there was a 20% decrease in the total uptake of ['4C]glucose into the whole cell wall fraction as compared with the S. pornbe. wild type. In order to determine whether the cw11+ gene is essential, a disruption mutation was generated, as shown in Figure 5A. The AsuII-AsuII 2.8 kb fragment containing the cw11+ gene (Figure 2A) was cloned into the pGEM3 (Promega), generating the plasmid pCG5. The HindIII-SulI fragment from this plasmid was replaced by the uru4+ gene cut with the same restriction enzymes. The plasmid thus obtained (pCG6) was BurnHI-EcoRI cut and the linear fragment was used to transform S. pombe leul-32 uvu4-Dl8 h . Integration and disruption at the cwll+ locus was confirmed by Southern blot analysis using two different probes (Figures 5B). None of the disruptant clones analysed showed a phenotype different from that of the wild type when they were grown either at 28°C or 37°C. DISCUSSION The morphogenesis and growth of yeast cells are necessarily related to the biosynthesis and degradation of the cell wall. Therefore, these processes must be strictly controlled and linked to the general signal transduction pathways of the cell (Cid et ul., 1995). We employed dominant genetics in an attempt to identify new genes directly or indirectly involved in cell wall biosynthesis. The gene cloned in this study, cwll+, showed no known homologs and the gene product contained no obvious motifs which might provide further insight into its possible function. An interesting feature of the cwll' gene is that transcription starts 452 nucleotides upstream from the ORF, while in S. pombe transcription generally starts within 200 nucleotides upstream from the O R F (Russell, 1989). However, there were no short ORFs in the cwlI+ leader sequence that might imply translational control of the gene. The morphological phenotype before lysis of cells overexpressing cwll+ under its own promoter is quite similar to that exhibited by S. pombe cells in which (1-3)P-~-glucan synthesis is inhibited (Varona et a/., 1983). When a stronger promoter ISOLATION OF c w l l f , A LYTIC GENE such as nmt is used, cells lyse more dramatically in the absence of an osmotic stabilizer, have a lower cell wall content and show a defect in cell separation after division. therefore, cwU+ might be directly involved in the regulation of the cell separation process. In budding yeasts, a burst of chitin synthesis at the end of mitosis deposits the primary septum on the mother cell side within the previously formed chitin ring. Deposition of additional cell wall material from both sides of the primary septum produces the secondary septum. An asymmetric degradation of the septum is required for cell separation and the chitinous septum remains with the mother cell as part of the bud scar. Cell separation depends on the CTSIcoded chitinase (Kuranda and Robbins, 1991) differentially regulated in mother and daughter cells by A CE2 (Dohrmann et al., 1982). It has been proposed that the chitin synthase Chslp exerts a repair function at the end of cytokinesis, counterbalancing the lytic effect of the chitinase (Cabib et al., 1992). Unfortunately, little is known about septum formation in fission yeast or about the lytic enzymes required to separate the two cells once the septum has been formed (Fankhauser and Simanis, 1994). On the other hand, disruption of the cwll+ gene caused no noticeable phenotype in S. pombe and hence further studies will be necessary to elucidate the possible role of cwlZ+ in morphogenesis and cell wall assembly. ACKNOWLEDGEMENTS We thank Dr Y. Sanchez and Dr J. C. Ribas for critical reading of the manuscript, C. Belinchon for photographic work and N. Skinner for correcting the English. M. Arellano acknowledges support from a fellowship granted by the University of Salamanca. This work was supported by grant B109 1-0437 from the Comision Interministerial de Ciencia y Tecnologia, Madrid, Spain. 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