JB
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cappellaro, C.
Right arrow Articles by Tanner, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cappellaro, C.
Right arrow Articles by Tanner, W.

 Previous Article  |  Next Article 

Journal of Bacteriology, October 1998, p. 5030-5037, Vol. 180, No. 19
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

New Potential Cell Wall Glucanases of Saccharomyces cerevisiae and Their Involvement in Mating

Corinna Cappellaro,dagger Vladimir Mrsa, and Widmar Tanner*

Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, 93040 Regensburg, Germany

Received 28 May 1998/Accepted 3 August 1998

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Biotinylation of intact Saccharomyces cerevisiae cells with a nonpermeant reagent (Sulfo-NHS-LC-Biotin) allowed the identification of seven cell wall proteins that were released from intact cells by dithiothreitol (DTT). By N-terminal sequencing, three of these proteins were identified as the known proteins beta -exoglucanase 1 (Exg1p), beta -endoglucanase (Bgl2p), and chitinase (Cts1p). One protein was related to the PIR protein family, whereas the remaining three (Scw3p, Scw4p, and Scw10p [for soluble cell wall proteins]) were found to be related to glucanases. Single knockouts of these three potential glucanases did not result in dramatic phenotypes. The double knockout of SCW4 and the homologous gene SCW10 resulted in slower growth, significantly increased release of proteins from intact cells by DTT, and highly decreased mating efficiency when these two genes were disrupted in both mating types. The synergistic behavior of the disruption of SCW4 and SCW10 was partly antagonized by the disruption of BGL2. The data are discussed in terms of a possible counterplay of transglucosidase and glucosidase activities.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Saccharomyces cerevisiae has become the best-known eucaryotic organism and serves as a model for the study of almost any problem in cell biology (2, 4). S. cerevisiae invests about 20% of its dry weight to build up the cell wall, but nevertheless, this extracellular organelle is not very well understood. Although great progress has recently been made in understanding the polysaccharide network of the wall (8), the proteins that contribute to this structure and amount to about 10% of the wall dry weight (7, 8, 16) have not been systematically and extensively studied. It has been shown that cell wall proteins can be subdivided into two classes: one class can be solubilized by sodium dodecyl sulfate (SDS) under reducing conditions; the other one is considered covalently linked to cell wall polysaccharides and is released only by beta -glucanase treatment (12, 23).

A major problem in definitely identifying cell wall proteins has been contamination with intracellular material. Recently, labeling procedures using a nonpermeant biotinylation reagent have been described (3, 14). With one of these methods, about 20 cell wall (cell surface) proteins could be labeled in S. cerevisiae (14). Among the covalently attached cell wall proteins (Ccw = covalently linked cell wall proteins), the proteins of the PIR family (proteins with internal repeats [22]) have been identified as wall components (14).

Here we describe the identification of seven biotinylated, SDS-soluble proteins (Scw = soluble cell wall proteins) by N-terminal sequencing. Three of them (beta -exoglucanase 1 [Exg1p], beta -endoglucanase [Bgl2p], and chitinase [Cts1p]) were already known (6, 9, 24), while one was identical to the recently identified covalently linked cell wall protein Ccw5p/Ccw11p, which belongs to the PIR protein family (14). The three previously unidentified proteins (Scw3p, Scw4p, and Scw10p) all show significant homology to glucanases. These gene products had not previously been identified as cell wall components. Single and double knockouts were made, defining synergistic and antagonistic functions of the described proteins.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Chemicals, strains, and growth conditions. Calcofluor white was obtained from Sigma, Sulfo-NHS-LC Biotin was from Pierce, and streptavidin-peroxidase was from Amersham. All of the S. cerevisiae strains used in this work were derived from SEY6210 (MATalpha leu2-3,112 ura3-52 his3-Delta 200 lys2-801 trp1-Delta 901 suc2-Delta 9 GAL) or SEY6211 (MATa ura3-52 leu2-3,112 his3-Delta 200 trp1-Delta 901 ade2-101 suc2-Delta 9 GAL) (19). Disruption of CCW5 was described by Mrsa et al. (14). All strains were grown in standard YPD medium (1% yeast extract, 2% peptone, 2% dextrose).

Isolation and purification of Scwps. Yeast cells (strain SEY6210) were harvested at an optical density at 578 nm (OD578) of 6 to 8 (1 U of OD578 corresponds to 107 cells), washed with water and with 25 mM Tris/HCl (pH 8.5) and finally resuspended in this buffer containing 2 mM dithiothreitol (DTT). Cells were shaken vigorously for 2 h at 4°C and then pelleted. The supernatant was concentrated by lyophilization, resuspended in water, and dialyzed against 2 mM Tris/HCl (pH 8.0)-1 mM EDTA. The extract was adjusted to 20 mM sodium acetate (pH 7.3)-150 mM NaCl-1 mM KCl-1 mM MnCl2-1 mM MgCl2 and bound to concanavalin A (ConA)-agarose (Sigma). After washing, the material bound to ConA was eluted with 500 mM alpha -methylmannoside. An amount equivalent to 15 g of cells was separated by SDS-polyacrylamide gel electrophoresis (PAGE) (10), blotted onto polyvinylidene difluoride membranes (Millipore), and stained with Coomassie, and the protein bands were cut out for sequencing.

For the purification of Scw4p and Scw10p, DTT extracts of mutant cells lacking one or the other of the two proteins were applied to a MonoQ fast protein liquid chromatography (FPLC) column (1 ml). Chromatography was performed in 20 mM Tris-HCl buffer (pH 6.0), and proteins were eluted in a 0 to 500 mM NaCl gradient (total volume, 25 ml) at a flow rate of 0.5 ml/min. Fractions (1 ml) were analyzed electrophoretically.

SDS-PAGE, blotting, and staining procedures. Cell surface labeling with Sulfo-NHS-LC-Biotin, SDS-PAGE, and blotting were carried out as previously described (14). For silver staining, the method of Morissey (13) was used, and the staining of gels for carbohydrates (with periodic acid-Schiff [PAS] stain) was done by the method of Zacharius et al. (25). Myosin (212 kDa), beta -galactosidase (116 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), catalase (57.5 kDA), and aldolase (40 kDa) were used as molecular mass standards.

DNA amplification and disruption of SCW genes. SCW genes were amplified by standard PCR methods using Taq polymerase. The identity of a gene was verified by restriction analysis. Disruptions were made by fusing N- and C-terminal parts of the gene of interest with marker genes. In particular, SCW4 (fragments -149 to -6 and 901 to 1166) was fused with LEU2, SCW10 (-180 to 130 and 958 to 1338) was fused with HIS3, SCW11 (-213 to 157 and 1435 to 1844) was fused with TRP1, and SCW3 (-181 to 139 and 1147 to 1490) was fused with TRP1 (numbers refer to the corresponding start ATG). The BGL2 disruption was done as previously described (6). Disruptions were tested by PCR and by streptavidin-peroxidase blots of protein extracts from Sulfo-NHS-LC-Biotin-labeled cells.

Calcofluor white treatment. YPD agar plates with 0 to 50-µg/ml calcofluor white were prepared. Yeast cells were diluted to 2 · 105/ml, 2 · 104/ml, and 2 · 103/ml, and 5 µl of each suspension was dropped onto the plates.

Mating test. Minimal medium agar plates lacking lysine and adenine, thus allowing the growth of diploid cells only, were prepared. Stationary-phase a and alpha  cells of the wild type and different mutants were mixed and diluted, and 5 µl of the suspension (corresponding to 104 cells) was dropped onto plates.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isolation and identification of cell wall proteins soluble under reducing conditions. Previous attempts to identify SDS-solubilized cell wall proteins prepared from isolated cell walls repeatedly led to the identification of glycolytic enzymes like enolase, 3-phosphoglyceraldehyde dehydrogenase, triosephosphate isomerase, etc. (14a, 20a). The alleged occurrence of glycolytic enzymes as cell wall components has, indeed, been reported in the literature (1). To avoid this probable artifact, surface proteins were labeled. Intact S. cerevisiae cells were treated with the biotin reagent sulfosuccinimidyl-6-(biotinamido)hexanoate, which is unable to penetrate the cell membrane. Subsequent extraction of purified cell walls with SDS under reducing conditions gave rise to nine major biotinylated proteins, which were visualized with streptavidin-horseradish peroxidase (14). Since the biotinylation reagent blocks N-terminal amino groups, purification of individual biotinylated cell surface proteins for sequencing required copurification of the corresponding nonbiotinylated material. When this procedure was followed, the first N-terminal sequence finally obtained again corresponded to enolase (2a). Thus, it became clear that although it was possible to label cell wall proteins specifically, after breaking the cells and separating the cell walls, these proteins were contaminated with glycolytic enzymes, which were present, of course, per cell in large excess. It was necessary, therefore, to release the SDS-soluble cell wall proteins without breaking the cells. As has often been shown before, several cell wall proteins could be released from intact cells with SH reagents (3, 17, 18). A comparison of patterns of biotinylated proteins from SDS-extracted cell walls with that of DTT-released proteins from intact cells (Fig. 1) strongly suggested that several major bands of the SDS extract could also be released under these milder conditions from intact cells. It is not certain, of course, whether the proteins with the same apparent molecular weight obtained under the two conditions are, indeed, identical. However, for reasons given above, further work was concentrated on the biotinylated proteins released from intact cells by DTT. They were purified via a ConA column. As expected, all of the proteins released from the column by alpha -methylmannoside gave positive PAS staining, indicating that they were glycoproteins (Fig. 2).


View larger version (35K):
[in this window]
[in a new window]
  		FIG. 1.   Comparison of two extraction methods for Sulfo-NHS-LC-Biotin-labeled cell wall proteins. This blot was stained with a streptavidin-peroxidase conjugate. (A) Extraction of intact cells with DTT. An amount of extract equivalent to 4 ml of cells with an OD578 of 5 was subjected to SDS-PAGE. (B) Cell walls isolated from broken cells were extracted by heating in Laemmli sample buffer under reducing conditions (2% SDS, 5% beta -mercaptoethanol). The equivalent of 200 µl of cells with an OD578 of 5 was applied. For the molecular weight standards used, see Materials and Methods. The values beside the gel are molecular sizes in kilodaltons.


View larger version (56K):
[in this window]
[in a new window]
  		FIG. 2.   Analysis of isolated proteins. (Left) Concentrated and ConA-purified DTT extract of intact cells stained for sugars (PAS) and protein (silver). (Right) Parallel extract isolated from biotinylated cells blotted and visualized with a streptavidin-peroxidase conjugate. The coincident patterns indicate that the purified materials are surface-exposed, glycosylated proteins. The names given resulted from N-terminal sequencing (Table 1). Cts1p, chitinase; Exg1p, exoglucanase 1; Bgl2p, beta -endoglucanase 2; Scw8p, protein with an unknown function; Scw3p, Scw4p, and Scw10p, soluble cell wall proteins 3, 4, and 10 (see text). ConA is a contamination due to the purification procedure.

When corresponding nonbiotinylated proteins obtained in an identical way were blotted onto Immobilon and N terminally sequenced, seven of them gave rise to informative sequences (Table 1). The 116-kD protein (Scw2p, according to Mrsa et al. [14]) turned out to be chitinase (9), and the 29-kD protein (Scw9p) and the 44-kD protein (Scw6p) were beta -endoglucanase (Bgl2p) and exoglucanase 1 (Exg1p), respectively (6, 23). The finding that these three known soluble cell wall proteins were found among the biotinylated protein bands indicated once more the high specificity of the labeling method. The Scw8p sequence has previously been found among the covalently linked cell wall proteins (14), which could be solubilized with laminarinase (Ccw5p), as well as with 30 mM NaOH (Ccw11p). The gene coding for this protein belongs to the PIR gene family, although the typical conserved repetitive sequence of this gene family (protein with internal repeats) occurs only once within this protein. The other three N-terminal sequences, corresponding to Scw3p (95 kDa, open reading frame [ORF] YNL066w), Scw4p (66 kDa, ORF YGR279c), and Scw10p (66 kDa, ORF YMR305c), were completed with the information from the yeast genome project. These genes code for previously unknown proteins, which, however, all show homology to glucanases. Since Scw4p and Scw10p are practically the same size, Scw10p could only be purified and sequenced after an scw4 mutant had been constructed (see below).

                              
View this table:
[in this window]
[in a new window]
  		TABLE 1.   N-terminal sequences of proteins shown in Fig. 2a

Three gene families of potential glucanases. The sequence of Scw4p was derived from the N-terminal sequence of the protein band with an apparent molecular mass of 66 kDa. This sequence was very similar to another one found in the yeast genome and designated Scw10p. Since both sequences have practically the same length and since the disruption of the SCW4 gene resulted only in partial disappearance of the protein band (see later), it was assumed that the remaining band corresponded to Scw10p. Therefore, the protein was extracted from the scw4 mutant cells, purified by MonoQ FPLC column chromatography, and sequenced. The sequence obtained, indeed, corresponded to that of Scw10p (Table 1). These two proteins are 63% identical; both contain a signal sequence, a Kex2p processing site, and no GPI anchoring sequence (Fig. 3). In addition, another ORF with significant homology to SCW4 and SCW10 has been found in the S. cerevisiae sequence and designated SCW11 (ORF YGL028c). A comparison of the three protein sequences, together with the known cell wall glucanase Bgl2p, is given in Fig. 3.


View larger version (54K):
[in this window]
[in a new window]
  		FIG. 3.   Scw4p and three related gene products are potential glucanases. Amino acid sequences of Scw4p (sequence 1), Scw10p (sequence 2), Scw11p (sequence 3), and Bgl2p (sequence 4) are compared. The Munich Information Center for Protein Sequences database was screened with the sequence of Scw4p (by using FASTA). The resulting ORFs were aligned by using the pileup function of The University of Wisconsin Genetics Computer Group package. ORFs are available from the Munich Information Center for Protein Sequences under the following accession numbers (PIR code): Scw4p, S64614; Scw10p, S53975; Scw11p, S64030, Bgl2p, A33499. Boldface letters are used when at least two amino acids are identical.

The Scw3p sequence was found to be most closely related to a beta -glucosidase of Candida wickerhamii (61% identity, 72% homology), which in this organism is required to ferment cellodextrins (21).

The four known S. cerevisiae glucanases are Exg1p, Exg2p, Bgl2p, and Ssg1p (6, 11, 20). They can be arranged according to the degree of homology, together with the new potential glucanases described in this work, as shown in Fig. 4. In Fig. 4, five additional ORFs, which may very well also code for glucanases or glucanase-related enzymes, were included according to their degrees of homology within the group of 13 genes. These putative glucanases can be subgrouped into three families as shown.


View larger version (14K):
[in this window]
[in a new window]
  		FIG. 4.   Grouping of known and potential glucanases. ORFs of gene products related to known glucanases were obtained from the Munich Information Center for Protein Sequences based on a FASTA search with the sequences of Exg1p, Scw4p, and Scw3p (S53916). Sequences were processed with the lineup function of the University of Wisconsin Genetics Computer Group package.

Phenotypes of SCW3, SCW4, SCW10, and SCW11 disruptants. Disruptions of genes SCW3, SCW4, SCW10, SCW11, and BGL2 were constructed and checked by PCR. As shown in Fig. 5, in the scw3 and scw8 mutants, the corresponding biotinylated protein bands were missing. The disruption of the SCW4 gene brought about a significant decrease in the intensity of the corresponding electrophoretic band but did not lead to its complete disappearance. Only when the SCW10 gene was also disrupted did the 66-kDa band seem to disappear; however, it was difficult to prove this since much more protein was extracted by DTT from the cell wall of the double mutant than from the wild type or single mutants (Fig. 5A).


View larger version (41K):
[in this window]
[in a new window]
  		FIG. 5.   scw4, scw10, scw8 and scw3 knockout mutants. The corresponding mutant cells were labeled with Sulfo-NHS-LC-biotin and extracted with DTT (scw4, scw10, and scw8), or whole cells were heated in Laemmli sample buffer (scw3). For the molecular size standards, see Materials and Methods. The arrows mark gene products missing in the mutants. Lane 5 in panel A contains 1/10 of the material in lane 4. WT, wild type.

Among the single mutants, only scw11 showed a growth or morphological phenotype. This mutant did not separate well after division and grew clumpy. Not even sonication could change this property significantly (data not shown).

In addition, double and triple disruptions were produced. Cells carrying the scw4 scw10 double disruption displayed morphological changes: irregular and frequently larger cells were seen (Fig. 6). The growth rate of this double mutant was also decreased, such that the doubling time increased from about 70 min (wild type) to 100 min. Surprisingly, the increase in doubling time of the scw4 scw10 double mutant was compensated if a third gene, BGL2, coding for a beta -endoglucanase (6), was also deleted. Corresponding behavior was observed when the amount of proteins released from intact cells with DTT was compared. As shown in Fig. 7, a large amount of protein could be released from the scw4 scw10 double mutant, which was not the case with the wild type or with any of the single mutants. Again, the additional deletion of the BGL2 gene significantly compensated the effect. The same behavior was observed when not the total protein but rather the biotinylated cell wall proteins were visualized by streptavidin (Fig. 5A). For both phenomena, increased doubling time and enhanced release of cell wall proteins, it was clear that the two gene products, Scw4p and Scw10p, act synergistically. The double mutants containing scw11 with scw4, scw10, or bgl2 did not show any synergism; they did not differ from the corresponding single knockouts (Fig. 7).


View larger version (91K):
[in this window]
[in a new window]
  		FIG. 6.   Morphology of wild-type (A) and scw4 scw10 double-mutant (B) cells.


View larger version (56K):
[in this window]
[in a new window]
  		FIG. 7.   Demonstration of cell wall lability. Log-phase cells were collected and extracted with DTT, and an aliquot was separated by SDS-PAGE and stained with Coomassie brilliant blue R-250. An amount corresponding to 6.6 ml of cells at an OD578 of 3.0 was applied to each lane of the gel. WT, wild type.

The synergism of Scw4p and Scw10p was also seen when the cells were tested for increased sensitivity to the cell wall-weakening agent calcofluor white (Fig. 8). Of the single knockouts, only scw10 showed somewhat increased sensitivity. Of the different double or multiple mutants tested, all combinations that included both scw4 and scw10 were found to be sensitive. The compensating effect of the BGL2 disruption was not observed in this case. To test if the observed properties of the scw4 scw10 double mutant were due to a change in the overall composition of the cell wall, the amounts of mannose, glucose, and N-acetylglucosamine were determined after a total wall hydrolysis with trifluoroacetic acid (4 M, 2 h). Cell walls of the wild type and scw4 and scw10 single mutants, the double mutant, and a triple mutant carrying an additional bgl2 mutation were tested, but no significant change in the carbohydrate composition of the walls could be detected. The percentage of mannose ranged between 56 and 58% in the stationary-phase cells or between 54 and 57% in the logarithmically growing strains. Glucose was present in amounts between 39 and 42% in stationary-phase cells and between 40 and 44% in logarithmic-phase cells, while the amount of N-acetylglucosamine was always between 2 and 3%. Such results indicate that the observed differences reflect finer structural changes in the wall rather than the overall carbohydrate composition.


View larger version (36K):
[in this window]
[in a new window]
  		FIG. 8.   Synergistic effect of the scw4 and scw10 mutations on calcofluor white sensitivity. Five-microliter volumes of different dilutions (2 × 105, 2 × 104, and 2 × 103 cells/ml) of stationary-phase cell cultures were dropped onto YPD plates containing 45 µg of calcofluor white and incubated for 5 days at 29°C. WT, wild type.

The potential glucanases encoded by SCW4 and SCW10 also play a role in the mating process. When the various single and multiple disruptions were introduced into a and alpha  cells and the cells were mixed for mating and incubated on agar plates selecting for diploids, the pattern shown in Fig. 9 was obtained. The double-mutant cross scw4 scw10 × scw4 scw10 showed poor mating. This phenotype was also partly compensated by deleting BGL2 in addition (Fig. 9), an effect that was not due simply to the introduction of the URA3 disruption marker, as shown by checking with a corresponding plasmid (data not shown). The synergism concerning the decrease in mating efficiency was again observed only with Scw4p and Scw10p, but not with Scw11p (data not shown). To study this effect in more detail, the mating of double mutant a and alpha  strains was observed microscopically. It could be seen that shmoo formation, as well as contact between cells of opposite mating types and the formation of agglutinates, was not affected in the mutants. However, the formation of zygotes was much less frequent than in the case of the mating of wild-type cells.


View larger version (33K):
[in this window]
[in a new window]
  		FIG. 9.   Requirement of Scw4p and Scw10p for mating. The mutants indicated in a Lys- background (SEY 6210, MATalpha ) were mated with mutants in an Ade- background (SEY6211, MATa). Portions (104 cells) of stationary-phase cell cultures were mixed and dropped onto Lys- Ade- minimal-medium plates for detection of diploid (Lys+ Ade+) cells. WT, wild type.

Finally, the effect of scw4 and scw10 mutations on the amount of proteins covalently linked in the cell wall was also tested. As can be seen in Fig. 10, the scw10 single mutant showed no difference, whereas scw4 had some effect on the incorporation of the 55 to 70-kDa material extractable by laminarinase. The double mutation, however, decreased the amount of all glucanase-extractable protein bands significantly, again demonstrating the synergistic activity of the two proteins.


View larger version (39K):
[in this window]
[in a new window]
  		FIG. 10.   Influence of Scw4p and Scw10p on the incorporation of covalently bound cell wall proteins. Cell wall proteins of the wild type (WT) and mutants were labeled by biotinylation (see Materials and Methods). Walls were purified, and the SDS-extractable proteins were extracted by Laemmli sample buffer. The remaining covalently attached proteins were then solubilized by laminarinase, subjected to electrophoresis, and blotted, and the blot was visualized by using a streptavidin-peroxidase conjugate. Each lane contains proteins obtained from cells corresponding to 25 U of OD600. The values on the right are molecular sizes in kilodaltons.

To investigate directly the potential exo- and/or endoglucosidase activity of Scw4p, the protein was extracted from the cell walls of the scw10 mutant and purified to homogeneity by using a MonoQ FPLC column. However, no hydrolytic activity of the purified protein could be detected by using p-nitrophenylglucoside, laminarin, yeast glucan, chitin, or pustulan as the substrate, perhaps indicating either a more complex activity of Scw4p or an inactivation caused, for example, by reduction.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The use of nonpermeant biotinylation agents to label cell surface proteins (3, 14) most likely allows the identification of the main protein components of the cell wall. In a recent study, it was shown for S. cerevisiae that 20 proteins labeled by biotin could be distinguished in this way (14). These proteins were solubilized from intensively washed, isolated cell walls either by SDS or, following SDS extraction, by laminarinase treatment or by mild alkaline treatment. Thus, in principle, the procedure first suggested by Valentin et al. (23) to distinguish between extractable and covalently bound cell wall proteins has been followed. This procedure has been extended due to the observation that part of the covalently bound proteins could be released from the walls by 30 mM NaOH. The latter, after SDS-PAGE, were pure enough to be sequenced and identified. They turned out to be the PIR gene products (14), a gene family originally conspicuous because of internal repeats in the ORFs (22).

As pointed out here, the SDS-extracted cell wall proteins could not be purified enough to be sequenced. However, since release of proteins from intact cells by DTT resulted in a pattern comparable to that of biotinylated proteins after SDS-PAGE, such DTT-released cell wall proteins were used for further characterization, although the method required about 20 times more cells than the SDS extraction. Of the seven N-terminal sequences obtained from this group of proteins (Table 1), three were well-known cell wall proteins, showing that the labeling procedure was specific. One protein, Scw8p, was found to be the same as one of the previously identified Pir proteins (Ccw5p), while the remaining three were related, but not identical, to previously described glucanases. Thus, the Scw4p and Scw10p proteins, as well as the related Scw11p protein, detected as a sequence contained in the yeast genome, were related to Bgl2p, an endo-beta -glucanase, with 49 to 56% similarity and 27 to 29% identity. The Scw4p, Scw10p, and Scw11p proteins are 52 to 75% similar and 36 to 63% identical (Fig. 3 and 4). This group of cell wall proteins, therefore most likely represents potential endoglucanases or possibly transglucosidases (5). Scw3p, on the other hand, is not related to any known S. cerevisiae glucanase but rather to a beta -glucosidase of C. wickerhamii (21) that degrades cellodextrins extracellularly. Since four additional S. cerevisiae ORFs detected in the yeast genome are related to the Scw3p protein identified here as a cell wall protein, this whole group of five gene products (Fig. 4) may constitute extracellular proteins involved in the degradation of polymeric glucans to make them available for fermentation. It seems possible that such proteins are substrate induced and therefore may not have been present on the cells used here.

Since processes like growth, budding, mating, ascospore formation and release, and polysaccharide fermentation most likely all require cell wall glucanases, it is not too surprising that as many as 13 genes seem to encode proteins with this or related enzymatic activities. In addition, not all of these enzymes need to be hydrolytic ones. As shown by Goldman et al. (5), the Bgl2p protein, under certain conditions, also shows transglucosylating activity in vitro. On the other hand, this cannot be taken as evidence that in vivo, this enzyme really acts as a transglucosidase. Furthermore, our attempts to demonstrate exo- or endoglucanase activity of Scw4p failed, suggesting that this protein might preferentially catalyze a transglucosylation reaction. However, the possibility that the enzyme was inactivated during the extraction with DTT cannot be excluded, either. The observation that in the scw4 scw10 double mutant, the amount of laminarinase-extractable proteins is significantly decreased suggests that the two proteins may play a direct or indirect role in the anchoring of proteins to beta -1,6-glucan, as has been suggested (8, 12). However, since some protein could still be released from the wall by glucanases even in the double mutant, a corresponding low activity must still be present in the cell wall. The fact that the mannose content of the mutant cell wall did not differ significantly from that of the wild type could be explained if proteins were attached to, or associated with, the wall by an alternative mechanism in a glucanase-insoluble form or if they could even become members of the cell wall SDS- or DTT-soluble proteins. The latter assumption would partially explain the increased amount of cell wall proteins released by DTT from the scw4 scw10 cells (Fig. 7). It is interesting that in a number of phenomena, the potential glycosidases Scw4p and Scw10p show synergistic effects, whereas Bgl2p acts antagonistically in relation to the same phenomena (growth rate, release of proteins, mating). The observation that much more cell wall proteins are released by DTT in an scw4 scw10 double mutant and significantly fewer are released when BGL2 is disrupted in addition might suggest that Scw4p and Scw10p are transglucosidases. Their disruption leads to a less stable cell wall, especially if the BGL2 gene product acts as an endoglucosidase and degrades nontransglucosylated glucan chains (see the effect of calcofluor white, Fig. 7). Disruption of BGL2 under these conditions would give rise to a less destabilized glucan layer. The decrease in mating efficiency in scw4 scw10 double mutants could be interpreted in an analogous way: transglucosylation of glucan polymers may be a prerequisite for stabilization of the fusing stage of the two haploid mating partners. Again, decreased glucan transglucosylation may favor the degradation of glucan chains by Bgl2p, thus further preventing successful mating. In any event, the cell wall proteins coded for by SCW4 and its homologue SCW10, which most likely represent glucanases or transglucosidases, are the first such proteins shown to be involved in mating. The only glucanase known that has a similar specific function for yeast cells is that encoded by the sporulation-specific glucanase gene SSG1 (15, 20). The precise role of the Scw4p-Scw10p-Bgl2p glucanase family, especially in mating, needs to be elucidated in future work.

    ACKNOWLEDGMENTS

We are grateful to Rainer Deutzmann, Regensburg, Germany, for sequencing the purified cell wall proteins, to Ludwig Lehle for determining the sugar composition, and to Ilja Hagen and Margit Ecker for experimental help.

This work has been supported by grants from the Deutsche Forschungsgemeinschaft (SFB 521) and from the Fonds der Chemischen Industrie.

    FOOTNOTES

* Corresponding author. Mailing address: Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, 93040 Regensburg, Germany. Phone: 49 941 943 3018. Fax: 49 941 943 3352. E-mail: widmar.tanner{at}biologie.uni-regensburg.de.

dagger Present address: Institut für molekulare Genetik, 37077 Göttingen, Germany.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1. Angiolella, L., M. Facchin, A. Stringaro, B. Maras, N. Simonetti, and A. Cassone. 1996. Identification of a glucan-associated enolase as a main cell wall protein of Candida albicans and an indirect target of lipopeptide antimycotics. J. Infect. Dis. 173:684-690[Medline].
2. Broach, J. R., E. W. Jones, and J. R. Pringle (ed.). 1991. The molecular and cellular biology of the yeast Saccharomyces, vol. 1. Genome dynamics, protein synthesis and energetics. Cold Spring Harbor Laboratory Press, Plainview, N.Y.
2a. Cappellaro, C. Unpublished data.
3. Casanova, M., J. L. Lopez-Ribot, J. P. Martinez, and R. Sentandreu. 1992. Characterization of cell wall proteins from yeast and mycelial cells of Candida albicans by labeling with biotin: comparison with other techniques. Infect. Immun. 60:4898-4906[Abstract/Free Full Text].
4. Goffeau, A., B. G. Barrell, H. Bussey, R. W. Davis, B. Dujon, et al. 1996. Life with 6000 genes. Science 274:546-567[Abstract/Free Full Text].
5. Goldman, R. C., P. A. Sullivan, D. Zakula, and J. O. Capobianco. 1995. Kinetics of beta-1,3 glucan interaction at the donor and acceptor sites of the fungal glucosyltransferase encoded by the BGL2 gene. Eur. J. Biochem. 227:372-378[Medline].
6. Klebl, F., and W. Tanner. 1989. Molecular cloning of a cell wall exo-beta -1,3-glucanase from Saccharomyces cerevisiae. J. Bacteriol. 171:6259-6264[Abstract/Free Full Text].
7. Klis, F. M. 1994. Review: cell wall assembly in yeast. Yeast 10:851-869[Medline].
8. Kollar, R., B. B. Reinhold, E. Petrakova, H. J. C. Yeh, G. Ashwell, J. Drgonova, J. C. Kapteyn, F. M. Klis, and E. Cabib. 1997. Architecture of the yeast cell wall. beta (1right-arrow6)-Glucan interconnects mannoprotein, beta (1right-arrow3)-glucan, and chitin. J. Biol. Chem. 272:17762-17775[Abstract/Free Full Text].
9. Kuranda, M. J., and P. W. Robbins. 1991. Chitinase is required for cell separation during growth of Saccharomyces cerevisiae. J. Biol. Chem. 266:19758-19767[Abstract/Free Full Text].
10. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685[Medline].
11. Larriba, G., E. Andaluz, R. Cueva, and R. D. Basco. 1995. Molecular biology of yeast exoglucanases. FEMS Microbiol. Lett. 125:121-126[Medline].
12. Montijn, R. C., J. van Rinsum, F. A. Van Schoger, and F. M. Klis. 1994. Glucomannoproteins in the cell wall of Saccharomyces cerevisiae contain a novel type of carbohydrate side-chain. J. Biol. Chem. 269:19338-19342[Abstract/Free Full Text].
13. Morissey, J. H. 1981. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal. Biochem. 117:307-310[Medline].
14. Mrsa, V., T. Seidl, M. Gentzsch, and W. Tanner. 1997. Specific labeling of cell wall proteins by biotinylation. Identification of four covalently linked O-mannosylated proteins of S. cerevisiae. Yeast 13:1145-1149[Medline].
14a. Mrsa, V., et al. Unpublished data.
15. Muthukumar, G., S. H. Suhng, P. T. Magee, R. D. Jewell, and D. A. Primerano. 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/Free Full Text].
16. Orlean, P. 1997. Biogenesis of yeast wall and surface components, p. 229-362. In J. R. Pringle, J. R. Broach, and E. W. Jones (ed.), The molecular and cellular biology of the yeast Saccharomyces, vol. 3. Cell cycle and cell biology. Cold Spring Harbor Laboratory Press, Plainview, N.Y.
17. Orlean, P., H. Ammer, M. Watzele, and W. Tanner. 1986. Synthesis of an O-glycosylated cell surface protein induced in yeast Saccharomyces cerevisiae by alpha factor. Proc. Natl. Acad. Sci. USA 83:6263-6266[Abstract/Free Full Text].
18. Pierce, M., and C. E. Ballou. 1983. Cell-cell recognition in yeast. Characterization of the sexual agglutination factors from Saccharomyces kluyveri. J. Biol. Chem. 258:3576-3582[Abstract/Free Full Text].
19. Robinson, J. S., D. J. Klionsky, L. M. Banta, and S. D. Emr. 1988. Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol. Cell. Biol. 8:4936-4948[Abstract/Free Full Text].
20. San Segundo, P., J. Correa, C. R. Vazquez de Aldana, and F. del Rey. 1993. SSG1, a gene encoding a sporulation-specific 1,3-beta -glucanase in Saccharomyces cerevisiae. J. Bacteriol. 175:3823-3837[Abstract/Free Full Text].
20a. Sharma, C. B. Unpublished data.
21. Skory, C. D., and S. N. Freer. 1995. Cloning and characterization of a gene encoding a cell-bound, extracellular beta -glucosidase in the yeast Candida wickerhamii. Appl. Environ. Microbiol. 61:518-525[Abstract].
22. Toh-e, A., S. Yasunaga, H. Nisogi, K. Tanaka, T. Oguchi, and Y. Matsui. 1993. Three yeast genes, PIR1, PIR2 and PIR3, containing internal tandem repeats, are related to each other, and PIR1 and PIR2 are required for tolerance to heat shock. Yeast 9:481-494[Medline].
23. Valentin, E., W. Herrero, J. F. I. Pastor, and R. Sentandreu. 1984. Solubilization and analysis of mannoprotein molecules from the cell wall of Saccharomyces cerevisiae. J. Gen. Microbiol. 130:1419-1428.
24. Vasquez de Aldana, C. R., J. Correa, P. San Segundo, A. Bueno, A. R. Nebreda, E. Mendez, and F. del Rey. 1991. Nucleotide sequence of the exo-1,3-beta-glucanase-encoding gene, EXG1, of the yeast Saccharomyces cerevisiae. Gene 97:173-182[Medline].
25. Zacharius, R. M., T. E. Zell, J. H. Morisson, and J. J. Woodlock. 1969. Glycoprotein staining following electrophoresis on acrylamide gels. Anal. Biochem. 30:148-152[Medline].

Journal of Bacteriology, October 1998, p. 5030-5037, Vol. 180, No. 19
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cappellaro, C.
Right arrow Articles by Tanner, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cappellaro, C.
Right arrow Articles by Tanner, W.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Appl. Environ. Microbiol. Infect. Immun. Eukaryot. Cell
Mol. Cell. Biol. J. Virol. Microbiol. Mol. Biol. Rev.
ALL ASM JOURNALS