Laboratory of Biochemistry, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10 000 Zagreb, Croatia
Correspondence
Vladimir Mra
vmrsa{at}pbf.hr
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ABSTRACT |
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INTRODUCTION |
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Since 1989, and the first report on cloning and partial characterization of a Saccharomyces cerevisiae integral cell wall protein, Bgl2p (Klebl & Tanner, 1989), later characterized in vitro as endoglucanase (Mr
a et al., 1993
), these efforts have resulted in the identification, cloning, characterization and often disruption of corresponding genes of over 30 different proteins isolated from the walls of many different yeasts, particularly S. cerevisiae (Van Der Vaart et al., 1995
; Cappellaro et al., 1998
; Mr
a et al., 1997
; Rodriguez-Pena et al., 2000
). In all cases the disruptants reported survived the lack of the depleted protein and required no osmotic stabilization for growth (Cappellaro et al., 1998
; Mr
a & Tanner, 1999
; Moukadiri et al., 1997
; Mr
a et al., 1999
; Rodriguez-Pena et al., 2000
). Since a significant genome redundancy was observed, particularly in S. cerevisiae, multiple gene deletions have been performed. These, however, had little apparent effect on cellular functions and no physiologically significant phenotype was observed (Mr
a & Tanner, 1999
), except for increased sensitivity to some chemical drugs like Calcofluor White (CFW) or Congo Red, and sometimes changed growth behaviour. As a consequence, the present understanding of the physiological functions of yeast cell wall proteins is rather poor, which is particularly intriguing bearing in mind their number.
In this work, additional phenotypic characteristics of different single and multiple cell wall protein mutants lacking either non-covalently or covalently attached proteins, respectively, were observed at the level of individual cells in an attempt to gain further insight into their significance for the cell.
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METHODS |
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CFW staining.
Cells were grown to mid-exponential phase and fixed by the addition of formaldehyde to a final concentration of 3·7 %. Cells were subsequently incubated for 1 h at 30 °C, washed with 0·05 M phosphate buffer, pH 8·0, and resuspended in the same buffer. CFW (Sigma) was added to a final concentration of 2 µg ml1. After 15 min incubation at room temperature, cells were washed in phosphate buffer. Chitin in the cell wall was visualized by fluorescence microscopy.
Nuclear DNA staining.
Exponential-phase cells were fixed by the addition of formaldehyde to a final concentration of 3·7 % and incubated for 1 h at 30 °C. After that cells were washed in 0·05 M phosphate buffer, pH 8·0, and fixed with 70 % ethanol for 20 min at room temperature. Cells were washed once and resuspended in the same buffer. Nuclear DNA was stained with 4,6-diamino-2-phenylindole (DAPI) by adding the dye into the cell suspension to a final concentration of 1 µg ml1. After 15 min incubation at room temperature, cells were washed in phosphate buffer. Samples were observed by fluorescence microscopy.
Methylene blue staining.
Cells were grown in standard YPD medium or in YPD medium with 1 M sorbitol. The cell suspension was mixed with an equal volume of methylene blue and incubated for 5 min at room temperature. Dead, blue-coloured cells were scored (Smart et al., 1999). Typically, about 1000 cells were counted for each strain in each experiment, and every experiment was repeated at least three times. Mean values, together with the obtained ranges, are presented in diagrams.
MTT assay.
Cells were grown in standard YPD medium to early exponential or stationary phase and washed with water. Cell density was adjusted to an OD600 of 2 (corresponding to approx. 2x107 cells ml1). Cells from 1 ml suspension were harvested and resuspended in 0·4 ml 10 % MTT [3-(4,5-dimethylthiazoyl-2-yl) 2,5-diphenyltetrazolium bromide]. The mixture was incubated at room temperature with shaking for 2 h. After that cells were harvested and resuspended in 1 ml acid 2-propanol (0·04 M HCl in 2-propanol). The suspension was agitated for 10 min, then centrifuged at 8000 r.p.m. The OD540 of the supernatant was then measured (Garlier & Thomasett, 1986).
Transformation of ccw5ccw6ccw7 with CCW5.
Mutant strain ccw5ccw6ccw7 was transformed with the CCW5 gene cloned into the SmaI restriction site of yeast episomal plasmid YEp351. Transformation was accomplished by the standard lithium-acetate method.
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RESULTS AND DISCUSSION |
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To investigate the role of yeast cell wall proteins of each of the three classes, namely SDS-extractable (Scw-proteins), glucanase-extractable (GPI-anchored proteins) and NaOH-extractable (Pir) proteins, different mutants were constructed lacking the most prominent and abundant of them (Table 1). Some of these mutants, such as the one lacking all four known Pir-proteins, have already been described and some of their phenotypic characteristics have been reported (Mr
a et al., 1997
; Mr
a & Tanner, 1999
). However, these mutants could not help us to estimate the physiological significance of these proteins. Therefore, we decided to investigate properties such as morphological changes and survival capacity of these mutants under physiological conditions. First, the mortality of the mutants grown under usual laboratory aerobic conditions in YPD medium was investigated. The number of dead cells was estimated by methylene blue staining (Smart et al., 1999
). Other methods currently used for this purpose, including simple counting of colony forming units, were found to be inappropriate, since some of the mutants exhibited clumpy growth. In most clumps both dead and living cells were found, thus direct microscopic investigation was required to obtain accurate data. To avoid the possibility of artefacts potentially caused by the staining procedure, the number of dead cells was also estimated by the MTT assay. Unlike methylene blue, MTT stains living cells and the assay is based on a different principle (Garlier &Thomasett, 1986
; Zheng & Ben, 1992
), but the data obtained by both methods were essentially the same. Results presented in Fig. 1
show that some mutants show much higher mortality in normal laboratory cultivation. Among different Scw (non-covalently linked, SDS-soluble wall proteins; Fig. 1a
) mutant scw4 had a somewhat increased mortality compared to wild-type, scw10 or scw11. All three proteins are homologues of known glucanases or transglucosidases and particularly SCW4 and SCW10 share a very high degree of identity (Cappellaro et al., 1998
). Therefore, it was not surprising that an scw4scw10 double mutant had a significantly increased mortality, reaching about 5 % dead cells in culture. Unexpectedly, an additional scw11 mutation led to suppression of the observed phenotype, indicating that Scw11p has an activity antagonistic to that of Scw4p and Scw10p. Further deletion of another known endoglucanase gene, BGL2, led to a further increase in the mortality rate and, consistently, the highest number of dead cells was found in a triple scw4scw10bgl2 mutant.
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As shown in Fig. 1(b), depletion of Pir-proteins from the cell wall had an even more pronounced effect on the mortality of cells. A single ccw5 mutation causes a significant increase in the number of dead cells and, as reported for other mutant features (Mr
a & Tanner, 1999
), the phenotype was more pronounced when more members of this protein family were depleted, reaching nearly 8 % dead cells in a ccw5ccw6ccw7ccw8 mutant. A decreased growth rate of about 2030 %, which could be a result of increased mortality, has been reported for Pir-protein mutants (Mr
a et al., 1997
), but the accurate estimation of growth rates of some cell wall protein mutants is often made difficult by their clumpy growth.
The influence of GPI-anchored proteins on the viability of cells was also investigated. Since there is no apparent protein family in this group of proteins, although in some cases pairs of partially homologous proteins can be observed, a mutant lacking the five proteins reported to be the most abundant in the wall was constructed. It was found that this mutant also had an increased mortality compared to wild-type, although not as high as for the other two groups of wall proteins (Fig. 1c).
To investigate the cause of death of cells in culture, cells were grown in medium containing sorbitol as osmotic stabilizer. Fig. 1 shows that in all cases significant suppression of the phenotype was achieved, suggesting that mutant cells died due to their osmotic instability. Electrophoretic analysis of proteins from the culture medium supported this finding, since in mutants with higher mortality more intracellular proteins were found in the medium (not shown). Thus, although the lack of single cell wall proteins showed no pronounced phenotype, mutations of entire protein families or groups of proteins affected the main physiological function of the wall, as observed by the staining of individual cells.
Under natural physiological conditions yeast cells probably stay in stationary phase for most of the time. Therefore, the importance of different groups of wall proteins for cell viability was also estimated when cells were allowed to enter the stationary phase of growth (typically after 48100 h cultivation in YPD). As shown in Fig. 2, the mortality rate of scw mutants was only slightly increased in stationary cells. In contrast, the lack of Pir-proteins, as well as GPI-anchored proteins, had a much more pronounced effect, leading to up to 25 % of dead cells in the culture. These results indicate that the functions of these proteins are related to this growth phase or at least that the disabilities of mutants become critical only when cells enter stationary phase. Pir-proteins belong to the same family and share high sequence homologies (Mr
a et al., 1997
). They also seem to have similar physiological role(s) as indicated previously (Mr
a & Tanner, 1999
) and in this study by the cumulative effect of multiple mutations and by the fact that transformation of the multiple mutants with just one of the wild-type genes (CCW5 expressed from a high-copy-number plasmid) significantly restored wild-type characteristics. In contrast, the selection of mutated GPI-anchored proteins was done simply by their relative abundance in the wall as they do not seem to belong to any particular protein family. Therefore, their individual contribution in the overall phenotype may be different. An example is Ccw12p which seems to have very little impact on exponential-phase cells but is very important for cells in the stationary phase.
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To prove that the observed phenotype was indeed a direct result of the depletion of wall proteins, the ccw5ccw6ccw7 mutant was transformed with a plasmid carrying CCW5. As shown in Fig. 2(b) (ccw5ccw6ccw7+CCW5), significant reversion to wild-type characteristics was achieved, indicating that members of the Pir-family can functionally complement each other.
An interesting observation was made upon closer observation of mutants lacking Pir-proteins. In many cases, particularly if cells were grown in minimal (yeast nitrogen base) medium, mother cells with two daughters attached could be seen. Usually, as shown in Fig. 3, both daughters stained with methylene blue, while the mother was still alive (in our laboratory jargon we called such structures Mickey Mouse cells' for reasons obvious in Fig. 3
). No mothers with more than two daughters were observed in the culture and the phenomenon was specific for Pir-protein mutants. It was observed neither in other mutants nor in very rare cases when dead wild-type cells were found in the medium (Fig. 3
). Staining with DAPI showed that all cells, including the dead daughters, contained nuclei, so death occurred at a later stage of development and was not connected to formation or transport of new chromosomes. The fact that mother and daughters of multiple Pir-protein mutants showed different staining indicated that the septum between the cells was already complete, preventing diffusion of mother enzymes to daughter cells, but detachment of daughters could not take place. CFW staining corroborated that since the chitin septum between the cells could clearly be seen and the staining pattern, as well as intensity, was very similar to that of the wild-type (not shown), no increase in the amount of chitin was observed in the mutant.
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Received 3 May 2004;
revised 30 June 2004;
accepted 8 July 2004.
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