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YEAST

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Cyclic Variations in the Permeability of the Cell Wall of Saccharomyces cerevisiae JOHANNES G. DE NOBEL, FRANS M. KLIS, ARTHUR RAM, HANS VAN UNEN, JAN PRIEM, TEUN MUNNIK AND HERMAN VAN DEN ENDE University oj’Amsterdam, Molecular Cell Biology, Biotechnology Center, Kruislaan 318, 1098 SM Amsterdam, The Netherlands

Received 15 January 1991; revised 9 March 1991

To study cell-cycle-related variations in wall permeability of Saccharomyces cerevbiae, two approaches were used. First, an asynchronous culture was fractionated by centrifugal elutriation into subpopulations containing cells of increasing size. The subpopulations representeddifferent stagesof the cell cycle as judged by light microscopy. Cell wall porosity increased when these subpopulationsbecame enriched with budded cells. Secondly, synchronous cultures were obtained by releasing MA Ta cells from alpha-factor induced GI-arrest. These cultures grew synchronously for at least two generations. The cell wall porosity increased sharply in these cultures, shortly before buds became visible and was maximal during the initial stagesof bud growth. I t decreased in cells which had completed nuclear migration and before abscission of the bud had occurred. The porosity reached its lowest value during abscission and in unbudded cells. We examined the incorporation of mannoproteinsinto the wall during the cell cycle. SDS-extractablemannoproteins were incorporated continuously. However, the incorporation of glucanase-extractable mannoproteins, which are known to affect cell wall porosity, showed cyclic oscillations and reached its maximum after nuclear migration. This coincided with a rapid decrease in cell wall porosity, indicating that glucanase-extractable mannoproteins might contribute to this decrease. KEY WORDS - Cell wall

porosity; cell cycle; centrifugal elutriation; synchronous growth.

INTRODUCTION permeability is an important property of the wall of Saccharomyces cerevisiae, because it limits the release of homologous (e.g. periplasmic proteins; De Nobel et al., 1989) and heterologous proteins into the medium, and the efficiency by which yeast cells are transformed by heterologous DNA (Brzobohaty and K o v k , 1986). Scherreretal. (1974)estimated the average pore size of walls of stationary-phase cells to beabout 0.89 nm; thislimits the passageofmolecules larger than 760 Da. However, exponentially growing cells have been shown to secrete and internalizecompounds of much larger molecular mass into or from the medium (Wood et al., 1985; Pentilla et al., 1988; MacKay et al., 1988; Makarow, 1985; De Nobel et al., 1989). This indicates that the permeability of growingcellsis much higher than in stationary-phase cells. Using different techniques, we found the cell wall of growing cells to be permeable to molecules with a hydrodynamic radius of up to 5.8 nm, which corresponds with globular proteins with a molecular mass of 400 000 (De Nobel et al., 1989, 1990a). In 0749 503X:91/060589 10 SO5.00

addition, it was shown that the porosity of the wall varies with growth phase and medium composition and isstronglyaffected by cell turgor(DeNobe1 etal., 1990a,b). The cell wall of yeast consists of almost equal amounts of glucan and mannoprotein, and a small amount of chitin (Fleet and PhaR, 1981; Wessels and Sietsma, 1981; Ballou, 1982; Cabib et al., 1982). The mannoproteins can be classified as (a) SDS-extractable mannoproteins, and (b) SDS-nonextractable, but glucanase-extractable mannoproteins (Valentin et al., 1984). We have shown that the glucanase-extractable mannoproteins limit the cell wall porosity, in particular by their large mannan side-chains (De Nobel et al., 1990b). In view of this, the following data indicate that cell wall porosity might vary during the cell cycle. First, the cell wall of the bud is thinner than that of the mother, which seems to result from differences in the mannoprotein layer (Sentandreu and Northcote, 1969; Linnemans et al., 1977). Hence, the permeabilityofthewallin thebudmightdifferfrom that in the mother. Secondly, assembly of the cell wall is

590 predominantly confined to the growing bud (Sloat et al., 1981; Schekman and Novick, 1982). In addition, several authors have found discontinuities in the rate of incorporation of glucan and mannan during cell-cycle progression. A considerable reduction in the rate of synthesis occurs during cytokinesis and the prebudding phase (Wiemken et al., 1970; Biely et al., 1973; Sierra et al., 1973;Hayashibe et al., 1970, 1977; Biely, 1978). We show here that the porosity of the cell wall varies during the cell cycle and that the lowest permeability is reached when the rate of incorporation of the glucanase-extractable mannoproteins is maximal. MATERIALS AND METHODS Yeast strain andgrowth conditions Saccharomyces cerevisiae strain X2 180-1A was obtained from the Yeast Genetic Stock Center, Berkeley, California, U.S.A. The cells were grown at 28°C in YPG medium 1% (w/v) yeast extract, Gibco; 1% (w/v) bactopeptone, Difco; 3% (w/v) glucose]. Labeling experiments were performed in minimal medium [0.67% (w/v) Yeast Nitrogen Base without amino acids, Difco; 2% (w/v) glucose]. Age fructionation by centrifugal elutriation

The cells were harvested in the early exponential phase (absorbance at 530 nm = 2; this corresponds with 1 mg fresh weight per ml). The cells (400mg fresh weight, which corresponds with 12x109cells) were washed three times with ice-cold distilled water and resuspended in 5 ml distilled water. Cycloheximide (10 pg/ml) and sodium azide (0.5 mg/ml) were added to inhibit cell growth. The washed cells were sonicated briefly to disperse aggregates. A fraction of these cells was stored at 4°C as a control to the remainder, which was loaded into the Beckman JE-6B elutriator rotor. Regulation of the counterflow with distilled water at 4°C was as described by Van Doorn et al. (1988). The rotor speed was kept at 3400 rpm. Loading of the cells was carried out at a flow rate of 10 ml/min and, subsequently, the loaded cells were equilibrated in the elutriation chamber at a flow rate of 12ml/min. The first cells were collected at a flow rate of 14ml/min, successive fractions were collected at increments in the flow rate of 2ml/min until a rate of 30ml/min was reached. The next two fractions were at 35 and 40 ml/min and, subsequently, the centrifuge was switched off to collect any remaining cells. The

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collected fractions each had a volume of l00ml. Samples of 1.5 ml were taken from the elutriation fractions and fixed by addition of formaldehyde to a final concentration of 0.13% (w/v). These samples were used for the assessment of the total cell number by counting in a hemocytometer. In addition, nuclear staining of the cells was performed (see below) to determine the relative abundance of different cell stages in the elutriation fractions. The cells in the remaining 98.5ml of the elutriation fractions were collected by filtration (SM I13 cellulose nitrate filters, 0.45 pm, Sartorius GmbH, Gottingen). The cells were resuspended and washed in I0 mM-Tris-HC1, pH 7.4, prior to determination of their fresh weight. Subsequently, the relative cell wall porosity was determined with the polycation assay (see below). Synchronous cultures Cells were cultured asynchronously a t 28°C to an absorbance at 530 nm of 1.5. Alpha-factor was added to a final concentration of 4 pg/ml, and after 2 hofcontinuedgrowthat28°C thecellswerewashed with pre-chilled YPG medium to remove the pheromone. Subsequently, the cells were resuspended in fresh YPG medium and culturing was continued at 20°C. This temperature was chosen to extend the duration ofthe cell cycle ofthe synchronized cells and to allow a more detailed analysis of the cell cycle. Samples were taken every 30 min. An aliquot from the cells was fixed with 0.13% (w/v) formaldehyde and sonicated briefly to break up aggregates. Subsequently, the total cell number and the relative abundance of the different cell stages were determined as described for the elutriation fractions. The remainder of the samples was washed with 10 mMTris-HCI, pH 7.4, and, subsequently, the porosity of the cell walls was determined with the polycation assay, or the cells were used for labeling experiments. In the case ofmannoprotein labeling, the cells (60 mg of fresh weight) were resuspended in 25 ml of minimal medium to which 5 pCi of a ['4C]amino-acid mixture was added. These cells were allowed to grow in this medium for another 30 min at 23°C prior to final harvesting. Subsequently, the cell walls were isolated and mannoproteins were extracted (see below). Incorporation of radioactive label into the various extracts was determined by scintillation counting. Nuclear staining The relative abundance of various cell types was determined in samples from the synchronized

CYCLIC VARIATIONS IN THE PERMEABILITY OF THE CELL WALL OF SACCHAROMYCES CEREVISIAE

cultures and from the elutriation fractions. Formaldehyde-fixed cells were spinned down and resuspended in 96% (v/v) ethanol. Cell nuclei were stained by incubating the cells for 15 min with 1 pg 4',6-diamidino-2-phenylindole(DAPI) per ml at 20°C in the dark. Subsequently, fluorescence microscopy was used to distinguish four different cell stages: unbudded cells, budded cells with a single nucleus, budded cells with a migrating nucleus, and binucleated cells. Stain ing of'cell ivalls

Staining of chitin with Calcofluor white (CFW) was performed according to Pringle et al. (1989). Fluorescence microscopy was used to visualize the stained cells. Poljca t ion assa,v

The polycation assay was performed as described by De Nobel et al. ( 1 990a). In principle, this assay is based on the observation that polybasic macromolecules interacting with the plasma membrane cause leakage of UV-absorbing material from the cytoplasm (Durr et al., 1975). Since the plasma membrane is enclosed by the cell wall, the sensitivity of yeast cells for polybasic macromolecules depends on the permeability of the yeast walls to these polymers (De Nobel et al., 1990a). Isolation of cell walls

Cells were washed with 10 mM-Tris-HCI, pH 7.4, containing 1 m~-phenylmethylsulfonyl fluoride (PMSF). Glass beads (0.5 mm diameter) were added to a concentration of 40 mg per mg fresh weight of cells. Cells were disrupted by vortexing for 15 s and subsequent chilling in melting ice for 5 s. This procedure was repeated 18 times. Walls were pelleted at 1000 x g for 5 min and washed three times with 1 MNaCl and thrce timeswith 10 mM-Tris-HC1, pH 7.4, containing 1 mM-PMSF. Mannoprotein extructions

The SDS-extractable mannoproteins were removed from isolated cell walls by boiling 10 mg of cell walls (fresh weight) for 5 min in 50 p1 10 mMTris --HCI,pH 7.4, containing 1 mM-PMSF and 2% (w:v) SDS (Valentin et al., 1984). Extraction with Zymolyase was performed as follows: 10 mg fresh weight of isolated walls were incubated at 36°C for

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2 h with 0.25 units Zymolyase 20T in 50 pl 20 m w sodium phosphate, pH 6.7, containing 5 mw-PMSF and 0.01% (w/v) NaN,. Chemicals

Poly-L-lysine(hydrobromide; 50.0 kDa), DEAEdextran (chloride form; 500 kDa), cycloheximide and DAPI were from Sigma. Zymolyase 20T was from Kirin Brewery. Alpha-factor was from Bachem Feinchemikalien AG. Calcofluor white M2RS New was from American Cyanamid Co. The ['4C]amino-acid mixture had a specific activity of 1.85 GBq/mmol(50 mCi/mmol) and was purchased from Amersham. RESULTS Cell wall porosity in successive age fractions Since buds have thinner walls than mother cells (Sentandreu and Northcote, 1969; Linnemans et al., 1977),we were interested in whether the fraction of budded cells might affect the average wall porosity in asynchronous cultures of Saccharomyces cerevisiae. Yeast cells can be separated in an elutriator rotor by size and thus by age in the cell cycle (Gordon and Elliott, 1977). We elutriated an asynchronous culture of batch-grown cells using a slightly modified procedure according to Van Doom et ul. ( 1 988). The fresh weight per cell in the collected fractions increased when the flow rate was accelerated (Figure I).Onaverage,the freshweight percellincreased bya factor of 3.0 from the first to the last fractions. Because balanced growth should result in a doubling ofcell volume (Campbell, 1957), the very small and/ or very large cells in the tails of the size distribution should be ignored in order to deduce the true cell cycle pattern (Mitchison, 1988).The best estimate of average birth volume V is the value where the enclosed area under the size distribution curve between V and 2 V is maximal (Salmon and Poole, 1983).According to this criterion, the fractions from I4 ml/min to 30 ml/min should represent the truecell cycle. At a flow rate of30 ml/min, already94Y0of the cells had been driven out of the rotor and the fresh weight per cell was approximately twice that of the first collected cells (Figure 1). Microscopical analysis showed that this elutriation fractionation resulted in partial separation of different stages of the cell cycle (Figure 2A). The first fractions (14 and 16 ml/min) consisted predominantly ofunbudded (G 1 phase)cells. In the following fractions, budded cells appeared. At flow rates of 20

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Figure 1. Size fractionation by centrifugal elutriation of an asynchronous population ofcells. The number ofcellscollected at the various flow rates was determined and presented as a percentage of the total number of elutriated cells (0).The fresh weight of the cells in the successive fractions was determined and, subsequently, the fresh weight per lo9 cells was calculated ( 0 ) .The results presented are the mean & SEM of five independent experiments.

and 22 ml/min, budded cells with a single nucleus dominated, representing S and G2 phase. Cells with migrating nuclei (M phase) never really dominated the fractions, possibly because it is a relatively short event during the cell cycle (Barford and Hall, 1976). The highest percentage of these cells was found at flow rates of 24 and 26ml/min. From a flow rate of 24 ml/min, budded cells with two nuclei, representing GI-phase cells which still had to undergo cytokinesis and abscission (G1*-phase; Barford and Hall, 1976; Pringle and Hartwell, 1981), dominated the fractions. At higher flow rates the heterogeneity of the fractions increased as demonstrated by an increasing amount of unbudded cells at flow rates of 2 30 ml/min (not shown). This is possibly due to a limited progression through the cell cycle, resulting in the abscission of daughter cells during the elutriation procedure even though it was executed at 4°C. The partially separated subpopulations were used to determine the relative wall porosity of the cells by means of the polycation assay. The sensitivity of the cells to poly-L-lysine, which is independent of wall porosity (De Nobel et al., 1990a), was constant for

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Figure 2. Cell wall porosity in successive age fractions as obtained by centrifugal elutriation of an asynchronous population of cells. The relative abundance of different cell stages (see inset) was determined after nuclear staining of the cells in the successive subpopulations (A). The relative wall porosity ( 0 )of the cells was determined and compared with the total amount of budded cells (0)in these subpopulations (B). The results presented are the mean SEM of five independent experiments.

the tested subpopulations. However, the sensitivity of the cells to DEAE-dextran, which depends on wall porosity (De Nobel et al., 1990a), was different for the various subpopulations. The ratio between these sensitivities can be used as a relative measure of wall porosity (De Nobel et al., 1990a). It appears that wall porosity increased when the fraction of unbudded cells decreased in favour of the fraction of budded cells (Figure 2B). The amount of cells collected at flow rates higher than 30ml/min was too low to be used for porosity determinations. The average wall porosity of the various subpopulations did not differ significantly from the porosity of the cells which were collected just before elutriation (data not shown).

CYCLIC VARIATIONS IN THE PERMEABILITY OF THE CELL WALL OF SACCHAROMYCES CEREVISIAE

Cell wall porosity in synchronously growing cultures As we have seen, the subpopulations obtained by centrifugal elutriation are only partially homogeneous. In the next experiment we made use of synchronously growing cells. Since we needed a high yield of cells, we applied induction instead of selection synchrony. Alpha-factor has been shown to arrest cells just prior to the initiation of DNA synthesis (Bucking-Throm et al., 1973). Removal of this block results in synchronous growth (Shulman, 1978). Preliminary experiments showed that maximal synchrony was obtained by releasing cells from a 2-h induction period with 4 pg alpha-factor per ml medium. This induction period resulted in a decrease of the percentage of budding cells from 60 to 10%. The remaining budding cells were all binucleated. After alpha-factor was washed away and cells were resuspended in fresh medium, the cells resumed division in a synchronous fashion for at least two generations, as observed by light microscopy (Figure 3). Initially, a temperature of 28°C was chosen, but in later experiments 20°C was used to allow a more extensive analysis of the cell cycle. At both temperatures similar results were obtained. Cells were sampled every 30min to determine their wall porosity with the polycation assay. In Figure 3, we present the results of such an experiment. During the first half hour of synchronous growth, the cells were unbudded and showed the typical shmoo morphology of pheromone-arrested cells (Thorner, 1981). These cells had a relative wall porosity of almost 40%, which is slightly higher than in non-induced cells from the same culture. After continued growth, the porosity of the cell walls increased suddenly, just before the new buds became visible (Figure 3A). The porosity remained at a high level during the initial stages of bud growth but decreased abruptly before abscission of the bud had occurred. A closer examination of the successive cell-cycle stages revealed that this decrease in porosity occurred near the time of nuclear migration (Figure 3B). In the subsequent cycle, the same oscillation in wall porosity was observed. The observed oscillation in wall porosity was typical for synchronous cultures. When we allowed cells to recover spontaneously from cell-cycle arrest, they showed no synchrony and no oscillations in wall porosity could be observed (data not shown). Mannoprotein incorporation during the cell cycle The porosity of yeast cell walls is limited by the mannoproteins in the walls (Zlotnik et al., 1984; De

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Time after removal of alpha-factor Ihl

Figure 3. Cell wall porosity during synchronous growth. Synchronous cells were obtained by releasing cells from alphaFdctor-induced G I-arrest. After induction, cells were grown at 20°C to extend the duration of the cell cycle. Cells were sampled and at 30-min intervals and the percentage of budded cells (0) the relative wall porosity (0) were determined (A). For a more detailed analysis of cell cycle stage, the cells were fixed and a nuclear staining was performed to determine the relative abundance of different cell stages (seeinset; B).

Nobel et al., 1989,1990b). Hence, we were interested in whether alterations in wall porosity during the cell cycle could be related to variations in incorporation of mannoproteins into the cell wall. In preliminary experiments, we pulse-labeled an asynchronous culture with a ['4C]amino-acid mixture for 10 min at 28°C. After fractionating the culture by centrifugal elutriation, the different fractions were analysed for protein incorporation into the cell wall. The results showed that the rate of incorporation of SDS-extractable mannoproteins into the cell wall remained constant during the cell cycle (data not shown). In the next experiment, we pulse-labeled synchronously growing cultures, released from alpha-pheromone arrest, with the same ["C] amino-

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acid mixture and analysed the incorporation of the label into the wall. This method allows a more pronounced separation of the different cell stages than can be obtained by centrifugal elutriation.

poration of SDS-extractable mannoproteins and a cell-cycle-dependent incorporation of glucanaseextractable mannoproteins into the cell wall.

Drflerences in cell wall staining patterns by CF W between mother and daughter cells

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Staining of cells with CFW, a chitin-specific stain (Pringle et al., 1989), revealed strong staining of the bud scars but also some staining of the lateral wall (Figure 5). This is in accordance with the observations of Molano et al. (1980) who showed that chitin was not restricted to the bud scar but was also present in the lateral walls. Staining of the lateral wall of mother cells was much denser than in the corresponding buds (Figure 5); this points to another difference in wall composition between mother and daughter cells.

Time after removal of alpha-factor [h]

Figure 4. Rate of incorporation of glucanase-extractable and SDS-extractable mannoproteins into the walls of synchronously growing cells. An asynchronous culture was synchronized with was followed alpha-factor and the percentage of budded cells (0) during continued growth in the absence of alpha-factor. Cells were sampled and labeled with a [“Clamino-acid mixture for 30 min. Isolated cell walls were extracted with Zymolyase (0)or SDS (0). Extracted label was presented as a percentage of label incorporated into the wall. The results presented are the mean of two independent experiments.

The release of radioactivity from isolated walls by extraction with SDS showed no cell-cycle-dependent variations (Figure 4). On average, 70% of the radioactivity of the walls was released by SDS. This is in accordance with the amount of SDS-extractable mannoproteins from asynchronous cells (Valentin et al., 1984). However, the amount of radioactivity released by digestion of the walls with Zymolyase depended on the stage of the cell cycle. The rate of incorporation of glucanase-extractable mannoproteins (measured as increased levels of glucanasereleased label) increased gradually during the early stages of bud growth. The highest level ofglucanasereleased label was obtained with wallsofbudded cells shortly before abscission (probably G 1 *-phase cells; see Figure 3B). After abscission of the bud, a reduction in the rate of incorporation of glucanaseextractable mannoproteins was observed. The same oscillation in release of glucanase-extractable mannoproteins was observed in the next cell cycle. The oscillations were absent in asynchronous cultures obtained by spontaneous recovery from pheromone-induced G 1-arrest (data not shown). These results suggest a constant rate of incor-

DISCUSSION Using elutriation fractions and synchronous cultures, we have demonstrated that budded cells show increased wall permeability compared with unbudded cells (Figures 2 and 3). A closer examination of the two approaches shows partially different results. The elutriation experiments revealed a correlation between the fraction of budded cells and the average permeability of the subpopulations (Figure 2). The permeability of the synchronously growing cells, however, increases before the new buds become visible and decreases abruptly in the older buds, before abscission has occurred. A comparable difference has been shown for cyclic variations in glucanase activity. On the one hand, several studies have shown that in synchronous cultures exo-p-( 1 -3)-glucanase activity increases sharplybeforethenew budsbecomevisible(Maddox and Hough, 1971; Cortat et al., 1972; Ruiz and Rodriguez, 1989). On the other hand, Rey et al. (l979), using a fractionated asynchronous culture, found an increase of exo-glucanase activity occurring somewhat later, apparently at the S to G2 transition. These results suggest that the differences in cyclic variations as observed for permeability as well as glucanase activity, depend on the technique used. Since synchronization of growth results in a much more pronounced separation of the cell stages (cf. Figures 2A and 3B), and allows successive cycles to be examined, we think that in our case this method provides a better approach to study cell-cycle events.

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Figure 5. Cell wall staining with Calcofluor white.

Cell wall changes related to cell wall porosity

Cell budding requires controlled hydrolysis of the walls (Phaff, 1977).Glucanases seem to play a major role in this process (Rey et al., 1979; Hien and Fleet. 1983). Like the sharp increase in wall porosity (Figure 3), a sudden increase of extracellular glucanase activity has been shown to precede bud emergence in synchronous cultures (Maddox and Hough, 1971; Cortat et af., 1972; Ruiz and Rodriguez, 1989), strongly suggesting a role in budding for this enzyme. Two possible working mechanisms seem possible. On the one hand, glucanase activity might result in stretching of the wall, becauseglucan determines the rigidity ofthecell wall (Duffus et al., 1982). On the other hand, glucanase activity might result in the release of glucanaseextractable mannoproteins from the yeast cell wall (Sanz et al., 1985). Both stretching of the wall and the removal of the mannoprotein layer have been shown to affect cell wall porosity (Zlotnik et al., 1984; De Nobel et al., 1990a,b). Calcofluor white staining of growing cells resulted in much denser staining of the lateral

walls of mother cells than of their buds (Figure 5 ) , suggesting that the amount of chitin in the lateral wall of mother cells is higher than in their buds. Horisberger and Clerc (1987) obtained similar results after labeling with wheat germ agglutinin, another chitin-specific label. In S. cerevisiae, an interaction between chitin and glucan in the lateral wall seems to be responsible for the alkaliinsolubility of part of the glucan (Sietsma and Wessels, 1981; Mol and Wessels, 1987). When buds grow older, the amount of alkali-soluble glucan decreases in favour of the amount of alkaliinsoluble glucan (Katohda et al., 1976: Hayashibe et al., 1977), suggesting an increased complexing between chitin and glucan during bud growth. The increased level of CFW staining of the lateral walls during growth might reflect this phenomenon. These results can be interpreted as follows in terms of cell wall porosity. Because the alkali-insoluble glucan provides the rigid framework of the yeast wall (Duffus et al., 1982), the wall of young buds, which contains less alkali-insoluble glucan than that of older buds (Katohda et al., 1976; Hayashibe et al., 1977), might be more elastic and might

596 become more stretched and thus more porous (De Nobel e t al., 1990a) than in old buds and mother cells. The incorporation of glucanase-extractable mannoproteins depends on the stage of the cell cycle in contrast to the incorporation of SDS-extractable mannoproteins (Figure 4). The rate of incorporation of glucanase-extractable mannoproteins reached a maximum near the time of nuclear migration. This result seems in agreement with immunolabeling studies on alpha-agglutinin, a glucanase-extractable mannoprotein (Ballou, 1988; Hauser and Tanner, 1989). Alpha-agglutinin is rarely detectable in young buds, whereas it is generally detectable in mother cells (Wojciechowicz and Lipke, 1989). The enhanced rate of incorporation of glucanaseextractable mannoproteins during nuclear migration (Figure 4) might contribute to the subsequent decrease in wall permeability (Figure 3A), because these mannoproteins have been shown to limit the permeability ofthecell wall (De Nobeletal., 1990b). Interestingly, in the maturation phase of bud growth, when the size of the bud is about two-thirds of that of the mother cell, polarized tip growth shuts down and subsequent expansion is completed by non-polarized spherical extension of the bud (Farkas el al., 1974: Staebell and Soll, 1985). Apparently, the incorporation of glucanase-extractable mannoproteins is maximal during this second phase of bud growth. The following picture emerges from the observations presented. After localized secretion of autolytic enzymes (Matile et al., 1971; Maddox and Hough, 1971; Cortat et al., 1972; Ruiz and Rodriguez, 1989), the cell wall becomes locally plasticized, the wall permeability of the budding cell increases and a new bud emerges (Nickerson, 1963; Phaff, 1977). During or shortly after nuclear migration, cell wall porosity decreases sharply. This seems to coincide with the transition from polarized tip growth of the bud to spherical expansion (Farkas et al., 1974; Staebell and Soll, 1985). During bud growth, the rate of incorporation of glucanase-extractable mannoproteins into the wall increases progressively and reaches its maximum when cell wall porosity begins to decrease. Similarly, the content of alkali-insoluble glucan in the wall increases, in particular, during the second half of bud growth (Katohda er al., 1976; Hayashibe et a f . , 1977). We propose that both events contribute to the decrease of porosity in the latter phases of bud growth.

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ACKNOWLEDGEMENT We are indebted to P. Huls for his technical assistance with the elutriation experiments. REFERENCES Ballou, C. E. (1982). Yeast cell wall and cell surface. In Strathem, J. N., Jones, E. W. and Broach, J. R. (Eds), The Molecular Biology of the Yeast Saccharomyces. Metabolism and Gene Expression. Cold Spring Harbor Laboratory, New York, pp. 335-360. Ballou, C. E. (1988). Organization of the Saccharomyces cerevisiae cell wall. In Varner, J. E. (Ed.), SeyAssembling Architecture. Alan R. Liss, Inc., New York, pp. 105-1 17. Barford, J. P. and Hall, R. J. (1976). Estimation of the length of cell cycle phases from asynchronous cultures of Saccharomyces cerevisiae. Exp. Cell Res. 102, 276-284. Biely, P., Kovarik, J. and Bauer, S. (1973). Cell wall formation in yeast. An electron microscopic autoradiographic study. Arch. Mikrobiol. 94,365-371. Biely, P. (1978). Changes in the rate of synthesis of wall polysaccharides during the cell cycle of yeast. Arch. Microbiol. 119,213-214. Brzobohaty, B. and Kovac, L. (1986). Factors enhancing genetic transformation of intact yeast cells modify cell wall porosity. J. Cen. Microbiol. 132,3089-3093. Bucking-Throm, E., Duntze, W., Hartwell, L. H. and Manney, T. R. (1973). Reversible arrest of haploid yeast cells at the initiation of DNA synthesis by a diffusible sex factor. Exp. Cell Res. 76,99-110. Cabib, E., Roberts, R. and Bowers, B. (1982). Synthesis of theyeast cell wall and its regulation. Ann. Rev. Biochem. 51,763-793. Campbell, A. (1957). Synchronization of cell division. Bacteriol. Rev. 21,263-272. Cortat, M., Matile, P. and Wiemken, A. (1972). Isolation of glucanase-containing vesicles from budding yeast. Arch. Mikrobiol. 82, 189-205. De Nobel, J. G., Dijkers, C., Hooijberg, E. and Klis, F. M. (1989). Increased cell wall porosity in Saccharomyces cerevisiae after treatment with dithiothreitol or EDTA. J. Gen. Microbiol. 135,2077-2084. De Nobel, J. G., Klis, F. M., Munnik, T., Priem, J. and Van den Ende, H. (1990a). An assay of relative cell wall porosity in Saccharomyces cerevisiae, Kluyveromyces lactis and Schizosaccharoml.ces pombe. Yeast 6, 483490. DeNobel, J. G., Klis, F. M., Priem, J., Munnik,T. and Van den Ende, H. (1990b). The glucanase-soluble mannoproteins limit cell-wall porosity in Saccharomyces cerevisiae. Yeast 6,491499. Duffus, J. H., Levi, C. and Manners, D. J. (1982). Yeast cell-wall glucans. Adv. Microb. Physiol. 23, 1 5 1- I8 1. Diirr, M., Boller, T. and Wiemken, A. (1975). Polybase induced lysis of yeast spheroplasts; a new gentle

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