The cell wall porosity of batch-grown Saccharomyces cerevisiae was maximal in the early exponential phase and fell off rapidly to lower levels in later growth phases. Treatment of stationary-phase cells with alpha-mannosidase restored wall porosity to the level of cells in early exponential phase. When cells in the early exponential phase were treated with alpha-mannosidase, or tunicamycin, an inhibitor of N-glycosylation, even higher porosities were obtained. Mutants with truncated mannan side-chains in their wall proteins also had very porous walls. The importance of the mannan side-chains for wall porosity was also seen during sexual induction. Treatment with alpha pheromone, which leads to the formation of wall proteins with shorter mannan side-chains, enhanced wall porosity. Disulphide bridges also affect cell wall porosity. They were predominantly found in the glucanase-soluble wall proteins. Because the main part of the mannan side-chains is also found in this family of wall proteins, our results demonstrate that the glucanase-soluble mannoproteins limit cell wall porosity in yeast.

We have developed a new assay to determine relative cell wall porosity in yeasts, which is based on polycation-induced leakage of UV-absorbing compounds. Polycations with a small hydrodynamic radius as measured by gel filtration (poly-L-lysine) caused cell leakage independent of cell wall porosity whereas polycations with a large hydrodynamic radius (DEAE-dextrans) caused only limited cell leakage due to limited passage through the cell wall. This allowed the ratio between DEAE-dextran- and poly-L-lysine-induced cell leakage to be used as a measure of cell wall porosity in Saccharomyces cerevisiae, Kluyveromyces lactis and Schizosaccharomyces pombe. Using this assay, we found that the composition of the growth medium affected cell wall porosity in S. cerevisiae. In addition, we could show that cell wall porosity is limited by the number of disulphide bridges in the wall and is dependent on cell turgor. It is argued that earlier methods to estimate cell wall porosity in S. cerevisiae resulted in large underestimations.

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Intact cells of Saccharomyces cerevisiae were able to endocytose FITC-dextrans of 70 kDa, but not of 150 kDa, whereas spheroplasts took up both components. The rate of uptake of 70 kDa dextrans by spheroplasts was about three times higher than that by intact cells. Pretreatment of intact cells with dithiothreitol (DTT) or EDTA increased the rate of uptake of 70 kDa dextrans considerably, but 150 kDa dextrans were still excluded. Release of periplasmic invertase activity into the medium by glucose-derepressed cells was negligible in control cell suspensions, but was strongly stimulated in the presence of DTT. The released invertase had an apparent molecular mass of 320 kDa, indicating that the dimeric form was released. In the presence of EDTA only a slight increase in the release of invertase was observed. Pretreatment with DTT was accompanied by an increased loss of cell wall proteins. This suggests that the loss of mannoproteins, in combination with a more general opening up of the wall by reducing disulphide bridges, increases cell wall porosity. It is argued from the Stokes radius of 70 kDa dextran (5.8 nm) that yeast cell walls are, in principle, permeable to globular proteins with a molecular mass up to 400 kDa. .466reviations: BSA, bovine serum albumin; FD, FITC-dextran ; FITC, fluorescein isothiocyanate.

To study cell-cycle-related variations in wall permeability of Saccharomyces cerevisiae, two approaches were used. First, an asynchronous culture was fractionated by centrifugal elutriation into subpopulations containing cells of increasing size. The subpopulations represented different stages of the cell cycle as judged by light microscopy. Cell wall porosity increased when these subpopulations became enriched with budded cells. Secondly, synchronous cultures were obtained by releasing MATa cells from alpha-factor induced G1-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 stages of bud growth. It 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 mannoproteins into the wall during the cell cycle. SDS-extractable mannoproteins 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.