The fungal cell wall protects the cell against lysis by internal turgor pressure and mechanical damage and is essential for survival of the organism. Because the wall grows with the cell and imparts shape to it, we have used it for many years as a model for morphogenesis. In Saccharomyces cerevisiae, the cell wall is made up of four different components: beta(1-3)glucan, the main structural component, beta(1-6)glucan, several mannoproteins and chitin. The strength of the cell wall is insured by cross-links between the different components. Thus, the proteins are attached to beta(1-6)glucan, which, in turn is linked to beta(1-3)glucan. Chitin, a beta(1-4)-linked acetylglucosamine polysaccharide, is attached to both beta(1-3)- and beta(1-6)glucan (1). Chitin accounts for only a few percent of the total cell wall, however it is essential. It is found in three locations in the cell. One is the primary septum laid down during cell division. Another is a chitin ring that forms in the neck between mother and daughter cell at bud emergence. In addition, some chitin is found dispersed all over the cell wall. During the cell cycle, new wall is continuously synthesized to keep up with bud growth, but there is an area that remains unchanged after bud emergence, the neck between mother and daughter cell, where cytokinesis eventually takes place. Two ring-shaped structures are present at the mother-bud neck. The above-mentioned chitin ring in the cell wall and a ring composed of 5 proteins, the septins, inside the plasma membrane. About 8 years ago we found that the chitin and septin rings were redundantly required for control of growth at the neck (2). When both rings were defective, necks grew wider and cytokinesis could not be completed. We suggested that the chitin ring would control growth at the neck through chemical linkages. We had shown previously that chitin attaches to beta(1-3)glucan at the same sites where beta(1-6)glucan is bound (1). We now proposed that the high concentration of chitin at the neck would compete out the beta(1-6)glucan, also preventing the attachment of mannoproteins to it and inhibiting wall growth (2). This hypothesis predicted that most of the chitin at the neck would be bound to beta(1-3)glucan, whereas that in the lateral wall it would be attached to beta(1-6)glucan, as was later confirmed by us (3). However, it was not clear how metabolism and growth of beta(1-3)glucan itself is controlled. It is possible that the binding of chitin at the reducing ends of beta(1-3)glucan, aside from preventing the addition of beta(1-6)glucan, also interferes with the continuous reorganization of beta(1-3)glucan necessary for growth. If this were true, one would expect the size distribution of the bulk polysaccharide, undergoing remodeling, to be different from that bound to chitin in the neck, where there is no growth. Nothing was known about the size distribution of beta(1-3)glucan. The only information available was the chain length of the polysaccharide (about 1,500 glucose units from methylation data) and the presence of 3% of beta(1-6) linkages that may represent cross-links. First, I devised a method for the isolation of beta(1-3)glucan. To this end, cells were labeled in vivo with 14C-glucose. Cell walls were prepared and treated with recombinant beta(1-6)glucanase to solubilize the beta(1-3)glucan and attached proteins. The water-insoluble residue, which contained beta(1-3)glucan and chitin, was carboxymethylated to make it soluble in water. Chromatography of this material on Sephacryl S-500 showed a fraction of very high molecular weight, emerging at the column void volume, followed by polydisperse material covering the remainder of the eluate. Treatment of the beta(1- 6)glucanase-resistant fraction with 1M NaOH at room temperature before carboxymethylation solubilized all the polydisperse material, as shown by subsequent carboxymethylation and chromatography. As for the excluded material, Sephacryl S-500 should exclude polysaccharides of molecular weight greater than 2 x 107, which would contain approximately 120,000 glucose units, almost a hundred times the length of a single chain. This results supports our previous proposal that beta(1-3)glucan chains are highly cross-linked through beta(1-6)linkages. This fraction probably represents the glucan fibrillar network seen on the yeast cell surface by electron microscopy after enzymatic treatments, whereas the polydisperse material belongs to glucan undergoing remodeling. To isolate the glucan attached to chitin, the beta(1-6)glucanase-resistant material was treated with 1 M NaOH at 80C. According to previous results, this treatment should solubilize only the free glucan, the chitin-bound glucan remaining in the insoluble fraction. Chitinase digestion of this fraction, followed by suspension in 1 M NaOH at room temperature resulted in solubilization of about two thirds of the radioactivity, which would correspond to glucan previously bound to chitin. However, a strain in which Crh1 and Crh2, the two transglycosylases necessary for the formation of chitin-glucan linkages, had been deleted showed a similar behavior, although the percentage of radioactivity solubilized by alkali after chitinase was lower. This was a surprising result, because I had previously found that such strains contain no beta(1-3) or beta(1-6) glucan linked to chitin (4). One possible explanation was that part of the glucan was noncovalently bound to chitin both in the wild type and mutant strain. To investigate this possibility, all the material insoluble in NaOH at 80C both from wild type and the crh1delta crh2delta mutant was solubilized by carboxymethylation. Since no chitinase digestion was performed, the solubilized mixture should contain CM (carboxymethyl)-glucan-chitin, plus free CM-chitin and CM-glucan, coming in part from the putative noncovalent complex and in part from polysaccharides that were already free. To separate the chitin-containing components from the CM-glucan, a wheat germ agglutinin (WGA)-agarose column was used. WGA specifically binds beta(1-4)linked acetylglucosamines. Part of the radioactivity did not bind to the WGA-agarose column, about 55% for wild type and 75% for the mutant. This material was characterized as CM-beta(1-3)glucan of high molecular weight. Subsequent elution with 0.1 M NaOH released more radioactivity. When these fractions were subjected to chromatography on Sephacryl S-500, that from wild type gave rise to two peaks, the first of which ran as a high molecular weight glucan, whereas the second was identified as free chitin. The mutant material only showed the free chitin peak. These results lead to the following conclusions: first, there is, both in wild type and in the mutant, a fraction of the beta(1-3)glucan that is physically, but not covalently, bound to chitin. The two polysaccharides can be separated after carboxymethylation, because the negative charges introduced by this procedure cause repulsion between the sugar chains. Second, there is a fraction of chitin-beta(1-3)glucan present in the wild type but not in the mutant, that is of high molecular weight and does not show the polydisperse material present in the free beta(1-3)glucan. These results support the notion that the beta(1-3)glucan bound to chitin is not being remodeled and therefore the corresponding area of the cell wall, i.e. the neck between mother and daughter cell, is not growing, as predicted by our hypothesis. (1) Cabib, E., Roh, D.-H., Schmidt, M., Crotti, L.B., and Varma, A. (2001) J. Biol. Chem. 276, 19679-19682. (2) Schmidt, M., Varma, A., Drgon, T., Bowers, B., and Cabib, E. (2003) Mol. Biol. Cell 14, 2128-2141. (3) Cabib, E. and Durn , A. (2005) J. Biol. Chem. 280, 9170-9179. (4) Cabib, E. (2009) Eukaryot. Cell 8, 1626-1636.

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