Work carried out in our laboratory in the previous period and published in the present one showed that in budding yeast the integrity of the neck region between mother and daughter cell depends on both the filamentous septin ring and the cell wall chitin ring, both formed at the base of the bud at the onset of budding. When both structures are defective, the neck grows and widens, leading to a cytokinesis defect and eventually to cell death. This was the first demonstration that the chitin ring, whose synthesis is catalyzed by chitin synthase III (CS III), has a physiological function. At cytokinesis, another chitin structure, the primary septum, is formed as a disk at the neck, in a process requiring a different chitin synthase, CS II. CS III is not necessary for septation in wild type cells; chs3 mutants, defective in the catalytic moiety of CS III, are viable. However, Chs3p is required for the formation of aberrant but functional remedial septa when Chs2p is missing. The question came up, whether Chs3p was necessary in this case because of its contribution to the formation of the chitin ring or of the rather abundant chitin found in the remedial septa or of both. To answer this question, we used synchronized, unbudded chs2 cells and inhibited chitin synthesis with nikkomycin Z, a specific inhibitor of CSIII, either during chitin ring formation or during subsequent biogenesis of the remedial septa. The inhibitor only prevented septum formation when it was present throughout the experiment or when it was added after chitin ring formation, but not when it was present only during ring biosynthesis. It was concluded that the chitin laid down in the aberrant septa and not that of the neck ring is necessary for remedial septa formation. A paper reporting these results was recently published. These findings clarify the three functions of CS III. This enzyme is required for the formation of the chitin ring that, together with the septin ring, prevents widening of the mother-bud neck during growth; for chitin dispersed in the cell wall, that may contribute to rigidity of the wall, and for deposition of chitin in remedial septa. In previous work, we demonstrated that chitin is covalently bound to two different polysaccharides in the yeast cell wall, beta(1-3)glucan, the main structural component of the wall, and beta(1-6)glucan, to which mannoproteins are attached. In turn, beta(1-6)glucan is linked to beta(1-3)glucan at the same position where, in other molecules, chitin is attached. Our results about function of the chitin ring suggested that chitin in the ring may be linked to beta(1-3)glucan, thus capping the ends to which beta(1-6)glucan-mannoprotein normally attaches and preventing further wall growth at the neck. On the other hand, the chitin interspersed in the cell wall may be linked to beta(1-6)glucan, a notion consistent with observations made in our laboratory and published more than 20 years ago. This hypothesis suggests the presence of two distinct regulatory systems for CS III activation and chitin transfer to each acceptor, at different stages of the cell cycle. To check the hypothesis, two different methods must be developed: one, to determine the amount of chitin bound to beta(1-3) or beta(1-6)glucan, the other, to manipulate the cell in such a way that [14C]glucosamine will be incorporated into chitin either in the neck ring or in the bulk of the cell wall. During my sabbatical at the University of Salamanca I have carried out preliminary experiments towards the development of the first methodology. The following steps were tentatively planned: 1) incorporation of [14C]glucosamine into chitin in vivo; 2) isolation of cell walls; 3) treatment of walls with either beta(1-3)glucanase or beta (1-6)glucanase to liberate the chitin linked to one or the other of the two polysaccharides; 4) solubilization of the cell walls; 5) separation of the cell wall components by column chromatography and evaluation of the liberated chitin in each case. The first three steps presented no special problems. The fourth step was more difficult. No solubilization of yeast cell walls without extensive breakage of chemical bonds has been achieved so far. In particular, chitin, the component to be determined, is extremely insoluble in water. However, it is soluble in dimethylacetamide-LiCl. Intact cell wall were insoluble in this solvent, but they went readily into solution after mild treatment with alkali to remove proteins and soluble glucan. Attempts were made to separate the cell wall components by HPLC on a Tosoh TSK-Gel GMHHR-N column, but the separation between chitin and the other components was insufficient for their individual determination. At present, different solvent combinations and column materials are tested, in an effort to improve separations.
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