Chitin Synthase 1, an Auxiliary Enzyme for Chitin Synthesis in Saccharomyces cerevisiae

Previously, we showed that chitin synthase 2 (Chs2) is required for septum formation in Saccharomyces cerevisiae, whereas chitin synthase 1 (Chsl) does not appear to be an essential enzyme. However, in strains carrying a disrupted CHS1 gene, frequent lysis of buds is observed. Lysis occurs after nuclear separation and appearg to result from damage to the cell wall, as indicated by osmotic stabilization and by a '~50-nm orifice at the center of the birth scar. Lysis occurs at a low pH and is prevented by buffering the medium above pH 5. A likely candidate for the lytic system is a previously described chitinase that is probably involved in cell separation. The chitinase has a very acidic pH optimum and a location in the periplasmic space that exposes it to external pH. Accordingly, allosamidin, a specific chitinase inhibitor, substantially reduced the number of lysed cells. Because the presence of Chsl in the cell abolishes lysis, it is concluded that damage to the cell wall is caused by excessive chitinase activity at acidic pH, which can normally be repaired through chitin synthesis by Chsl. The latter emerges as an auxiliary or emergency enzyme. Other experiments suggest that both Chsl and Chs2 collaborate in the repair synthesis of chitin, whereas Chsl cannot substitute for Chs2 in septum formation. T hE primary septum of the yeast, Saccharomyces cerevisiae, consists largely of the polysaccharide chitin (15, 17). The biosynthesis of chitin has been extensively studied, since the septum is both a useful model for morphogenesis and a potential target for antifungal agents (3, 4). Cloning of the structural gene for chitin synthase 1 (Chsl), ~ at that time the only chitin synthase known in yeast, followed by disruption of the gene, produced the unexpected result that Chsl is not essential for chitin synthesis in vivo (2). Shortly thereafter, a new chitin synthase activity, chitin synthase 2 (Chs2), was detected in yeast (16, 22). Subsequent cloning of CHS2 and its disruption showed unequivocally that this gene is essential for septum formation and chitin synthesis (24). In the presence of a CHS2 disruption, Chsl could not substitute for Chs2 and its function remained obscure. In previous work (2) it was ascertained that cells carrying a disrupted CHS1 gene frequently give rise to lysing buds when grown in minimal medium. Starting from this fact, we have accumulated evidence to indicate that Chsl, although not required under optimal conditions, can serve as an auxiliary or repair enzyme in certain limiting situations. 1. Abbreviations used in this paper: Chsl, chitin synthase 1; Chs2, chitin synthase 2; DAPI, 4',6-diamidino-2-phenyl-indole; MU(Ch)3, 4-methylumbelliferyl-13-o-N,N',N "-triacetylchitotriose. Materials and Methods Reagents and Enzymes [3HlChitin was prepared by acetylation of chitosan as described (14). 4-Methylumbelliferyl-f3-o-N,N',N"-triacetylchitotriose (MU[Ch]3) was obtained from Calbiochem-Behring Corp. (La Jolla, CA). [2-14C] Propionic acid (sp. act. 50 mCi/mmol) was from Amersham Corp. (Arlington Heights, IL). Deoxyribonuclease I was purchased from Sigma Chemical Co. (St. Louis, MO). Polyoxin D was kindly provided by Schering-Plough Corp. (Bloomfield, N J) and by Kaken Chemical Company (Tokyo, Japan). A1losamidin was a generous gift of A. Isogai (The University of Tokyo, Tokyo, Japan) and of P. Somers (Eli Lilly and Company, Indianapolis, IN). The product from Eli Lilly, which was used in most experiments because of the larger amount available, was named A82516, but appears to be identical to allosamidin. Purified yeast chitinase was from an earlier preparation obtained in this laboratory (5). Strains and Cell Growth The strains of S. cerevisiae used in most experiments were D3A (MATt~, ade2-101, ura3-52, chsl: : URA3), D3B (MATa, his4, ura3-52, chsl: : URA3), D3C (MATer, ura3-52), and D3D (MATa, ade2-101, his4, ura3-52), all from the same tetrad. In some experiments, a diploid (prb1-1122/prb1-1122, see reference 5) was used to measure chitinase activity in intact cells. Yeast was grown at 30°C either in YEPD (2% glucose, 2% peptone, I% yeast extract) or in minimal medium (2 % glucose, 0.7 % yeast nitrogen base) plus nutritional supplements. When a buffer at pH 5.8 was added to minimal medium, the final concentration was 85 mM for succinate and 100 mM for 2-(N-morpholino)ethane sulfonic acid (MES) or phthalate. Counting of Refractile Cells Counting of normal and refractile cells was performed with a Universal © The Rockefeller University Press, 0021-9525/89/05/1665/8 $2.00 The Journal of Cell Biology, Volume 108, May 1989 1665-1672 1665 on M ay 1, 2017 D ow nladed fom Published May 1, 1989