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J. Biol. Chem., Vol. 280, Issue 10, 9170-9179, March 11, 2005

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Synthase III-dependent Chitin Is Bound to Different Acceptors Depending on Location on the Cell Wall of Budding Yeast^*

Enrico Cabib¶ and Angel Durán

From the Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20852 and the Instituto de Microbiología Bioquímica, CSIC/Universidad de Salamanca, 37007 Salamanca, Spain

In yeast, chitin is laid down at three locations: a ring at the mother-bud neck, the primary septum and, after cytokinesis, the cell wall of the daughter cell. Some of the chitin is free and the remainder attached to (1–3)glucan or (1–6)glucan. We recently reported that the chitin ring contributes to the prevention of growth at the mother-bud neck and hypothesized that this inhibition is achieved by a preferential binding of chitin to (1–3)glucan at that site. Here, we devised a novel strategy for the analysis of chitin cross-links in [¹⁴C]glucosamine-labeled cell walls, involving solubilization in water of alkali-treated walls by carboxymethylation. Intact cell walls or their digestion products with (1–3)glucanase or (1–6)glucanase were carboxymethylated and fractionated on size columns, and the percentage of chitin bound to different polysaccharides was calculated. Chitin dispersed in the wall was labeled in maturing unbudded cells and that of the ring in early budding cells. The former was mostly attached to (1–6)glucan and the latter to (1–3)glucan. This confirmed our hypothesis and indicated that the cell has mechanisms to attach chitin, a water-insoluble substance, synthesized here through chitin synthase III, to different acceptors, depending on location. In contrast, most of the chitin synthase II-dependent chitin of the primary septum was free, with the remainder linked to (1–3)glucan.

Received for publication, December 13, 2004

^* This work was supported in part by Grant BIO2001-2048 of the Comisión Interministerial de Ciencia y Tecnología. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This article was selected as a paper of the week.

^¶ E. C. carried out part of this work during a sabbatical sponsored by the Spanish Ministerio de Educación, Cultura y Deporte (SAB 2000-0336), which was also part of a PHS Foreign Work/Study program. To whom correspondence should be addressed: NIDDK, National Institutes of Health, Bldg. 8, Rm. 403, Bethesda, MD 20854. Tel.: 301-496-1008; Fax: 301-496-9431; E-mail: enricoc{at}bdg10.niddk.nih.gov.

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