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© The Rockefeller University Press, 0021-9525/1997//75 $5.00
The Journal of Cell Biology, Volume 139, Number 1, , 1997 75-93

Article

A Septin-based Hierarchy of Proteins Required for Localized Deposition of Chitin in the Saccharomyces cerevisiae Cell Wall



Douglas J. DeMarini*, Alison E.M. Adams{ddagger}, Hanna Fares*, Claudio De Virgilio*, Giorgio Valle§, John S. Chuang||, and John R. Pringle*

* Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280; {ddagger} Department of Molecular and Cellular Biology, University of Arizona, Tucson, Arizona 85721; § Departimento di Biologia and CRIBI Biotechnology Centre, Universitá degli Studi di Padova, Padova, Italy I-35121; and || Department of Molecular and Cell Biology, University of California, Berkeley, California 94720

Just before bud emergence, a Saccharomyces cerevisiae cell forms a ring of chitin in its cell wall; this ring remains at the base of the bud as the bud grows and ultimately forms part of the bud scar marking the division site on the mother cell. The chitin ring seems to be formed largely or entirely by chitin synthase III, one of the three known chitin synthases in S. cerevisiae. The chitin ring does not form normally in temperature-sensitive mutants defective in any of four septins, a family of proteins that are constituents of the "neck filaments" that lie immediately subjacent to the plasma membrane in the mother-bud neck. In addition, a synthetic-lethal interaction was found between cdc12-5, a temperature-sensitive septin mutation, and a mutant allele of CHS4, which encodes an activator of chitin synthase III. Two-hybrid analysis revealed no direct interaction between the septins and Chs4p but identified a novel gene, BNI4, whose product interacts both with Chs4p and Cdc10p and with one of the septins, Cdc10p; this analysis also revealed an interaction between Chs4p and Chs3p, the catalytic subunit of chitin synthase III. Bni4p has no known homologues; it contains a predicted coiled-coil domain, but no other recognizable motifs. Deletion of BNI4 is not lethal, but causes delocalization of chitin deposition and aberrant cellular morphology. Overexpression of Bni4p also causes delocalization of chitin deposition and produces a cellular morphology similar to that of septin mutants. Immunolocalization experiments show that Bni4p localizes to a ring at the mother-bud neck that lies predominantly on the mother-cell side (corresponding to the predominant site of chitin deposition). This localization depends on the septins but not on Chs4p or Chs3p. A GFP-Chs4p fusion protein also localizes to a ring at the mother-bud neck on the mother-cell side. This localization is dependent on the septins, Bni4p, and Chs3p. Chs3p, whose normal localization is similar to that of Chs4p, does not localize properly in bni4, chs4, or septin mutant strains or in strains that accumulate excess Bni4p. In contrast, localization of the septins is essentially normal in bni4, chs4, and chs3 mutant strains and in strains that accumulate excess Bni4p. Taken together, these results suggest that the normal localization of chitin synthase III activity is achieved by assembly of a complex in which Chs3p is linked to the septins via Chs4p and Bni4p.

Abbreviations used in this paper: 5-FOA, 5-fluoroorotic acid; AD, activation domain; DBD, DNA-binding domain; DIC, differential-interference-contrast; GFP, green fluorescent protein; GST, glutathione-S-transferase; ORF, open reading frame; SC, synthetic complete.

The present address of Douglas J. DeMarini is Department of Comparative Genetics, Smithkline Beecham Pharmaceuticals, King of Prussia, PA 19406.

The present address of Hanna Fares is Department of Biochemistry, Columbia University, New York, NY 10032.

The present address of Claudio De Virgilio is Botanisches Institut der Universität Basel, Basel, Switzerland, CH-4056.

Address all correspondence to John R. Pringle, Department of Biology, CB #3280 Coker Hall, University of North Carolina, Chapel Hill, NC 27599-3280. Phone: 919-962-2293; FAX: 919-962-0320; E-mail: jpringle @email.unc.edu

2. Fares, H., M.S. Longtine, and J.R. Pringle, manuscript in preparation.


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