A SEPTIN-BASED HIERARCHY OF PROTEINS REQUIRED FOR LOCALIZED DEPOSITION OF CHITIN IN THE SACCHAROMYCES-CEREVISIAE CELL-WALL

Just before bud emergence, a Saccharomyces cerevisiae cell forms a rin g 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 th e 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 ch itin synthases in S. cerevisiae. The chitin ring does not form normall y in temperature-sensitive mutants defective in any of four septins, a family of proteins that are constituents of the ''neck filaments'' th at lie immediately subjacent to the plasma membrane in the mother-bud neck. In addition, a synthetic-lethal interaction was found between cd c12-5, a temperature-sensitive septin mutation, and a mutant allele of CHS4, which encodes an activator of chitin synthase III. Two-hybrid a nalysis revealed no direct interaction between the septins and Chs4p b ut identified a novel gene, BNI4, whose product interacts both with Ch s4p and Cdc10p and with one of the septins, Cdc10p; this analysis also revealed an interaction between Chs4p and Chs3p, the catalytic subuni t of chitin synthase III. Bni4p has no known homologues; it contains a predicted coiled-coil domain, but no other recognizable motifs. Delet ion of BNI4 is not lethal, but causes delocalization of chitin deposit ion and aberrant cellular morphology. Overexpression of Bni4p also cau ses delocalization of chitin deposition and produces a cellular morpho logy 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 predominan t 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 localiz ation is dependent on the septins, Bni4p, and Chs3p. Chs3p, whose norm al localization is similar to that of Chs4p, does not localize properl y in bni4, chs4, or septin mutant strains or in strains that accumulat e excess Bni4p. In contrast, localization of the septins is essentiall y normal in bni4, chs4, and chs3 mutant strains and in strains that ac cumulate excess Bni4p. Taken together, these results suggest that the normal localization of chitin synthase III activity is achieved by ass embly of a complex in which Chs3p is linked to the septins via Chs4p a nd Bni4p.