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* Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280; 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.
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
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