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A septin-based hierarchy of proteins required for localized deposition of chitin in the Saccharomyces cerevisiae cell wall.

DeMarini DJ, Adams AE, Fares H, De Virgilio C, Valle G, Chuang JS, Pringle JR - J. Cell Biol. (1997)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina, Chapel Hill 27599-3280, USA.

ABSTRACT
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.

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Immunolocalization of Cdc11p in wild-type and mutant strains. Exponentially growing cells of strains (A) YEF473 (wild-type); (B) DDY174 (chs4-Δ1/chs4-Δ1); (C) DDY175 (bni4-Δ1/bni4-Δ1); (D) DDY179 (chs4-Δ1/chs4-Δ1 bni4-Δ1/bni4-Δ1); (E) DDY186  (chs3-Δ1/chs3-Δ1); and (F) DDY175 containing plasmid p356 (high-copy BNI4) were examined by immunofluorescence microscopy using Cdc11p-specific antibodies.
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Figure 9: Immunolocalization of Cdc11p in wild-type and mutant strains. Exponentially growing cells of strains (A) YEF473 (wild-type); (B) DDY174 (chs4-Δ1/chs4-Δ1); (C) DDY175 (bni4-Δ1/bni4-Δ1); (D) DDY179 (chs4-Δ1/chs4-Δ1 bni4-Δ1/bni4-Δ1); (E) DDY186 (chs3-Δ1/chs3-Δ1); and (F) DDY175 containing plasmid p356 (high-copy BNI4) were examined by immunofluorescence microscopy using Cdc11p-specific antibodies.

Mentions: The results presented above indicate that the localization of Bni4p, Chs4p, and Chs3p depends upon the septins. To determine if the localization of the septins depends upon these other proteins, chs4-Δ1, bni4-Δ1, chs4-Δ1 bni4-Δ1, chs3-Δ1, and Bni4p-overexpressing strains were examined by immunofluorescence using Cdc11p-specific antibodies. Cdc11p localized nearly normally in the mutant strains (Fig. 9). However, the band of septin staining appeared somewhat narrower (not extending as far into either mother cell or bud) in the mutants (compare Fig. 9, B–E, to Fig. 9 A). This effect was not apparent in the Bni4p-overexpressing strain (Fig. 9 F). It is not clear whether the altered appearance of the septin band is a real change in the structure of the septin assembly or a visual artifact resulting from a change in neck shape and/or dimensions. In any case, it appears that Chs4p, Bni4p, and Chs3p have, at most, a modest influence on septin organization and are not required to localize the septins to the mother-bud neck.


A septin-based hierarchy of proteins required for localized deposition of chitin in the Saccharomyces cerevisiae cell wall.

DeMarini DJ, Adams AE, Fares H, De Virgilio C, Valle G, Chuang JS, Pringle JR - J. Cell Biol. (1997)

Immunolocalization of Cdc11p in wild-type and mutant strains. Exponentially growing cells of strains (A) YEF473 (wild-type); (B) DDY174 (chs4-Δ1/chs4-Δ1); (C) DDY175 (bni4-Δ1/bni4-Δ1); (D) DDY179 (chs4-Δ1/chs4-Δ1 bni4-Δ1/bni4-Δ1); (E) DDY186  (chs3-Δ1/chs3-Δ1); and (F) DDY175 containing plasmid p356 (high-copy BNI4) were examined by immunofluorescence microscopy using Cdc11p-specific antibodies.
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Related In: Results  -  Collection

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Figure 9: Immunolocalization of Cdc11p in wild-type and mutant strains. Exponentially growing cells of strains (A) YEF473 (wild-type); (B) DDY174 (chs4-Δ1/chs4-Δ1); (C) DDY175 (bni4-Δ1/bni4-Δ1); (D) DDY179 (chs4-Δ1/chs4-Δ1 bni4-Δ1/bni4-Δ1); (E) DDY186 (chs3-Δ1/chs3-Δ1); and (F) DDY175 containing plasmid p356 (high-copy BNI4) were examined by immunofluorescence microscopy using Cdc11p-specific antibodies.
Mentions: The results presented above indicate that the localization of Bni4p, Chs4p, and Chs3p depends upon the septins. To determine if the localization of the septins depends upon these other proteins, chs4-Δ1, bni4-Δ1, chs4-Δ1 bni4-Δ1, chs3-Δ1, and Bni4p-overexpressing strains were examined by immunofluorescence using Cdc11p-specific antibodies. Cdc11p localized nearly normally in the mutant strains (Fig. 9). However, the band of septin staining appeared somewhat narrower (not extending as far into either mother cell or bud) in the mutants (compare Fig. 9, B–E, to Fig. 9 A). This effect was not apparent in the Bni4p-overexpressing strain (Fig. 9 F). It is not clear whether the altered appearance of the septin band is a real change in the structure of the septin assembly or a visual artifact resulting from a change in neck shape and/or dimensions. In any case, it appears that Chs4p, Bni4p, and Chs3p have, at most, a modest influence on septin organization and are not required to localize the septins to the mother-bud neck.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina, Chapel Hill 27599-3280, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus