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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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Phenotypic analysis of chs4 and bni4 mutants and of a BNI4-overexpressing strain. Shown are DIC micrographs (A–C, G–I)  and fluorescence micrographs of Calcofluor-stained cells (D–F, J–L) of strains (A and D) YEF473 (wild-type); (B and E) DDY174  (chs4-Δ1/chs4-Δ1); (C and F) DDY175 (bni4-Δ1/bni4-Δ1); (G and J) DDY179 (chs4-Δ1/chs4-Δ1 bni4-Δ1/bni4-Δ1); (H and K) DDY175  containing plasmid p356 (high-copy BNI4); and (I and L) DDY185-1A (cdc10-Δ1). All strains were grown at 30°C except DDY185-1A,  which was grown at 23°C. Arrowheads indicate protuberances at previous division sites; arrows indicate larger-than-normal bud necks.
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Figure 4: Phenotypic analysis of chs4 and bni4 mutants and of a BNI4-overexpressing strain. Shown are DIC micrographs (A–C, G–I) and fluorescence micrographs of Calcofluor-stained cells (D–F, J–L) of strains (A and D) YEF473 (wild-type); (B and E) DDY174 (chs4-Δ1/chs4-Δ1); (C and F) DDY175 (bni4-Δ1/bni4-Δ1); (G and J) DDY179 (chs4-Δ1/chs4-Δ1 bni4-Δ1/bni4-Δ1); (H and K) DDY175 containing plasmid p356 (high-copy BNI4); and (I and L) DDY185-1A (cdc10-Δ1). All strains were grown at 30°C except DDY185-1A, which was grown at 23°C. Arrowheads indicate protuberances at previous division sites; arrows indicate larger-than-normal bud necks.

Mentions: To examine the phenotypes resulting from the loss of CHS4 and/or BNI4 function, these ORFs were deleted (Fig. 2 A) in the same genetic background. DNA–DNA blot-hybridization analyses confirmed the success of these deletions (Fig. 2 B). For both DDY172 (CHS4/chs4-Δ1) and DDY173 (BNI4/bni4-Δ1) (Table I), viability segregated 4+:0−, and TRP1 segregated 2:2 at 22, 30, 37, and 39°C, indicating that neither CHS4 nor BNI4 is essential for viability. chs4-Δ1 and bni4-Δ1 haploids were mated, and the resulting diploid (DDY176) was subjected to tetrad analysis. Segregants that contained both mutations were viable at temperatures from 22–37°C and had growth rates similar to those of their CHS4 BNI4 siblings. DIC microscopy revealed that some chs4-Δ1 cells had enlarged bud necks and that many had protuberances at previous division sites (Fig. 4 B; cf. wild-type cells in Fig. 4 A), as noted previously for chs1 chs3 mutants by Shaw et al. (1991). Similarly, the majority of bni4-Δ1 cells had enlarged bud necks, and some had protuberances at previous division sites (Fig. 4 C), and the majority of chs4-Δ1 bni4-Δ1 cells had both enlarged bud necks and protuberances (Fig. 4 G). The double-mutant cells also were extensively vacuolated.


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)

Phenotypic analysis of chs4 and bni4 mutants and of a BNI4-overexpressing strain. Shown are DIC micrographs (A–C, G–I)  and fluorescence micrographs of Calcofluor-stained cells (D–F, J–L) of strains (A and D) YEF473 (wild-type); (B and E) DDY174  (chs4-Δ1/chs4-Δ1); (C and F) DDY175 (bni4-Δ1/bni4-Δ1); (G and J) DDY179 (chs4-Δ1/chs4-Δ1 bni4-Δ1/bni4-Δ1); (H and K) DDY175  containing plasmid p356 (high-copy BNI4); and (I and L) DDY185-1A (cdc10-Δ1). All strains were grown at 30°C except DDY185-1A,  which was grown at 23°C. Arrowheads indicate protuberances at previous division sites; arrows indicate larger-than-normal bud necks.
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Related In: Results  -  Collection

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Figure 4: Phenotypic analysis of chs4 and bni4 mutants and of a BNI4-overexpressing strain. Shown are DIC micrographs (A–C, G–I) and fluorescence micrographs of Calcofluor-stained cells (D–F, J–L) of strains (A and D) YEF473 (wild-type); (B and E) DDY174 (chs4-Δ1/chs4-Δ1); (C and F) DDY175 (bni4-Δ1/bni4-Δ1); (G and J) DDY179 (chs4-Δ1/chs4-Δ1 bni4-Δ1/bni4-Δ1); (H and K) DDY175 containing plasmid p356 (high-copy BNI4); and (I and L) DDY185-1A (cdc10-Δ1). All strains were grown at 30°C except DDY185-1A, which was grown at 23°C. Arrowheads indicate protuberances at previous division sites; arrows indicate larger-than-normal bud necks.
Mentions: To examine the phenotypes resulting from the loss of CHS4 and/or BNI4 function, these ORFs were deleted (Fig. 2 A) in the same genetic background. DNA–DNA blot-hybridization analyses confirmed the success of these deletions (Fig. 2 B). For both DDY172 (CHS4/chs4-Δ1) and DDY173 (BNI4/bni4-Δ1) (Table I), viability segregated 4+:0−, and TRP1 segregated 2:2 at 22, 30, 37, and 39°C, indicating that neither CHS4 nor BNI4 is essential for viability. chs4-Δ1 and bni4-Δ1 haploids were mated, and the resulting diploid (DDY176) was subjected to tetrad analysis. Segregants that contained both mutations were viable at temperatures from 22–37°C and had growth rates similar to those of their CHS4 BNI4 siblings. DIC microscopy revealed that some chs4-Δ1 cells had enlarged bud necks and that many had protuberances at previous division sites (Fig. 4 B; cf. wild-type cells in Fig. 4 A), as noted previously for chs1 chs3 mutants by Shaw et al. (1991). Similarly, the majority of bni4-Δ1 cells had enlarged bud necks, and some had protuberances at previous division sites (Fig. 4 C), and the majority of chs4-Δ1 bni4-Δ1 cells had both enlarged bud necks and protuberances (Fig. 4 G). The double-mutant cells also were extensively vacuolated.

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