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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 Chs3p in wild-type and mutant strains. (A–G) Exponentially growing cells of strains (A) YEF473  (wild-type); (B) DDY186 (chs3-Δ1/chs3-Δ1); (C) DDY174 (chs4-Δ1/chs4-Δ1); (D) DDY175 (bni4-Δ1/bni4-Δ1); (E) DDY179 (chs4-Δ1/ chs4-Δ1 bni4-Δ1/bni4-Δ1); (F) DDY175 containing plasmid p356 (high-copy BNI4); and (G) DDY185-1A (cdc10-Δ1) were examined by  immunofluorescence microscopy using Chs3p-specific antibodies. (H–K) Cells of strain JPTA1493-HO1 (cdc12-6/cdc12-6) growing exponentially at 23°C (H and J) or collected 5 min after a shift to 37°C (I and K) were examined by immunofluorescence microscopy using  Chs3p-specific antibodies (H and I) or Cdc11p-specific antibodies (J and K). Arrowheads in some panels indicate rings at the bases of  tiny buds; arrows indicate patches of staining at presumptive bud sites.
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Figure 8: Immunolocalization of Chs3p in wild-type and mutant strains. (A–G) Exponentially growing cells of strains (A) YEF473 (wild-type); (B) DDY186 (chs3-Δ1/chs3-Δ1); (C) DDY174 (chs4-Δ1/chs4-Δ1); (D) DDY175 (bni4-Δ1/bni4-Δ1); (E) DDY179 (chs4-Δ1/ chs4-Δ1 bni4-Δ1/bni4-Δ1); (F) DDY175 containing plasmid p356 (high-copy BNI4); and (G) DDY185-1A (cdc10-Δ1) were examined by immunofluorescence microscopy using Chs3p-specific antibodies. (H–K) Cells of strain JPTA1493-HO1 (cdc12-6/cdc12-6) growing exponentially at 23°C (H and J) or collected 5 min after a shift to 37°C (I and K) were examined by immunofluorescence microscopy using Chs3p-specific antibodies (H and I) or Cdc11p-specific antibodies (J and K). Arrowheads in some panels indicate rings at the bases of tiny buds; arrows indicate patches of staining at presumptive bud sites.

Mentions: The evidence presented above suggests that the localization of Chs3p (and hence of chitin deposition) may depend upon Chs4p, Bni4p, and the septins. To test this hypothesis, Chs3p-specific antibodies (Chuang and Schekman, 1996) were used to localize this protein in wild-type, chs4-Δ1, bni4-Δ1, chs4-Δ1 bni4-Δ1, Bni4p-overexpressing, and septin mutant strains. As found previously (Chuang and Schekman, 1996; Santos and Snyder, 1997), Chs3p was observed in wild-type cells as a patch (presumably at the incipient bud site) in many unbudded cells (Fig. 8 A, arrow), as a distinct ring on the mother-cell side of the mother-bud neck in many tiny-budded cells (Fig. 8 A, arrowheads), and as a band on both sides of the neck in some cells with large buds (data not shown). In addition, punctate staining, which was also seen (although to a lesser extent) in chs3-Δ1 control cells (Fig. 8 B), was visible throughout the cells. In contrast, in chs4-Δ1, bni4-Δ1, and chs4-Δ1 bni4-Δ1 strains, Chs3p was typically found to be localized diffusely throughout the bud and in the vicinity of the mother-bud neck in tiny-budded cells, but it did not form a distinct ring at the neck like those seen in wild-type cells (Fig. 8, C–E). In addition, some unbudded cells of the mutant strains displayed patches of staining resembling, but typically less distinct than, those seen in wild-type cells (Fig. 7, C–E, arrows). In Bni4p-overexpressing and cdc10-Δ1 strains, Chs3p was observed in a ring in a few cells (Fig. 8, F and G, arrowheads) but was more commonly found diffusely throughout the cells. In a cdc12-6 strain grown at 23°C, Chs3p localized normally (Fig. 8 H), but normal localization was lost within 5 min after a shift to restrictive temperature (Fig. 8 I), paralleling the loss of septin localization (Fig. 8, J and K). Taken together, the data suggest that the normal localization of Chs3p to its major site of action (i.e., the site of chitin-ring formation) is dependent upon Chs4p, Bni4p, and the septins, and that the localization of Chs3p and Chs4p is interdependent (see also above). In the absence of Chs4p and/or Bni4p, the cell appears to recruit Chs3p to the correct general location but fails to assemble it into the well-defined ring seen in wild-type cells.


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 Chs3p in wild-type and mutant strains. (A–G) Exponentially growing cells of strains (A) YEF473  (wild-type); (B) DDY186 (chs3-Δ1/chs3-Δ1); (C) DDY174 (chs4-Δ1/chs4-Δ1); (D) DDY175 (bni4-Δ1/bni4-Δ1); (E) DDY179 (chs4-Δ1/ chs4-Δ1 bni4-Δ1/bni4-Δ1); (F) DDY175 containing plasmid p356 (high-copy BNI4); and (G) DDY185-1A (cdc10-Δ1) were examined by  immunofluorescence microscopy using Chs3p-specific antibodies. (H–K) Cells of strain JPTA1493-HO1 (cdc12-6/cdc12-6) growing exponentially at 23°C (H and J) or collected 5 min after a shift to 37°C (I and K) were examined by immunofluorescence microscopy using  Chs3p-specific antibodies (H and I) or Cdc11p-specific antibodies (J and K). Arrowheads in some panels indicate rings at the bases of  tiny buds; arrows indicate patches of staining at presumptive bud sites.
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Related In: Results  -  Collection

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Figure 8: Immunolocalization of Chs3p in wild-type and mutant strains. (A–G) Exponentially growing cells of strains (A) YEF473 (wild-type); (B) DDY186 (chs3-Δ1/chs3-Δ1); (C) DDY174 (chs4-Δ1/chs4-Δ1); (D) DDY175 (bni4-Δ1/bni4-Δ1); (E) DDY179 (chs4-Δ1/ chs4-Δ1 bni4-Δ1/bni4-Δ1); (F) DDY175 containing plasmid p356 (high-copy BNI4); and (G) DDY185-1A (cdc10-Δ1) were examined by immunofluorescence microscopy using Chs3p-specific antibodies. (H–K) Cells of strain JPTA1493-HO1 (cdc12-6/cdc12-6) growing exponentially at 23°C (H and J) or collected 5 min after a shift to 37°C (I and K) were examined by immunofluorescence microscopy using Chs3p-specific antibodies (H and I) or Cdc11p-specific antibodies (J and K). Arrowheads in some panels indicate rings at the bases of tiny buds; arrows indicate patches of staining at presumptive bud sites.
Mentions: The evidence presented above suggests that the localization of Chs3p (and hence of chitin deposition) may depend upon Chs4p, Bni4p, and the septins. To test this hypothesis, Chs3p-specific antibodies (Chuang and Schekman, 1996) were used to localize this protein in wild-type, chs4-Δ1, bni4-Δ1, chs4-Δ1 bni4-Δ1, Bni4p-overexpressing, and septin mutant strains. As found previously (Chuang and Schekman, 1996; Santos and Snyder, 1997), Chs3p was observed in wild-type cells as a patch (presumably at the incipient bud site) in many unbudded cells (Fig. 8 A, arrow), as a distinct ring on the mother-cell side of the mother-bud neck in many tiny-budded cells (Fig. 8 A, arrowheads), and as a band on both sides of the neck in some cells with large buds (data not shown). In addition, punctate staining, which was also seen (although to a lesser extent) in chs3-Δ1 control cells (Fig. 8 B), was visible throughout the cells. In contrast, in chs4-Δ1, bni4-Δ1, and chs4-Δ1 bni4-Δ1 strains, Chs3p was typically found to be localized diffusely throughout the bud and in the vicinity of the mother-bud neck in tiny-budded cells, but it did not form a distinct ring at the neck like those seen in wild-type cells (Fig. 8, C–E). In addition, some unbudded cells of the mutant strains displayed patches of staining resembling, but typically less distinct than, those seen in wild-type cells (Fig. 7, C–E, arrows). In Bni4p-overexpressing and cdc10-Δ1 strains, Chs3p was observed in a ring in a few cells (Fig. 8, F and G, arrowheads) but was more commonly found diffusely throughout the cells. In a cdc12-6 strain grown at 23°C, Chs3p localized normally (Fig. 8 H), but normal localization was lost within 5 min after a shift to restrictive temperature (Fig. 8 I), paralleling the loss of septin localization (Fig. 8, J and K). Taken together, the data suggest that the normal localization of Chs3p to its major site of action (i.e., the site of chitin-ring formation) is dependent upon Chs4p, Bni4p, and the septins, and that the localization of Chs3p and Chs4p is interdependent (see also above). In the absence of Chs4p and/or Bni4p, the cell appears to recruit Chs3p to the correct general location but fails to assemble it into the well-defined ring seen in wild-type cells.

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