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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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Localization of Bni4p. Cells were grown in rich (YM-P) medium except for those shown in B, which were grown in SC medium. (A–G) Exponentially growing cells of strains (A and B) YEF473 (wild-type); (C) DDY175 (bni4-Δ1/bni4-Δ1); (D) DDY175 containing plasmid p356 (high-copy BNI4); (E) DDY174 (chs4-Δ1/chs4-Δ1); (F) DDY186 (chs3-Δ1/chs3-Δ1); and (G) DDY185-1A (cdc10-Δ1) were examined by immunofluorescence microscopy using Bni4p-specific antibodies. (H–K) Cells of strain JPTA1493-H01 (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 Bni4p-specific antibodies (H and I) or Cdc11p-specific antibodies (J and K). Arrows and arrowheads indicate  structures discussed in the text.
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Figure 6: Localization of Bni4p. Cells were grown in rich (YM-P) medium except for those shown in B, which were grown in SC medium. (A–G) Exponentially growing cells of strains (A and B) YEF473 (wild-type); (C) DDY175 (bni4-Δ1/bni4-Δ1); (D) DDY175 containing plasmid p356 (high-copy BNI4); (E) DDY174 (chs4-Δ1/chs4-Δ1); (F) DDY186 (chs3-Δ1/chs3-Δ1); and (G) DDY185-1A (cdc10-Δ1) were examined by immunofluorescence microscopy using Bni4p-specific antibodies. (H–K) Cells of strain JPTA1493-H01 (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 Bni4p-specific antibodies (H and I) or Cdc11p-specific antibodies (J and K). Arrows and arrowheads indicate structures discussed in the text.

Mentions: In immunofluorescence experiments on wild-type cells grown in rich medium, Bni4p was detected as a single ring on most unbudded cells and as a single ring on the mother-cell side of the mother-bud neck in nearly all small-budded cells and most medium-budded cells (Fig. 6 A). In some medium-budded and many large-budded cells, Bni4p appeared as a double ring in the mother-bud neck, although with the signal on the bud side generally weaker than that on the mother-cell side (Fig. 6 A, arrowheads). The similarity in appearance between most of the Bni4p rings seen on unbudded cells and those seen at the necks of small-budded cells suggests that the former represent sites of incipient bud emergence. However, some of the Bni4p rings on unbudded cells appear to represent residual Bni4p at the preceding division site, a hypothesis supported by the observation of some cells with Bni4p rings near both poles (Fig. 6 A, arrows). In wild-type cells grown in synthetic complete medium, the Bni4p signal was also asymmetric, but the signal on the bud side typically appeared earlier in the cell cycle and subsequently reached an intensity apparently equivalent to that on the mother-cell side (Fig. 6 B). No signal was observed in a bni4-Δ1 strain (Fig. 6 C), confirming the specificity of the staining. Thus, Bni4p appears to localize to a ring at the presumptive bud site before bud emergence and is present asymmetrically on the mother-cell side of the neck early in the cell cycle, a pattern that correlates well with the localization and timing of bud-scar chitin deposition. Later in the cell cycle, Bni4p appears to accumulate also on the bud side of the neck, and a symmetric distribution of Bni4p across the neck was typical even in cells with small buds in a Bni4p-overproducing strain (Fig. 6 D). The overproducing cells also appeared to have Bni4p distributed throughout the cell or plasma membrane, and some unbudded cells displayed an intensely staining disc or ring (Fig. 6 D, arrow). Localization of Bni4p to the mother-bud neck is consistent with the two-hybrid data showing its interactions with Cdc10p and Chs4p, two other proteins that localize to this region (see introduction and below).


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)

Localization of Bni4p. Cells were grown in rich (YM-P) medium except for those shown in B, which were grown in SC medium. (A–G) Exponentially growing cells of strains (A and B) YEF473 (wild-type); (C) DDY175 (bni4-Δ1/bni4-Δ1); (D) DDY175 containing plasmid p356 (high-copy BNI4); (E) DDY174 (chs4-Δ1/chs4-Δ1); (F) DDY186 (chs3-Δ1/chs3-Δ1); and (G) DDY185-1A (cdc10-Δ1) were examined by immunofluorescence microscopy using Bni4p-specific antibodies. (H–K) Cells of strain JPTA1493-H01 (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 Bni4p-specific antibodies (H and I) or Cdc11p-specific antibodies (J and K). Arrows and arrowheads indicate  structures discussed in the text.
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Related In: Results  -  Collection

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Figure 6: Localization of Bni4p. Cells were grown in rich (YM-P) medium except for those shown in B, which were grown in SC medium. (A–G) Exponentially growing cells of strains (A and B) YEF473 (wild-type); (C) DDY175 (bni4-Δ1/bni4-Δ1); (D) DDY175 containing plasmid p356 (high-copy BNI4); (E) DDY174 (chs4-Δ1/chs4-Δ1); (F) DDY186 (chs3-Δ1/chs3-Δ1); and (G) DDY185-1A (cdc10-Δ1) were examined by immunofluorescence microscopy using Bni4p-specific antibodies. (H–K) Cells of strain JPTA1493-H01 (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 Bni4p-specific antibodies (H and I) or Cdc11p-specific antibodies (J and K). Arrows and arrowheads indicate structures discussed in the text.
Mentions: In immunofluorescence experiments on wild-type cells grown in rich medium, Bni4p was detected as a single ring on most unbudded cells and as a single ring on the mother-cell side of the mother-bud neck in nearly all small-budded cells and most medium-budded cells (Fig. 6 A). In some medium-budded and many large-budded cells, Bni4p appeared as a double ring in the mother-bud neck, although with the signal on the bud side generally weaker than that on the mother-cell side (Fig. 6 A, arrowheads). The similarity in appearance between most of the Bni4p rings seen on unbudded cells and those seen at the necks of small-budded cells suggests that the former represent sites of incipient bud emergence. However, some of the Bni4p rings on unbudded cells appear to represent residual Bni4p at the preceding division site, a hypothesis supported by the observation of some cells with Bni4p rings near both poles (Fig. 6 A, arrows). In wild-type cells grown in synthetic complete medium, the Bni4p signal was also asymmetric, but the signal on the bud side typically appeared earlier in the cell cycle and subsequently reached an intensity apparently equivalent to that on the mother-cell side (Fig. 6 B). No signal was observed in a bni4-Δ1 strain (Fig. 6 C), confirming the specificity of the staining. Thus, Bni4p appears to localize to a ring at the presumptive bud site before bud emergence and is present asymmetrically on the mother-cell side of the neck early in the cell cycle, a pattern that correlates well with the localization and timing of bud-scar chitin deposition. Later in the cell cycle, Bni4p appears to accumulate also on the bud side of the neck, and a symmetric distribution of Bni4p across the neck was typical even in cells with small buds in a Bni4p-overproducing strain (Fig. 6 D). The overproducing cells also appeared to have Bni4p distributed throughout the cell or plasma membrane, and some unbudded cells displayed an intensely staining disc or ring (Fig. 6 D, arrow). Localization of Bni4p to the mother-bud neck is consistent with the two-hybrid data showing its interactions with Cdc10p and Chs4p, two other proteins that localize to this region (see introduction and below).

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