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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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Delocalization of chitin deposition in septin mutants.  Cells were examined during growth at 23°C (A–D) or after a shift  to 37°C for 80–120 min (E–I). (A and E) cdc3 strain LH104-HO1;  (B and F) cdc10 strain LH17012-HO1; (C and G) cdc11 strain  JPT194-HO1; (D and H) cdc12 strain JPTA1493-HO1; (I) cdc4  strain 314D5. Arrows in A–D indicate growing buds; arrows in E–H  indicate the mother-bud necks lacking normal chitin rings; bud  scars formed during earlier growth at permissive temperature are  also visible on these cells.
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Figure 1: Delocalization of chitin deposition in septin mutants. Cells were examined during growth at 23°C (A–D) or after a shift to 37°C for 80–120 min (E–I). (A and E) cdc3 strain LH104-HO1; (B and F) cdc10 strain LH17012-HO1; (C and G) cdc11 strain JPT194-HO1; (D and H) cdc12 strain JPTA1493-HO1; (I) cdc4 strain 314D5. Arrows in A–D indicate growing buds; arrows in E–H indicate the mother-bud necks lacking normal chitin rings; bud scars formed during earlier growth at permissive temperature are also visible on these cells.

Mentions: The septin proteins form a ring at the presumptive bud site shortly before the chitin ring is formed in the overlying cell wall (see above). These observations suggested that the septins might be involved in the localization of chitin deposition. To address this possibility, temperature-sensitive septin mutants (cdc3, cdc10, cdc11, and cdc12) were shifted from permissive to restrictive temperature, and cells were examined by fluorescence microscopy after staining with Calcofluor. In each mutant, the chitin rings and bud scars formed during growth at permissive temperature appeared normal (Fig. 1, A–D), and their appearance did not change after the shift to restrictive temperature (Fig. 1, E–H). The first buds formed during growth at restrictive temperature, however, did not have discrete chitin rings at their bases. Instead, the Calcofluor-stainable material formed a diffuse band whose intensity decreased with distance from the mother-bud neck (Fig. 1, E–H). In contrast, several other temperature-sensitive cell-cycle mutants (cdc7, cdc13, cdc21, cdc31) displayed apparently normal chitin rings at the bases of the buds formed during cell-cycle arrest at restrictive temperature (data not shown). Of particular interest was the cdc4 mutant, which forms abnormally elongated buds like those of the septin mutants (Hartwell, 1971b) but maintains its septin organization (Byers, B., and L. Goetsch. 1976. J. Cell Biol. 70:35a; Haarer and Pringle, 1987; Kim et al., 1991) and forms normal-looking chitin rings (Fig. 1 I) at restrictive temperature. Thus, it appears that the delocalization of chitin deposition in the septin mutants results specifically from the loss of septin function and is not simply a consequence of cell-cycle arrest or abnormal bud shape.


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)

Delocalization of chitin deposition in septin mutants.  Cells were examined during growth at 23°C (A–D) or after a shift  to 37°C for 80–120 min (E–I). (A and E) cdc3 strain LH104-HO1;  (B and F) cdc10 strain LH17012-HO1; (C and G) cdc11 strain  JPT194-HO1; (D and H) cdc12 strain JPTA1493-HO1; (I) cdc4  strain 314D5. Arrows in A–D indicate growing buds; arrows in E–H  indicate the mother-bud necks lacking normal chitin rings; bud  scars formed during earlier growth at permissive temperature are  also visible on these cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139831&req=5

Figure 1: Delocalization of chitin deposition in septin mutants. Cells were examined during growth at 23°C (A–D) or after a shift to 37°C for 80–120 min (E–I). (A and E) cdc3 strain LH104-HO1; (B and F) cdc10 strain LH17012-HO1; (C and G) cdc11 strain JPT194-HO1; (D and H) cdc12 strain JPTA1493-HO1; (I) cdc4 strain 314D5. Arrows in A–D indicate growing buds; arrows in E–H indicate the mother-bud necks lacking normal chitin rings; bud scars formed during earlier growth at permissive temperature are also visible on these cells.
Mentions: The septin proteins form a ring at the presumptive bud site shortly before the chitin ring is formed in the overlying cell wall (see above). These observations suggested that the septins might be involved in the localization of chitin deposition. To address this possibility, temperature-sensitive septin mutants (cdc3, cdc10, cdc11, and cdc12) were shifted from permissive to restrictive temperature, and cells were examined by fluorescence microscopy after staining with Calcofluor. In each mutant, the chitin rings and bud scars formed during growth at permissive temperature appeared normal (Fig. 1, A–D), and their appearance did not change after the shift to restrictive temperature (Fig. 1, E–H). The first buds formed during growth at restrictive temperature, however, did not have discrete chitin rings at their bases. Instead, the Calcofluor-stainable material formed a diffuse band whose intensity decreased with distance from the mother-bud neck (Fig. 1, E–H). In contrast, several other temperature-sensitive cell-cycle mutants (cdc7, cdc13, cdc21, cdc31) displayed apparently normal chitin rings at the bases of the buds formed during cell-cycle arrest at restrictive temperature (data not shown). Of particular interest was the cdc4 mutant, which forms abnormally elongated buds like those of the septin mutants (Hartwell, 1971b) but maintains its septin organization (Byers, B., and L. Goetsch. 1976. J. Cell Biol. 70:35a; Haarer and Pringle, 1987; Kim et al., 1991) and forms normal-looking chitin rings (Fig. 1 I) at restrictive temperature. Thus, it appears that the delocalization of chitin deposition in the septin mutants results specifically from the loss of septin function and is not simply a consequence of cell-cycle arrest or abnormal bud shape.

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