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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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Immunoblot analysis of Bni4p-specific antibodies. Extracts of bni4-Δ1/bni4-Δ1 strain DDY175 (lane 1);  wild-type strain YEF473 (lane  2); and strain YEF473 containing the high-copy BNI4 plasmid p356 (lane 3) were analyzed using affinity-purified  antibodies to Bni4p (see Materials and Methods). The mobilities of molecular mass markers are indicated.
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Figure 5: Immunoblot analysis of Bni4p-specific antibodies. Extracts of bni4-Δ1/bni4-Δ1 strain DDY175 (lane 1); wild-type strain YEF473 (lane 2); and strain YEF473 containing the high-copy BNI4 plasmid p356 (lane 3) were analyzed using affinity-purified antibodies to Bni4p (see Materials and Methods). The mobilities of molecular mass markers are indicated.

Mentions: To determine the effects of Bni4p overexpression, a high-copy BNI4 plasmid (p356) was transformed into BNI4 (YEF473) and bni4-Δ1 (DDY175) strains. The resulting transformants were viable at 23, 30, and 37°C. However, the majority of cells grew with multiple, elongated buds with enlarged bud necks and displayed delocalized chitin deposition (Fig. 4, H and K). This phenotype was similar to that of cdc10-Δ1 mutants (Fig. 4, I and L; Flescher et al. [1993]). Immunoblot analysis (see Fig. 5) confirmed that Bni4p indeed accumulates to higher-than-normal levels when plasmid p356 is present. To confirm that the abnormal morphology was indeed due to BNI4 overexpression (and not to some other element in p356), a high-copy BNI4 plasmid (p372) and a derivative that contained the same DNA except for a fragment internal to the BNI4 ORF (p374) were constructed and transformed into wild-type strain YEF473. Transformants that carried p372 had the abnormal morphology, whereas those that carried p374 had normal morphology (data not shown). In addition, a high-copy plasmid that encodes only the NH2-terminal 508 amino acids of Bni4p (p367) caused the same morphology as did the plasmids carrying full-length BNI4, although Bni4pΔ508 was unable to complement the bni4-Δ1 allele when expressed from either low-copy (p368) or high-copy (p367) plasmids (data not shown). Thus, the abnormal morphology seen in strains carrying multiple copies of BNI4 is in fact due to overaccumulation of Bni4p, which suggests that excess Bni4p can titrate a factor or factors required for normal chitin deposition and cytokinesis. The NH2-terminal 508 amino acids of Bni4p appear to be sufficient for this titration.


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)

Immunoblot analysis of Bni4p-specific antibodies. Extracts of bni4-Δ1/bni4-Δ1 strain DDY175 (lane 1);  wild-type strain YEF473 (lane  2); and strain YEF473 containing the high-copy BNI4 plasmid p356 (lane 3) were analyzed using affinity-purified  antibodies to Bni4p (see Materials and Methods). The mobilities of molecular mass markers are indicated.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Immunoblot analysis of Bni4p-specific antibodies. Extracts of bni4-Δ1/bni4-Δ1 strain DDY175 (lane 1); wild-type strain YEF473 (lane 2); and strain YEF473 containing the high-copy BNI4 plasmid p356 (lane 3) were analyzed using affinity-purified antibodies to Bni4p (see Materials and Methods). The mobilities of molecular mass markers are indicated.
Mentions: To determine the effects of Bni4p overexpression, a high-copy BNI4 plasmid (p356) was transformed into BNI4 (YEF473) and bni4-Δ1 (DDY175) strains. The resulting transformants were viable at 23, 30, and 37°C. However, the majority of cells grew with multiple, elongated buds with enlarged bud necks and displayed delocalized chitin deposition (Fig. 4, H and K). This phenotype was similar to that of cdc10-Δ1 mutants (Fig. 4, I and L; Flescher et al. [1993]). Immunoblot analysis (see Fig. 5) confirmed that Bni4p indeed accumulates to higher-than-normal levels when plasmid p356 is present. To confirm that the abnormal morphology was indeed due to BNI4 overexpression (and not to some other element in p356), a high-copy BNI4 plasmid (p372) and a derivative that contained the same DNA except for a fragment internal to the BNI4 ORF (p374) were constructed and transformed into wild-type strain YEF473. Transformants that carried p372 had the abnormal morphology, whereas those that carried p374 had normal morphology (data not shown). In addition, a high-copy plasmid that encodes only the NH2-terminal 508 amino acids of Bni4p (p367) caused the same morphology as did the plasmids carrying full-length BNI4, although Bni4pΔ508 was unable to complement the bni4-Δ1 allele when expressed from either low-copy (p368) or high-copy (p367) plasmids (data not shown). Thus, the abnormal morphology seen in strains carrying multiple copies of BNI4 is in fact due to overaccumulation of Bni4p, which suggests that excess Bni4p can titrate a factor or factors required for normal chitin deposition and cytokinesis. The NH2-terminal 508 amino acids of Bni4p appear to be sufficient for this titration.

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