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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 GFP-Chs4p. Cells were grown, fixed, and observed as described in Materials and Methods. (A–F) Cells of the  indicated strains were collected during growth at 23°C: (A) DDY197 (chs4-Δ1/chs4-Δ1 [p326]); (B) DDY217 (chs4-Δ1/chs4-Δ1 bni4-Δ1/ bni4-Δ1 [p326]) containing pRS315 (control plasmid); (C) DDY217 containing p366 (low-copy, BNI4); (D) DDY197 containing p365  (high-copy, BNI4); (E) DDY244 (chs4-Δ1/chs4-Δ1 chs3-Δ1/chs3-Δ1 [p326]); and (F) DDY244 containing p408 (low-copy, CHS3). (G–J)  Cells of strain DDY218 (chs4-Δ1/chs4-Δ1 cdc12-6/cdc12-6 [p326]) containing either (G and H) YCplac111 (control plasmid) or (I and J)  p422 (low-copy, CDC12) were collected during growth at 23°C (G and I) or 20 min after a shift to 37°C (H and J). Small arrows indicate  GFP-Chs4p at presumptive bud sites or previous sites of cell division; small arrowheads indicate rings of Chs4p at the bases of tiny buds;  large arrows indicate mother-bud necks lacking GFP-Chs4p fluorescence; large arrowheads indicate mislocalized Chs4p; the asterisk indicates Chs4p on either side of the mother-bud neck (see text for details).
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Figure 7: Localization of GFP-Chs4p. Cells were grown, fixed, and observed as described in Materials and Methods. (A–F) Cells of the indicated strains were collected during growth at 23°C: (A) DDY197 (chs4-Δ1/chs4-Δ1 [p326]); (B) DDY217 (chs4-Δ1/chs4-Δ1 bni4-Δ1/ bni4-Δ1 [p326]) containing pRS315 (control plasmid); (C) DDY217 containing p366 (low-copy, BNI4); (D) DDY197 containing p365 (high-copy, BNI4); (E) DDY244 (chs4-Δ1/chs4-Δ1 chs3-Δ1/chs3-Δ1 [p326]); and (F) DDY244 containing p408 (low-copy, CHS3). (G–J) Cells of strain DDY218 (chs4-Δ1/chs4-Δ1 cdc12-6/cdc12-6 [p326]) containing either (G and H) YCplac111 (control plasmid) or (I and J) p422 (low-copy, CDC12) were collected during growth at 23°C (G and I) or 20 min after a shift to 37°C (H and J). Small arrows indicate GFP-Chs4p at presumptive bud sites or previous sites of cell division; small arrowheads indicate rings of Chs4p at the bases of tiny buds; large arrows indicate mother-bud necks lacking GFP-Chs4p fluorescence; large arrowheads indicate mislocalized Chs4p; the asterisk indicates Chs4p on either side of the mother-bud neck (see text for details).

Mentions: To localize Chs4p, we constructed plasmid p326, a low-copy plasmid encoding a GFP-Chs4p fusion protein. chs4-Δ1 strains harboring this plasmid (DDY172-2AX and DDY197) displayed normal Calcofluor staining and normal morphology (data not shown), indicating that the GFP-Chs4p fusion protein can supply Chs4p function. Examination of DDY197 cells by fluorescence microscopy revealed that GFP-Chs4p localized to a patch or ring at the presumptive bud site in many unbudded cells (Fig. 7 A, small arrows), to a ring at the base of the bud in most (if not all) cells with tiny buds (Fig. 7 A, arrowheads), and to both sides of the neck in many cells with large buds (Fig. 7 A, asterisk); localized signal was rarely detectable in cells with medium-sized buds (Fig. 7 A, large arrow). Some of the GFP-Chs4p fluorescence on unbudded cells appeared to represent residual protein at the preceding division site, a hypothesis supported by the observation that some cells displayed patches or rings of fluorescence near both poles (Fig. 7 A and G, small arrows). The localization observed for GFP-Chs4p is very similar to that observed for Chs3p (Chuang and Schekman, 1996; Santos and Snyder, 1997; also see below) in that both proteins are localized to the mother-bud neck early and late in the cell cycle but appear to be absent from the neck during intermediate stages of the cell cycle.


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 GFP-Chs4p. Cells were grown, fixed, and observed as described in Materials and Methods. (A–F) Cells of the  indicated strains were collected during growth at 23°C: (A) DDY197 (chs4-Δ1/chs4-Δ1 [p326]); (B) DDY217 (chs4-Δ1/chs4-Δ1 bni4-Δ1/ bni4-Δ1 [p326]) containing pRS315 (control plasmid); (C) DDY217 containing p366 (low-copy, BNI4); (D) DDY197 containing p365  (high-copy, BNI4); (E) DDY244 (chs4-Δ1/chs4-Δ1 chs3-Δ1/chs3-Δ1 [p326]); and (F) DDY244 containing p408 (low-copy, CHS3). (G–J)  Cells of strain DDY218 (chs4-Δ1/chs4-Δ1 cdc12-6/cdc12-6 [p326]) containing either (G and H) YCplac111 (control plasmid) or (I and J)  p422 (low-copy, CDC12) were collected during growth at 23°C (G and I) or 20 min after a shift to 37°C (H and J). Small arrows indicate  GFP-Chs4p at presumptive bud sites or previous sites of cell division; small arrowheads indicate rings of Chs4p at the bases of tiny buds;  large arrows indicate mother-bud necks lacking GFP-Chs4p fluorescence; large arrowheads indicate mislocalized Chs4p; the asterisk indicates Chs4p on either side of the mother-bud neck (see text for details).
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Related In: Results  -  Collection

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Figure 7: Localization of GFP-Chs4p. Cells were grown, fixed, and observed as described in Materials and Methods. (A–F) Cells of the indicated strains were collected during growth at 23°C: (A) DDY197 (chs4-Δ1/chs4-Δ1 [p326]); (B) DDY217 (chs4-Δ1/chs4-Δ1 bni4-Δ1/ bni4-Δ1 [p326]) containing pRS315 (control plasmid); (C) DDY217 containing p366 (low-copy, BNI4); (D) DDY197 containing p365 (high-copy, BNI4); (E) DDY244 (chs4-Δ1/chs4-Δ1 chs3-Δ1/chs3-Δ1 [p326]); and (F) DDY244 containing p408 (low-copy, CHS3). (G–J) Cells of strain DDY218 (chs4-Δ1/chs4-Δ1 cdc12-6/cdc12-6 [p326]) containing either (G and H) YCplac111 (control plasmid) or (I and J) p422 (low-copy, CDC12) were collected during growth at 23°C (G and I) or 20 min after a shift to 37°C (H and J). Small arrows indicate GFP-Chs4p at presumptive bud sites or previous sites of cell division; small arrowheads indicate rings of Chs4p at the bases of tiny buds; large arrows indicate mother-bud necks lacking GFP-Chs4p fluorescence; large arrowheads indicate mislocalized Chs4p; the asterisk indicates Chs4p on either side of the mother-bud neck (see text for details).
Mentions: To localize Chs4p, we constructed plasmid p326, a low-copy plasmid encoding a GFP-Chs4p fusion protein. chs4-Δ1 strains harboring this plasmid (DDY172-2AX and DDY197) displayed normal Calcofluor staining and normal morphology (data not shown), indicating that the GFP-Chs4p fusion protein can supply Chs4p function. Examination of DDY197 cells by fluorescence microscopy revealed that GFP-Chs4p localized to a patch or ring at the presumptive bud site in many unbudded cells (Fig. 7 A, small arrows), to a ring at the base of the bud in most (if not all) cells with tiny buds (Fig. 7 A, arrowheads), and to both sides of the neck in many cells with large buds (Fig. 7 A, asterisk); localized signal was rarely detectable in cells with medium-sized buds (Fig. 7 A, large arrow). Some of the GFP-Chs4p fluorescence on unbudded cells appeared to represent residual protein at the preceding division site, a hypothesis supported by the observation that some cells displayed patches or rings of fluorescence near both poles (Fig. 7 A and G, small arrows). The localization observed for GFP-Chs4p is very similar to that observed for Chs3p (Chuang and Schekman, 1996; Santos and Snyder, 1997; also see below) in that both proteins are localized to the mother-bud neck early and late in the cell cycle but appear to be absent from the neck during intermediate stages of the cell cycle.

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