Limits...
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.

Show MeSH

Related in: MedlinePlus

Amino acid sequences of Chs4p and Bni4p. Asterisks  represent the termination codons. (A) Portions of the predicted  amino acid sequence of Chs4p that include the possible calcium-binding domain (amino acids 237–250; underlined) and the  COOH terminus including the CAAX motif (amino acids 693– 696; underlined). (B) Predicted sequence of Bni4p. The predicted  coiled-coil domain (amino acids 106–139) is underlined, and the  first residue translated from the BNI4-containing AD fusion  clone is overlined.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139831&req=5

Figure 3: Amino acid sequences of Chs4p and Bni4p. Asterisks represent the termination codons. (A) Portions of the predicted amino acid sequence of Chs4p that include the possible calcium-binding domain (amino acids 237–250; underlined) and the COOH terminus including the CAAX motif (amino acids 693– 696; underlined). (B) Predicted sequence of Bni4p. The predicted coiled-coil domain (amino acids 106–139) is underlined, and the first residue translated from the BNI4-containing AD fusion clone is overlined.

Mentions: To identify the gene of interest in p196, various fragments were subcloned into pRS315 (low copy, LEU2) or pRS316 (low-copy, URA3) and transformed into strain DDY156 or 124Y03A. A 3.4-kb XbaI–HindIII fragment (Fig. 2 A, plasmid p206) was sufficient for complementation of the synthetic lethality. Partial sequence analysis of this fragment (data not shown) and comparison to the full sequence of this chromosome region (Scherens et al., 1993; accession number Z23261) revealed that the fragment contains two truncated ORFs. One of these contains all but the COOH-terminal 18 codons of YBL0519 or CHS4/ CSD4/CAL2/SKT5 (Bulawa, 1992, 1993; Kawamoto et al., 1992; Trilla et al. 1997; Greene, J., and B. DiDomenico, personal communication; Bulawa, C., personal communication), a gene involved in chitin synthesis (see introduction), and the other contains the NH2-terminal 272 codons of YBL0517, which encodes a 687–amino acid protein of unknown function. A 3.8-kb BglII–EcoRV fragment (Fig. 2 A, plasmid p267), which contains all of CHS4 but only 103 codons of YLB0517, was also sufficient for complementation of the synthetic lethality, suggesting that CHS4 was the gene responsible for the complementation. To confirm this conclusion, restriction enzyme digestion and an exonuclease procedure (see Materials and Methods) were used to truncate CHS4 further. Plasmids that contained as little as 560 codons of CHS4 still complemented well (Fig. 2 A, plasmid p376), but a plasmid that contained only 79 codons of CHS4 (together with the same portion of YBL0517) failed to complement (Fig. 2 A, plasmid p377). Thus, CHS4 indeed appears to be responsible for the observed complementation. Because the two reported sequences for the CHS4 region differ (Kawamoto et al., 1992; Scherens et al., 1993), we resequenced the regions of discrepancy (nucleotides −20–80 and 1655–1755 of YBL0519). Our sequence agrees with that of Scherens et al. (1993), which predicts that CHS4 encodes a protein of 696 amino acids that contains a putative calcium-binding domain (amino acids 237–250) and terminates in a probable prenylation site (CAAX motif) (Fig. 3 A).


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)

Amino acid sequences of Chs4p and Bni4p. Asterisks  represent the termination codons. (A) Portions of the predicted  amino acid sequence of Chs4p that include the possible calcium-binding domain (amino acids 237–250; underlined) and the  COOH terminus including the CAAX motif (amino acids 693– 696; underlined). (B) Predicted sequence of Bni4p. The predicted  coiled-coil domain (amino acids 106–139) is underlined, and the  first residue translated from the BNI4-containing AD fusion  clone is overlined.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Amino acid sequences of Chs4p and Bni4p. Asterisks represent the termination codons. (A) Portions of the predicted amino acid sequence of Chs4p that include the possible calcium-binding domain (amino acids 237–250; underlined) and the COOH terminus including the CAAX motif (amino acids 693– 696; underlined). (B) Predicted sequence of Bni4p. The predicted coiled-coil domain (amino acids 106–139) is underlined, and the first residue translated from the BNI4-containing AD fusion clone is overlined.
Mentions: To identify the gene of interest in p196, various fragments were subcloned into pRS315 (low copy, LEU2) or pRS316 (low-copy, URA3) and transformed into strain DDY156 or 124Y03A. A 3.4-kb XbaI–HindIII fragment (Fig. 2 A, plasmid p206) was sufficient for complementation of the synthetic lethality. Partial sequence analysis of this fragment (data not shown) and comparison to the full sequence of this chromosome region (Scherens et al., 1993; accession number Z23261) revealed that the fragment contains two truncated ORFs. One of these contains all but the COOH-terminal 18 codons of YBL0519 or CHS4/ CSD4/CAL2/SKT5 (Bulawa, 1992, 1993; Kawamoto et al., 1992; Trilla et al. 1997; Greene, J., and B. DiDomenico, personal communication; Bulawa, C., personal communication), a gene involved in chitin synthesis (see introduction), and the other contains the NH2-terminal 272 codons of YBL0517, which encodes a 687–amino acid protein of unknown function. A 3.8-kb BglII–EcoRV fragment (Fig. 2 A, plasmid p267), which contains all of CHS4 but only 103 codons of YLB0517, was also sufficient for complementation of the synthetic lethality, suggesting that CHS4 was the gene responsible for the complementation. To confirm this conclusion, restriction enzyme digestion and an exonuclease procedure (see Materials and Methods) were used to truncate CHS4 further. Plasmids that contained as little as 560 codons of CHS4 still complemented well (Fig. 2 A, plasmid p376), but a plasmid that contained only 79 codons of CHS4 (together with the same portion of YBL0517) failed to complement (Fig. 2 A, plasmid p377). Thus, CHS4 indeed appears to be responsible for the observed complementation. Because the two reported sequences for the CHS4 region differ (Kawamoto et al., 1992; Scherens et al., 1993), we resequenced the regions of discrepancy (nucleotides −20–80 and 1655–1755 of YBL0519). Our sequence agrees with that of Scherens et al. (1993), which predicts that CHS4 encodes a protein of 696 amino acids that contains a putative calcium-binding domain (amino acids 237–250) and terminates in a probable prenylation site (CAAX motif) (Fig. 3 A).

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