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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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(A) Physical maps of the CHS4 and BNI4 regions and  structures of the chs4-Δ1 and bni4-Δ1 deletion alleles. Open  boxes represent open reading frames, and stippled boxes represent the TRP1 gene plus flanking sequences. Arrows indicate the  directions of transcription. Solid black bars represent the fragments used to probe DNA blots. Lines beneath the CHS4 map  represent segments of DNA assayed (in the indicated low-copy  plasmids) for complementation of the synthetic lethality of strain  DDY156 or 124Y03A (SL) and of the chitin synthesis defect of  the chs4-Δ1 strain DDY174 (CS); AA indicates the COOH-terminal Chs4p amino acid encoded by the corresponding DNA.  Restriction enzymes: G, BglII; H, HindIII; N, NsiI; R, EcoRI; S,  SalI; Sa, Sau3AI; Sp, SphI; V, EcoRV; X, XbaI. There are other  Sau3AI sites in the BNI4 region; those indicated represent the  ends of the insert in plasmid p356 (see text). (B) DNA–DNA  blot-hybridization analyses of chs4-Δ1 and bni4-Δ1 strains. Genomic DNAs were digested with HindIII. Lanes 1–6 were probed  with a 2.9-kb BglII–HindIII fragment that contains most of CHS4  and some upstream DNA (see A). Lanes 7–12 were probed with  a 2-kb EcoRV fragment that contains a portion of BNI4 and  some downstream DNA (see A) together with a portion of the  tetR gene of YEp13. Lanes 1 and 7, YEF473 (wild-type); lanes 2  and 8, DDY176 (chs4-Δ1/CHS4 bni4-Δ1/BNI4); lanes 3–6 and 9–12,  the four segregants from one tetratype tetrad from DDY176.  Fragments of the predicted sizes were visualized in all lanes (note  the presence of a HindIII site in TRP1); their molecular weights  are indicated in kb.
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Figure 2: (A) Physical maps of the CHS4 and BNI4 regions and structures of the chs4-Δ1 and bni4-Δ1 deletion alleles. Open boxes represent open reading frames, and stippled boxes represent the TRP1 gene plus flanking sequences. Arrows indicate the directions of transcription. Solid black bars represent the fragments used to probe DNA blots. Lines beneath the CHS4 map represent segments of DNA assayed (in the indicated low-copy plasmids) for complementation of the synthetic lethality of strain DDY156 or 124Y03A (SL) and of the chitin synthesis defect of the chs4-Δ1 strain DDY174 (CS); AA indicates the COOH-terminal Chs4p amino acid encoded by the corresponding DNA. Restriction enzymes: G, BglII; H, HindIII; N, NsiI; R, EcoRI; S, SalI; Sa, Sau3AI; Sp, SphI; V, EcoRV; X, XbaI. There are other Sau3AI sites in the BNI4 region; those indicated represent the ends of the insert in plasmid p356 (see text). (B) DNA–DNA blot-hybridization analyses of chs4-Δ1 and bni4-Δ1 strains. Genomic DNAs were digested with HindIII. Lanes 1–6 were probed with a 2.9-kb BglII–HindIII fragment that contains most of CHS4 and some upstream DNA (see A). Lanes 7–12 were probed with a 2-kb EcoRV fragment that contains a portion of BNI4 and some downstream DNA (see A) together with a portion of the tetR gene of YEp13. Lanes 1 and 7, YEF473 (wild-type); lanes 2 and 8, DDY176 (chs4-Δ1/CHS4 bni4-Δ1/BNI4); lanes 3–6 and 9–12, the four segregants from one tetratype tetrad from DDY176. Fragments of the predicted sizes were visualized in all lanes (note the presence of a HindIII site in TRP1); their molecular weights are indicated in kb.

Mentions: A plasmid shuffle was performed in strain 124Y03A (Table I) to replace plasmid pB12G with plasmid YEp24(CDC12)N (Table II), thus creating strain DDY156. This strain was transformed with a yeast genomic DNA library in plasmid YCp50-LEU2 (kindly provided by F. Spencer and P. Hieter, Johns Hopkins University, Baltimore, MD), and the transformants were replica-plated to SC-Leu supplemented with 0.1% 5-fluoroorotic acid (5-FOA) to select against plasmid YEp24(CDC12)N. A complementing plasmid, p196, contained CHS4 (see Results). A 3.4-kb XbaI–HindIII fragment, which encodes amino acids 1–678 of CHS4 (Scherens et al., 1993; see Fig. 2 A), was subcloned from p196 into pRS315 (Table II), thus creating p206. Unidirectional deletions of DNA were made in this plasmid using appropriate restriction enzymes and a modified exonuclease III procedure (Henikoff, 1984; Beltzer et al., 1986), thus creating a series of plasmids containing CHS4 truncated to different extents at the 3′ end of the coding region. Included in this series of plasmids are p377 and p376, which contain alleles chs4Δ79 and chs4Δ560, which encode the NH2-terminal 79 and 560 amino acids of Chs4p, respectively. The complete CHS4 gene was subcloned from p196 into pRS316 as a BglII–EcoRV fragment that contained ∼1 kb on either side of the CHS4 open reading frame (ORF; see Fig. 2 A), thus creating p267. p267 was digested with EcoRI (one site in CHS4 and the other in vector sequences) and religated to remove the 3′ 259 nucleotides of the CHS4 ORF and all of the cloned DNA downstream of CHS4; the resulting plasmid, p357, contains the chs4Δ610 allele, which encodes amino acids 1–610 of Chs4p. p361 was constructed by simultaneously ligating a 2.7-kb XbaI–EcoRI fragment of p267 (XbaI site from vector sequences) and a 270-bp EcoRI–XhoI fragment of p348 (see below) into XbaI/XhoI-digested pRS316; this plasmid contains the chs4C693S allele.


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)

(A) Physical maps of the CHS4 and BNI4 regions and  structures of the chs4-Δ1 and bni4-Δ1 deletion alleles. Open  boxes represent open reading frames, and stippled boxes represent the TRP1 gene plus flanking sequences. Arrows indicate the  directions of transcription. Solid black bars represent the fragments used to probe DNA blots. Lines beneath the CHS4 map  represent segments of DNA assayed (in the indicated low-copy  plasmids) for complementation of the synthetic lethality of strain  DDY156 or 124Y03A (SL) and of the chitin synthesis defect of  the chs4-Δ1 strain DDY174 (CS); AA indicates the COOH-terminal Chs4p amino acid encoded by the corresponding DNA.  Restriction enzymes: G, BglII; H, HindIII; N, NsiI; R, EcoRI; S,  SalI; Sa, Sau3AI; Sp, SphI; V, EcoRV; X, XbaI. There are other  Sau3AI sites in the BNI4 region; those indicated represent the  ends of the insert in plasmid p356 (see text). (B) DNA–DNA  blot-hybridization analyses of chs4-Δ1 and bni4-Δ1 strains. Genomic DNAs were digested with HindIII. Lanes 1–6 were probed  with a 2.9-kb BglII–HindIII fragment that contains most of CHS4  and some upstream DNA (see A). Lanes 7–12 were probed with  a 2-kb EcoRV fragment that contains a portion of BNI4 and  some downstream DNA (see A) together with a portion of the  tetR gene of YEp13. Lanes 1 and 7, YEF473 (wild-type); lanes 2  and 8, DDY176 (chs4-Δ1/CHS4 bni4-Δ1/BNI4); lanes 3–6 and 9–12,  the four segregants from one tetratype tetrad from DDY176.  Fragments of the predicted sizes were visualized in all lanes (note  the presence of a HindIII site in TRP1); their molecular weights  are indicated in kb.
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Related In: Results  -  Collection

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

Figure 2: (A) Physical maps of the CHS4 and BNI4 regions and structures of the chs4-Δ1 and bni4-Δ1 deletion alleles. Open boxes represent open reading frames, and stippled boxes represent the TRP1 gene plus flanking sequences. Arrows indicate the directions of transcription. Solid black bars represent the fragments used to probe DNA blots. Lines beneath the CHS4 map represent segments of DNA assayed (in the indicated low-copy plasmids) for complementation of the synthetic lethality of strain DDY156 or 124Y03A (SL) and of the chitin synthesis defect of the chs4-Δ1 strain DDY174 (CS); AA indicates the COOH-terminal Chs4p amino acid encoded by the corresponding DNA. Restriction enzymes: G, BglII; H, HindIII; N, NsiI; R, EcoRI; S, SalI; Sa, Sau3AI; Sp, SphI; V, EcoRV; X, XbaI. There are other Sau3AI sites in the BNI4 region; those indicated represent the ends of the insert in plasmid p356 (see text). (B) DNA–DNA blot-hybridization analyses of chs4-Δ1 and bni4-Δ1 strains. Genomic DNAs were digested with HindIII. Lanes 1–6 were probed with a 2.9-kb BglII–HindIII fragment that contains most of CHS4 and some upstream DNA (see A). Lanes 7–12 were probed with a 2-kb EcoRV fragment that contains a portion of BNI4 and some downstream DNA (see A) together with a portion of the tetR gene of YEp13. Lanes 1 and 7, YEF473 (wild-type); lanes 2 and 8, DDY176 (chs4-Δ1/CHS4 bni4-Δ1/BNI4); lanes 3–6 and 9–12, the four segregants from one tetratype tetrad from DDY176. Fragments of the predicted sizes were visualized in all lanes (note the presence of a HindIII site in TRP1); their molecular weights are indicated in kb.
Mentions: A plasmid shuffle was performed in strain 124Y03A (Table I) to replace plasmid pB12G with plasmid YEp24(CDC12)N (Table II), thus creating strain DDY156. This strain was transformed with a yeast genomic DNA library in plasmid YCp50-LEU2 (kindly provided by F. Spencer and P. Hieter, Johns Hopkins University, Baltimore, MD), and the transformants were replica-plated to SC-Leu supplemented with 0.1% 5-fluoroorotic acid (5-FOA) to select against plasmid YEp24(CDC12)N. A complementing plasmid, p196, contained CHS4 (see Results). A 3.4-kb XbaI–HindIII fragment, which encodes amino acids 1–678 of CHS4 (Scherens et al., 1993; see Fig. 2 A), was subcloned from p196 into pRS315 (Table II), thus creating p206. Unidirectional deletions of DNA were made in this plasmid using appropriate restriction enzymes and a modified exonuclease III procedure (Henikoff, 1984; Beltzer et al., 1986), thus creating a series of plasmids containing CHS4 truncated to different extents at the 3′ end of the coding region. Included in this series of plasmids are p377 and p376, which contain alleles chs4Δ79 and chs4Δ560, which encode the NH2-terminal 79 and 560 amino acids of Chs4p, respectively. The complete CHS4 gene was subcloned from p196 into pRS316 as a BglII–EcoRV fragment that contained ∼1 kb on either side of the CHS4 open reading frame (ORF; see Fig. 2 A), thus creating p267. p267 was digested with EcoRI (one site in CHS4 and the other in vector sequences) and religated to remove the 3′ 259 nucleotides of the CHS4 ORF and all of the cloned DNA downstream of CHS4; the resulting plasmid, p357, contains the chs4Δ610 allele, which encodes amino acids 1–610 of Chs4p. p361 was constructed by simultaneously ligating a 2.7-kb XbaI–EcoRI fragment of p267 (XbaI site from vector sequences) and a 270-bp EcoRI–XhoI fragment of p348 (see below) into XbaI/XhoI-digested pRS316; this plasmid contains the chs4C693S allele.

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