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cdc12p, a protein required for cytokinesis in fission yeast, is a component of the cell division ring and interacts with profilin.

Chang F, Drubin D, Nurse P - J. Cell Biol. (1997)

Bottom Line: Here we show that cdc12p is a member of a family of proteins including Drosophila diaphanous, Saccharomyces cerevisiae BNI1, and S. pombe fus1, which are involved in cytokinesis or other actin-mediated processes.Based on these patterns, we present a model in which ring assembly originates from a single point on the cortex and in which a molecular pathway for the functions of cytokinesis proteins is suggested.Finally, we found that cdc12 and cdc3 mutants show a synthetic-lethal genetic interaction, and a proline-rich domain of cdc12p binds directly to profilin cdc3p in vitro, suggesting that one function of cdc12p in ring assembly is to bind profilin.

View Article: PubMed Central - PubMed

Affiliation: Imperial Cancer Research Fund, London, United Kingdom. fc99@columbia.edu

ABSTRACT
As in many other eukaryotic cells, cell division in fission yeast depends on the assembly of an actin ring that circumscribes the middle of the cell. Schizosaccharomyces pombe cdc12 is an essential gene necessary for actin ring assembly and septum formation. Here we show that cdc12p is a member of a family of proteins including Drosophila diaphanous, Saccharomyces cerevisiae BNI1, and S. pombe fus1, which are involved in cytokinesis or other actin-mediated processes. Using indirect immunofluorescence, we show that cdc12p is located in the cell division ring and not in other actin structures. When overexpressed, cdc12p is located at a medial spot in interphase that anticipates the future ring site. cdc12p localization is altered in actin ring mutants. cdc8 (tropomyosin homologue), cdc3 (profilin homologue), and cdc15 mutants exhibit no specific cdc12p staining during mitosis. cdc4 mutant cells exhibit a medial cortical cdc12p spot in place of a ring. mid1 mutant cells generally exhibit a cdc12p spot with a single cdc12p strand extending in a random direction. Based on these patterns, we present a model in which ring assembly originates from a single point on the cortex and in which a molecular pathway for the functions of cytokinesis proteins is suggested. Finally, we found that cdc12 and cdc3 mutants show a synthetic-lethal genetic interaction, and a proline-rich domain of cdc12p binds directly to profilin cdc3p in vitro, suggesting that one function of cdc12p in ring assembly is to bind profilin.

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cdc12 encodes a protein similar to the dia/BNI1 family of proteins. (A) Localization of the cdc12 gene. Restriction map of the  original 6-kb genomic clone insert (pFC103) that rescues cdc12-112 mutant cells. (Arrow) Extent and orientation of the single large  open reading frame in the region. Truncated versions of the clone and their ability to complement a cdc12-112 mutant; ++, full rescue;  +,  partial rescue. cdc12::ura4 deletion construct used in deleting cdc12. Much of the cdc12 open reading frame is replaced with a 1.6-kb  ura4 insert. (B) cdc12 open reading frame. Regions of predicted coiled-coil are underlined (Lupas et al., 1991), and the polyproline regions are boxed. (C) Similarity in gene domain structure. Proteins share a central FH1 polyproline rich domain (black box), an FH2  domain (stripe box), and a larger COOH-terminal area of homology (gray) also diagrammed in D. (D) Similarity of the proteins in a  COOH-terminal region. Proteins were aligned using Megalign in DNA Star. Residues identical to a calculated consensus are colored in  black, and conserved residues are colored in gray. These sequence data are available from GenBank/EMBL/DDBJ under accession  number Q10059.
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Figure 1: cdc12 encodes a protein similar to the dia/BNI1 family of proteins. (A) Localization of the cdc12 gene. Restriction map of the original 6-kb genomic clone insert (pFC103) that rescues cdc12-112 mutant cells. (Arrow) Extent and orientation of the single large open reading frame in the region. Truncated versions of the clone and their ability to complement a cdc12-112 mutant; ++, full rescue; +, partial rescue. cdc12::ura4 deletion construct used in deleting cdc12. Much of the cdc12 open reading frame is replaced with a 1.6-kb ura4 insert. (B) cdc12 open reading frame. Regions of predicted coiled-coil are underlined (Lupas et al., 1991), and the polyproline regions are boxed. (C) Similarity in gene domain structure. Proteins share a central FH1 polyproline rich domain (black box), an FH2 domain (stripe box), and a larger COOH-terminal area of homology (gray) also diagrammed in D. (D) Similarity of the proteins in a COOH-terminal region. Proteins were aligned using Megalign in DNA Star. Residues identical to a calculated consensus are colored in black, and conserved residues are colored in gray. These sequence data are available from GenBank/EMBL/DDBJ under accession number Q10059.

Mentions: The cdc12 gene was cloned by complementation of the temperature-sensitive lethal phenotype of cdc12-112. A pUR19 (ura4+)-based S. pombe genomic library (Barbet et al., 1992) was transformed into ura4D18 cdc12112 (FC112), and 5 × 104 ura+ transformant colonies were screened by replica printing onto YE5S with phyloxin for growth at 36°C. Four colonies showed plasmid-dependent rescue. Isolation of plasmid DNA from two of these transformants identified two related plasmids, pFC103 and pFC104. pFC103 complemented a cdc12-112 strain upon retransformation and contained a 6-kb insert in pUR19. Deletion analysis showed that a central region was critical for activity (see Fig. 1 A). Three genetic criteria demonstrated that complementing activity was the cdc12 gene: first, hybridization to an ordered cosmid blot (Hoheisel et al., 1993) showed that pFC103 mapped to left tip of chromosome I, at the corresponding location to which cdc12 has been previously mapped genetically. Second, pFC110, which contains a 3-kb EcoRI cdc12 insert from pFC103 and a sup3-5 marker (from pSTA12), was integrated into the genome by homologous recombination. Random spore analysis showed that the plasmid integrated at the cdc12 locus. Third, introduction of the COOH-terminal half of cdc12 gene into cdc12-112 does not confer rescue at 36°C in the majority of the cells, but confers rare stable gene conversion of the cdc12-112 allele, suggesting that the genetic defect of cdc12-112 resides in the COOHterminal half of the protein.


cdc12p, a protein required for cytokinesis in fission yeast, is a component of the cell division ring and interacts with profilin.

Chang F, Drubin D, Nurse P - J. Cell Biol. (1997)

cdc12 encodes a protein similar to the dia/BNI1 family of proteins. (A) Localization of the cdc12 gene. Restriction map of the  original 6-kb genomic clone insert (pFC103) that rescues cdc12-112 mutant cells. (Arrow) Extent and orientation of the single large  open reading frame in the region. Truncated versions of the clone and their ability to complement a cdc12-112 mutant; ++, full rescue;  +,  partial rescue. cdc12::ura4 deletion construct used in deleting cdc12. Much of the cdc12 open reading frame is replaced with a 1.6-kb  ura4 insert. (B) cdc12 open reading frame. Regions of predicted coiled-coil are underlined (Lupas et al., 1991), and the polyproline regions are boxed. (C) Similarity in gene domain structure. Proteins share a central FH1 polyproline rich domain (black box), an FH2  domain (stripe box), and a larger COOH-terminal area of homology (gray) also diagrammed in D. (D) Similarity of the proteins in a  COOH-terminal region. Proteins were aligned using Megalign in DNA Star. Residues identical to a calculated consensus are colored in  black, and conserved residues are colored in gray. These sequence data are available from GenBank/EMBL/DDBJ under accession  number Q10059.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139860&req=5

Figure 1: cdc12 encodes a protein similar to the dia/BNI1 family of proteins. (A) Localization of the cdc12 gene. Restriction map of the original 6-kb genomic clone insert (pFC103) that rescues cdc12-112 mutant cells. (Arrow) Extent and orientation of the single large open reading frame in the region. Truncated versions of the clone and their ability to complement a cdc12-112 mutant; ++, full rescue; +, partial rescue. cdc12::ura4 deletion construct used in deleting cdc12. Much of the cdc12 open reading frame is replaced with a 1.6-kb ura4 insert. (B) cdc12 open reading frame. Regions of predicted coiled-coil are underlined (Lupas et al., 1991), and the polyproline regions are boxed. (C) Similarity in gene domain structure. Proteins share a central FH1 polyproline rich domain (black box), an FH2 domain (stripe box), and a larger COOH-terminal area of homology (gray) also diagrammed in D. (D) Similarity of the proteins in a COOH-terminal region. Proteins were aligned using Megalign in DNA Star. Residues identical to a calculated consensus are colored in black, and conserved residues are colored in gray. These sequence data are available from GenBank/EMBL/DDBJ under accession number Q10059.
Mentions: The cdc12 gene was cloned by complementation of the temperature-sensitive lethal phenotype of cdc12-112. A pUR19 (ura4+)-based S. pombe genomic library (Barbet et al., 1992) was transformed into ura4D18 cdc12112 (FC112), and 5 × 104 ura+ transformant colonies were screened by replica printing onto YE5S with phyloxin for growth at 36°C. Four colonies showed plasmid-dependent rescue. Isolation of plasmid DNA from two of these transformants identified two related plasmids, pFC103 and pFC104. pFC103 complemented a cdc12-112 strain upon retransformation and contained a 6-kb insert in pUR19. Deletion analysis showed that a central region was critical for activity (see Fig. 1 A). Three genetic criteria demonstrated that complementing activity was the cdc12 gene: first, hybridization to an ordered cosmid blot (Hoheisel et al., 1993) showed that pFC103 mapped to left tip of chromosome I, at the corresponding location to which cdc12 has been previously mapped genetically. Second, pFC110, which contains a 3-kb EcoRI cdc12 insert from pFC103 and a sup3-5 marker (from pSTA12), was integrated into the genome by homologous recombination. Random spore analysis showed that the plasmid integrated at the cdc12 locus. Third, introduction of the COOH-terminal half of cdc12 gene into cdc12-112 does not confer rescue at 36°C in the majority of the cells, but confers rare stable gene conversion of the cdc12-112 allele, suggesting that the genetic defect of cdc12-112 resides in the COOHterminal half of the protein.

Bottom Line: Here we show that cdc12p is a member of a family of proteins including Drosophila diaphanous, Saccharomyces cerevisiae BNI1, and S. pombe fus1, which are involved in cytokinesis or other actin-mediated processes.Based on these patterns, we present a model in which ring assembly originates from a single point on the cortex and in which a molecular pathway for the functions of cytokinesis proteins is suggested.Finally, we found that cdc12 and cdc3 mutants show a synthetic-lethal genetic interaction, and a proline-rich domain of cdc12p binds directly to profilin cdc3p in vitro, suggesting that one function of cdc12p in ring assembly is to bind profilin.

View Article: PubMed Central - PubMed

Affiliation: Imperial Cancer Research Fund, London, United Kingdom. fc99@columbia.edu

ABSTRACT
As in many other eukaryotic cells, cell division in fission yeast depends on the assembly of an actin ring that circumscribes the middle of the cell. Schizosaccharomyces pombe cdc12 is an essential gene necessary for actin ring assembly and septum formation. Here we show that cdc12p is a member of a family of proteins including Drosophila diaphanous, Saccharomyces cerevisiae BNI1, and S. pombe fus1, which are involved in cytokinesis or other actin-mediated processes. Using indirect immunofluorescence, we show that cdc12p is located in the cell division ring and not in other actin structures. When overexpressed, cdc12p is located at a medial spot in interphase that anticipates the future ring site. cdc12p localization is altered in actin ring mutants. cdc8 (tropomyosin homologue), cdc3 (profilin homologue), and cdc15 mutants exhibit no specific cdc12p staining during mitosis. cdc4 mutant cells exhibit a medial cortical cdc12p spot in place of a ring. mid1 mutant cells generally exhibit a cdc12p spot with a single cdc12p strand extending in a random direction. Based on these patterns, we present a model in which ring assembly originates from a single point on the cortex and in which a molecular pathway for the functions of cytokinesis proteins is suggested. Finally, we found that cdc12 and cdc3 mutants show a synthetic-lethal genetic interaction, and a proline-rich domain of cdc12p binds directly to profilin cdc3p in vitro, suggesting that one function of cdc12p in ring assembly is to bind profilin.

Show MeSH
Related in: MedlinePlus