| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1996: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1995: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
Cofilin is a low-molecular weight, pH-regulated actin-binding and actin-depolymerizing protein. Cofilin is widely distributed among eukaryotes from the budding yeast S.cerevisiae to higher plants and mammals. In the budding yeast, the cfilin gene, COF1, is essential for cell growth (Iida, K.et al., Gene, 124,115-120,1993 ; Moon, A.L.et al., J.Cell Biol., 120,421-435,1993). The amino acid sequence of cofilin is well conserved among various species, and the porcine cofilin cDNA complements the lethality caused by the disruption of COF1 in the budding yeast. To clarify tye function of cofilin in the budding yeast, we isolated temperature-sensitive mutants of COF1 by in vitro random mutagenesis with hydroxylamine. All the four ts mutants obtained had amino acid changes in or very close to the dodecapeptide sequence, which had been shown to be an actin-binding region by in vitro studies (Yonezawa, N.et al., J.Biol. Chem., 266,10485-10489,1991). NMR analysis of the tertialy structure of porc
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ine destrin, an isoform of cofilin, confirmed this notion (Hatanaka, H.et al, Cell, 85,1047-1055,1996). The strains which have the ts alleles in place of the wild type COF1 showed temprtature sensitive growth. When shifted to the non-permissive temperature, the cells stopped, growth at a small-budded stage. Immunofluorescent satining showed that dofilin is localized to the actinpatches of the buds in the wild type cells. When the ts mutant strain was shifted to the nonpermissive temperature, the actin patches were dissapeared and actin-containing thick aggregates were formed in the cytopolasm. These results suggest that cofilin might be involved in the enlargement of the buds by regulating the organization of actin filaments. We next isolated a multicopy suppressor, SCF1, of the ts allele of COF1. SCF1 encodes a proetin of 615 amino acid residues. Disruption of SCF1 is synthetic lethal with the ts allele of COF1, although SCF1 is non-essential gene. This result suggests that Scflp plays important roles when the function of cofilin is imperfect. Disruption of SCF1 affects the localization of cofilin ; immunofluorescence staining with anti-cofilin antibody of SCF1-disrupted cells revealed cytoplasmic thin fibers in addition to the action patches. Scflp might be involved in the organization of actin filaments through its functions on cofilin and/or actin. Less
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