Molecular genetic analysis of factors which interact with an actin-binding protein cofilin of S.cerevisiae.

Cofilin是一种低分子量、pH调节的肌动蛋白结合和肌动蛋白解聚蛋白。切丝蛋白广泛分布于从芽殖酵母酿酒酵母到高等植物和哺乳动物的真核生物中。在芽殖酵母中，cfilin基因COF 1是细胞生长所必需的（Iida，K.et al.，Gene，124，115 - 120，1993; Moon，A.L.et等人，J.Cell Biol.，120，421 - 435，1993）。cofilin的氨基酸序列在各种物种中是很保守的，并且猪cofilin cDNA补充了芽殖酵母中COF 1破坏所引起的致死性。为了阐明cofilin在芽殖酵母中的功能，我们采用羟胺体外随机诱变的方法分离了COF 1的温度敏感突变体。获得的所有四种ts突变体在十二肽序列中或非常接近十二肽序列处具有氨基酸变化，所述十二肽序列已通过体外研究显示为肌动蛋白结合区域（Yonezawa，N.et al.，J. Biol. Chem.，266，10485 - 10489，1991）。核磁共振法分析聚氨酯的三级结构 ...更多信息 cofilin的一种同种型，inedestrin证实了这一观点（Hatanaka，H.et，Cell，85，1047 - 1055，1996）。具有ts等位基因取代野生型COF 1的菌株表现出温度敏感性生长。当温度升高到非允许温度时，细胞停止生长，处于小出芽阶段。免疫荧光染色显示dofilin定位于野生型细胞芽的肌动蛋白斑。当ts突变株被转移到非允许的温度，肌动蛋白补丁消失，并形成含有肌动蛋白的厚聚集体中的细胞溶解。这些结果表明，cofilin可能通过调节肌动蛋白丝的组织参与芽的扩大。接下来，我们分离了COF 1的ts等位基因的多拷贝抑制基因SCF 1。SCF 1编码615个氨基酸残基的蛋白质。尽管SCF 1是非必需基因，但SCF 1的破坏与COF 1的ts等位基因一起是合成致死的。这一结果表明，Scflp发挥重要作用时，cofilin的功能是不完善的。SCF 1的破坏影响cofilin的定位;用SCF 1破坏的细胞的抗cofilin抗体的免疫荧光染色显示除了动作补丁之外的细胞质细纤维。Scflp可能通过对cofilin和/或actin的作用参与肌动蛋白丝的组织。少

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 … More 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