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Molecular genetic analysis of the in vivo function of a low molecular weight actin-binding protein, cofilin, of S.cerevisiae.

酿酒酵母低分子量肌动蛋白结合蛋白 cofilin 体内功能的分子遗传学分析。

基本信息

批准号：
04833029
负责人：
IIDA Kazuko
金额：
$ 1.34万
依托单位：
The Tokyo Metropolitan Institute of Medical Science.
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

项目摘要

Cofilin is a low-MW pH-regulated actin-binding and actin-depolymerizing protein. Its activities on actin-filaments are inhibited by polyphosphoinositides. Cofilin is widely distributed among eukaryotes from the budding yeast to higher eukaryotes such as mammals, birds and plants.We have cloned a gene encoding yeast cofilin, COF1, from a genomic library of S.cerevisiae. COF1 is an essential gene for yeast cell growth. The amino acid sequence of cofilin is well conserved among various species. The recombinant yeast cofilin purified from E.coli exhibited in vitro activities on actin filaments similar to the activities of cofilins from higher eukaryotes. The porcine cofilin cDNA complements the disrupted cofilin gene in the budding yeast. These results indicate that the function of cofilin is well conserved among evolution.Temperature sensitive (ts) mutants of yeast cofilin were obtained by treating a COF1-carring plasmid with hydroxylamine. All the four mutants obtained had amino acid changes in or very close to the dodecapeptide sequence important for interaction with actin and polyphosphoinositides. The strain which has the ts mutant gene of cofilin in place of the wild type COF1 showed temperature sensitive growth. When shifted to nonpermissive temperatute, the cells stopped growth at a small-budded stage and then lost viabilty.The actin patches of the buds were stained specifically with anti-cofilin antibody. When the ts mutant strain was shifted to nonpermissive temperature, the actin patches were disspeared and actin-containing thick aggregates were formed in the cytoplasm. Cofilin might be involved in enlargement of the buds by regulating the organization of actin filaments.We cloned a multicopy suppressor (SCF1) of the cofilin ts mutant from a yeast genomic library. SCF1 is a nobel gene which encodes a polypeptide of 615 amino acid residues.

Cofilin是一种低分子量的pH调节的肌动蛋白结合和肌动蛋白解聚蛋白。它对肌动蛋白丝的活性被多磷酸肌醇抑制。Cofilin广泛分布于从芽殖酵母到哺乳动物、鸟类和植物等高等真核生物中，我们从酵母菌基因组文库中克隆了编码酵母Cofilin的基因COF1。COF1是酵母细胞生长所必需的基因。cofilin的氨基酸序列在各种物种中是非常保守的。从大肠杆菌中纯化的重组酵母cofilin在体外对肌动蛋白丝具有类似于高等真核生物cofilin的活性。猪cofilin cDNA补充了芽殖酵母中被破坏的cofilin基因。用羟胺处理携带COF1的酵母cofilin质粒，获得了温度敏感突变体（ts）。获得的所有四个突变体的氨基酸变化或非常接近的十二肽序列与肌动蛋白和聚磷酸肌醇的相互作用是重要的。具有cofilin的ts突变体基因代替野生型COF1的菌株表现出温度敏感性生长。当温度升至非允许温度时，细胞在小芽阶段停止生长，然后失去活力。芽的肌动蛋白斑块被抗cofilin抗体特异性染色。当ts突变株被转移到非允许的温度下，肌动蛋白补丁消失，并在细胞质中形成含有肌动蛋白的厚聚集体。我们从酵母基因组文库中克隆了一个cofilin ts突变体的多拷贝抑制子（SCF 1）。SCF 1是一个编码615个氨基酸残基的多肽的基因。