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MW pH调节的肌动蛋白结合和肌动蛋白 - 脱聚合蛋白。其对肌动蛋白丝的活性受到多磷酸肌醇的抑制。 Cofilin广泛分布在从萌芽的酵母到较高的真核生物（例如哺乳动物，鸟类和植物）中的真核生物中，我们从S. cerevisiae的基因组学库中克隆了一个编码酵母菌Cofilin的基因。 COF1是酵母细胞生长的必要基因。 Cofilin的氨基酸序列在各种物种中都充分保守。从大肠杆菌中纯化的重组酵母菌cofilin在类似于高等真核生物的cofilins的肌动蛋白丝上表现出体外活性。猪Cofilin cDNA补充了萌芽酵母中破坏的cofilin基因。这些结果表明，在进化中，Cofilin的功能是充分保守的。通过用羟基胺处理COF1的质粒，获得了酵母菌的温度敏感（TS）突变体。所有获得的四个突变体的变化或非常接近与肌动蛋白和聚磷酸肌醇相互作用的十二肽序列的变化。具有Cofilin的TS突变基因代替野生型COF1的菌株显示温度敏感的生长。当转移到非验证温度时，细胞在小批准的阶段停止生长，然后失去了纤维，然后用抗cofilin抗体特别染色了芽的肌动蛋白斑块。当TS突变菌株转移到非耐药温度时，将肌动蛋白斑块散发，并在细胞质中形成含有肌动蛋白的厚聚集体。 Cofilin可能通过调节肌动蛋白丝的组织来涉及芽的扩大。我们从酵母基因组文库中克隆了Cofilin TS突变体的多拷贝抑制器（SCF1）。 SCF1是一个编码615个氨基酸残基的多肽的诺贝尔基因。