Molecular genetic analysis of the in vivo function of a low molecular weight 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。其在肌动蛋白丝上的活性受到多磷酸肌苷的抑制。Cofilin广泛分布于真核生物中，从芽殖酵母到高等真核生物，如哺乳动物、鸟类和植物。我们从酿酒酵母基因组文库中克隆了一个编码酵母菌cofilin的基因COF1。COF1是酵母细胞生长的重要基因。cofilin的氨基酸序列在不同物种间具有较好的保守性。从大肠杆菌中纯化的重组酵母cofilin在肌动蛋白上的体外活性与高等真核生物cofilin的活性相似。猪cofilin cDNA补充了出芽酵母中被破坏的cofilin基因。这些结果表明，cofilin的功能在进化过程中具有较好的保守性。用羟胺处理携带cof1的质粒，获得了酵母cofilin的温敏突变体。所有获得的四种突变体的氨基酸都发生了变化，或非常接近与肌动蛋白和多磷酸肌苷相互作用重要的十二肽序列。用cofilin的ts突变基因代替野生型COF1的菌株表现出温敏生长。当转移到非允许温度时，细胞在小芽期停止生长，然后失去活力。用抗cofilin抗体特异性染色芽的肌动蛋白斑块。当ts突变菌株转移到非允许温度时，肌动蛋白斑块消失，细胞质中形成含肌动蛋白的厚聚集体。Cofilin可能通过调节肌动蛋白丝的组织参与芽的扩大。我们从酵母基因组文库中克隆了cofilin突变体的多拷贝抑制子（SCF1）。SCF1是一个诺贝尔基因，它编码615个氨基酸残基的多肽。

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.