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

点击翻译按钮获取中文摘要

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.