IDENTIFICATION, CHARACTERIZATION, AND INTRACELLULAR-DISTRIBUTION OF COFILIN IN DICTYOSTELIUM-DISCOIDEUM
IDENTIFICATION, CHARACTERIZATION, AND INTRACELLULAR-DISTRIBUTION OF COFILIN IN DICTYOSTELIUM-DISCOIDEUM
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DOI：
10.1074/jbc.270.18.10923
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发表时间：
1995-05-05
影响因子：
4.8
通讯作者：
YAHARA, I
中科院分区：
文献类型：
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作者：
AIZAWA, H;SUTOH, K;YAHARA, I
我们从盘基网眼菌中分离纯化了一个表观分子量为15,000的肌动蛋白单体结合蛋白。15 kDa的蛋白质以pH依赖的方式解聚肌动蛋白细丝。该蛋白还具有降低肌动蛋白溶液表观粘度的活性，且呈剂量依赖关系。这种活性被磷脂酰肌醇抑制。编码该蛋白的基因的分子克隆显示，该蛋白与酵母cofilin的初级序列有42%的同源性。我们得出结论，该15 kDa蛋白是该生物的衣丝蛋白。盘状芽孢杆菌含有两个cofilin基因(DCOF1和DCOF2)，它们的外显子核苷酸序列完全相同，但启动子和内含子区域不同。启动子实验表明，DCOF1在营养细胞和分化细胞中都有表达，而DCOF2在任何条件下都不表达。基因破坏实验表明，DCOF1可能是盘状芽孢杆菌细胞增殖所必需的，而DCOF2的破坏被证明不会改变任何表型。间接免疫荧光显微镜观察显示，Cofilin弥漫分布于营养细胞的细胞质中。在饥饿应激下的扁平细胞中，Cofilin定位于前沿褶皱膜中的肌动蛋白细胞骨架的显著重组，而不是定位于局灶性粘连斑块中的刚性肌动蛋白网络。这些结果提示，cofilin可能参与了膜肌动蛋白细胞骨架的动态重组。
We identified and purified an actin monomer-binding protein of apparent molecular weight of 15,000 from Dictyostelium discoideum. The 15-kDa protein depolymerized actin filaments in a pH-dependent manner. The protein also had an activity to decrease apparent viscosity of actin solutions in a dose dependent manner. This activity was inhibited by phosphatidyl inositides. Molecular cloning of genes encoding this protein revealed that the protein is 42% identical in its primary sequence to yeast cofilin. We concluded that the 15-kDa protein is cofilin of this organism. D. discoideum cells contain two cofilin genes (DCOF1 and DCOF2) whose nucleotide sequences were entirely identical in their exsons while the promoter and intron regions were different. Promoter assay experiments revealed that DCOF1 is expressed both in vegetative and differentiating cells and that DCOF2 is not expressed under any conditions examined. Gene disruption experiments suggested that DCOF1 might be essential for the proliferation of D. discoideum cells whereas the disruption of DCOF2 was proven not to alter any phenotypes. Indirect immunofluorescence microscopic observations showed that cofilin is distributed diffusely throughout cytoplasm in vegetative cells. In flattened cells under starvation stress, cofilin localized at dramatically reorganizing actin cytoskeletons in ruffling membranes of the leading edge, but not at rigid actin meshwork in focal adhesion plaques. These results suggest that cofilin may be involved in dynamic reorganization of membranous actin cytoskeletons.