Brush border myosin-I truncated in the motor domain impairs the distribution and the function of endocytic compartments in an hepatoma cell line
Brush border myosin-I truncated in the motor domain impairs the distribution and the function of endocytic compartments in an hepatoma cell line
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DOI：
10.1073/pnas.93.14.7053
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发表时间：
1996-07-09
影响因子：
11.1
通讯作者：
Coudrier, E
中科院分区：
文献类型：
--
作者：
Durrbach, A;Collins, K;Coudrier, E
肌球蛋白I是一种普遍存在的单体类肌球蛋白，具有基于肌动蛋白的运动特性，与血浆和/或囊泡膜相关，并被认为是细胞表面和细胞内膜结构之间运输事件的参与者。为了研究肌球蛋白I的功能，黑麦用编码鸡刷状边界肌球蛋白I （BBMI）的cdna转染了小鼠肝癌细胞系（BWTG3），并在运动域截断了两个变体。一种变体删除了前446个氨基酸，因此缺乏ATP结合位点，而另一种变体删除了整个马达结构域，缺乏ATP和肌动蛋白结合位点。我们观察到(i)在细胞分离后，大量截断的变体被膜组分恢复，（ii）它们与含有α 2-巨球蛋白的室共分布，通过荧光显微镜确定，内化30分钟，（iii） bbmi截断的变体的产生损害了酸性室和配体的分布内化30分钟。（iv）含有肌动蛋白结合位点的截断变体的产生降低了α 2-巨球蛋白的降解率，而缺乏ATP结合位点和肌动蛋白结合位点的变体的产生增加了α 2-巨球蛋白的降解率。这些观察结果表明，两个截断的变异对内吞区室的分布和功能有主要的负面影响。我们提出一种未知的肌球蛋白- 1可能有助于BWTG3细胞在核旁位置的内吞区室分布和/或调节配体向降解区室的递送。
Myosins I, a ubiquitous monomeric class of myosins that exhibits actin-based motor properties, are associated with plasma and/or vesicular membranes and have been suggested as players for trafficking events between cell surface and intracellular membranous structures, To investigate the function of myosins I, rye have transfected a mouse hepatoma cell line (BWTG3) with cDNAs encoding the chicken brush border myosin-I (BBMI) and two variants truncated in the motor domain, One variant is deleted of the first 446 amino acids and thereby lacks the ATP binding site, whereas the other is deleted of the entire motor domain and lacks the ATP and actin binding sites. We have observed (i) that significant amounts of the truncated variants are recovered with membrane fractions after cell fractionation, (ii) that they codistribute with a compartment containing alpha 2-macroglobulin internalized for 30 min as determined by fluorescent microscopy, (iii) that the production of BBMI-truncated variants impairs the distribution of the acidic compartment and ligands internalized for 30 min, and (iv) that the production of the truncated variant containing the actin binding site decreases the rate of alpha 2-macroglobulin degradation whereas the production of the variant lacking the ATP binding site and the actin binding site increases the rate of alpha 2-macroglobulin degradation. These observations indicate that the two truncated variants have a dominant negative effect on the distribution and the function of the endocytic compartments. We propose that an unidentified myosin-I might contribute to the distribution of endocytic compartments in a juxtanuclear position and/or to the regulation of the delivery of ligands to the degradative compartment in BWTG3 cells.