IDENTIFICATION OF A NONAPEPTIDE MOTIF IN THE VIMENTIN HEAD DOMAIN INVOLVED IN INTERMEDIATE FILAMENT ASSEMBLY
IDENTIFICATION OF A NONAPEPTIDE MOTIF IN THE VIMENTIN HEAD DOMAIN INVOLVED IN INTERMEDIATE FILAMENT ASSEMBLY
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DOI：
10.1016/0022-2836(92)90980-x
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发表时间：
1992-02-05
影响因子：
5.6
通讯作者：
FRANKE, WW
中科院分区：
文献类型：
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作者：
HERRMANN, H;HOFMANN, I;FRANKE, WW
可溶性波形蛋白亚基组装成中间丝 (IF) 取决于位于氨基末端结构域的信息。利用非洲爪蟾波形蛋白 cDNA 的定点诱变和大肠杆菌生产系统获得纯突变蛋白，我们在头域中鉴定了一个从两栖动物到人类进化上保守的九个氨基酸基序 (SSYRRIFGG)，它在 IF 的有序形成中发挥着重要作用。中心二精氨酸和两个芳香残基中的交换干扰波形蛋白的体外 IF 组装：在标准组装条件 (160 mm-NaCl) 下组装时，大部分蛋白质包含在致密聚集体中，其中 IF 比例可变且较小，而在较低离子浓度下，形成短且不完整的 IF 样结构。整个基序的删除导致蛋白质在标准组装条件（例如 160 mm-NaCl）下主要快速沉淀成非 IF 材料的大聚集体，而在较低离子强度（例如 50 mm-NaCl）下，IF 和致密聚集体同时形成。我们的结果表明，突变的蛋白质可以在相同的时间和相同的条件下呈现不同的形式。该基序本身不足以形成正常的 IF，正如突变体所证明的，其中通过删除 55 个内部氨基酸残基，该基序更接近 α-螺旋杆结构域。在转染缺乏波形蛋白 IF 的培养上皮细胞后，通过免疫荧光显微镜进行了相应的观察。讨论了头域基序对于 IF 组装和高阶排列的重要性。
The assembly of soluble vimentin subunits into intermediate filaments (IFs) is dependent on information located in the amino-terminal domain. Using site-directed mutagenesis of aXenopus laevisvimentin cDNA and anEscherichia coliproduction system to obtain pure mutated protein, we have identified, in the head domain, a nine amino acid motif (SSYRRIFGG), evolutionarily conserved from amphibia to man, which plays an important role in the orderly formation of IFs. Exchanges in the central di-arginine and in the two aromatic residues interfere with IF assembly of vimentinin vitro: on assembly under standard assembly conditions (160 mm-NaCl) most of the protein is included in dense aggregates, with a variable and minor proportion of IFs, whereas at lower ion concentrations short and incomplete IF-like structures are formed. The deletion of the whole motif results in a protein that under standard assembly conditions (e.g. 160 mm-NaCl) predominantly and rapidly precipitates into large aggregates of non-IF material, whereas at lower ionic strength (e.g. 50 mm-NaCl) both IFs and dense aggregates are formed simultaneously. Our results show that the mutated protein can assume different forms at the same time and under the same conditions. This motif alone is insufficient for the formation of normal IFs as demonstrated by a mutant in which the motif has been brought closer to the α-helical rod domain by deletion of 55 internal amino acid residues. Corresponding observations have been made, by immunofluorescence microscopy, upon transfection of cultured epithelial cells lacking vimentin IFs. The importance of the head domain motif for the assembly and higher-order arrangement of IFs is discussed.