Wiche G, Gromov D, Donovan A, Castañón M J, Fuchs E
Institute of Biochemistry and Molecular Cell Biology, University of Vienna, Austria.
J Cell Biol. 1993 May;121(3):607-19. doi: 10.1083/jcb.121.3.607.
Plectin is an intermediate filament (IF) binding protein of exceptionally large size. Its molecular structure, revealed by EM and predicted by its sequence, indicates an NH2-terminal globular domain, a long rodlike central domain, and a globular COOH-terminal domain containing six highly homologous repeat regions. To examine the role of the various domains in mediating plectin's interaction with IFs, we have constructed rat cDNAs encoding truncated plectin mutants under the control of the SV-40 promoter. Mutant proteins expressed in mammalian COS and PtK2 cells could be distinguished from endogenous wild type plectin by virtue of a short carboxy-terminal antigenic peptide (P tag). As shown by conventional and confocal immunofluorescence microscopy, the transient expression of plectin mutants containing all six or the last four of the repeat regions of the COOH-terminus, or the COOH-terminus and the rod, associated with IF networks of both the vimentin and the cytokeratin type and eventually caused their collapse into perinuclear aggregates. Similar effects were observed upon expression of a protein encoded by a full length cDNA construct. Microtubules and microfilaments were unaffected. Unexpectedly, mutants containing the rod without any of the COOH-terminal repeats, accumulated almost exclusively within the nuclei of cells. When the rod was extended by the first one and a half of the COOH-terminal repeats, mutant proteins showed a partial cytoplasmic distribution, although association with intermediate filaments was not observed. Nuclear and diffuse cytoplasmic distribution was also observed upon expression of the NH2-terminal domain without rod. These results indicate that sequences located roughly within the last two thirds of the globular COOH-terminus are indispensable for association of plectin with intermediate filaments in living cells.
网蛋白是一种超大尺寸的中间丝（IF）结合蛋白。通过电子显微镜揭示并由其序列预测的分子结构表明，它有一个NH2末端球状结构域、一个长杆状中央结构域以及一个包含六个高度同源重复区域的球状COOH末端结构域。为了研究各个结构域在介导网蛋白与中间丝相互作用中的作用，我们构建了在SV - 40启动子控制下编码截短网蛋白突变体的大鼠cDNA。在哺乳动物COS和PtK2细胞中表达的突变蛋白可凭借短的羧基末端抗原肽（P标签）与内源性野生型网蛋白区分开来。如传统免疫荧光显微镜和共聚焦免疫荧光显微镜所示，含有COOH末端所有六个或最后四个重复区域，或COOH末端和杆状结构的网蛋白突变体的瞬时表达，与波形蛋白型和细胞角蛋白型的中间丝网络相关，并最终导致它们塌陷成核周聚集体。在全长cDNA构建体编码的蛋白表达时也观察到了类似的效果。微管和微丝未受影响。出乎意料的是，含有杆状结构但没有任何COOH末端重复序列的突变体几乎完全聚集在细胞核内。当杆状结构由COOH末端的前一个半重复序列延伸时，突变蛋白呈现部分细胞质分布，尽管未观察到与中间丝的关联。在表达没有杆状结构的NH2末端结构域时也观察到了核内和弥漫性细胞质分布。这些结果表明，大致位于球状COOH末端最后三分之二区域内的序列对于网蛋白在活细胞中与中间丝的关联是不可或缺的。
