Coulombe P A, Chan Y M, Albers K, Fuchs E
Howard Hughes Medical Institute, Departments of Molecular Genetics, Chicago, Illinois.
J Cell Biol. 1990 Dec;111(6 Pt 2):3049-64. doi: 10.1083/jcb.111.6.3049.
To investigate the sequences important for assembly of keratins into 10-nm filaments, we used a combined approach of (a) transfection of mutant keratin cDNAs into epithelial cells in vivo, and (b) in vitro assembly of mutant and wild-type keratins. Keratin K14 mutants missing the nonhelical carboxy- and amino-terminal domains not only integrated without perturbation into endogenous keratin filament networks in vivo, but they also formed 10-nm filaments with K5 in vitro. Surprisingly, keratin mutants missing the highly conserved L L E G E sequence, common to all intermediate filament proteins and found at the carboxy end of the alpha-helical rod domain, also assembled into filaments with only a somewhat reduced efficiency. Even a carboxy K14 mutant missing approximately 10% of the rod assembled into filaments, although in this case filaments aggregated significantly. Despite the ability of these mutants to form filaments in vitro, they often perturbed keratin filament organization in vivo. In contrast, small truncations in the amino-terminal end of the rod domain more severely disrupted the filament assembly process in vitro as well as in vivo, and in particular restricted elongation. For both carboxy and amino rod deletions, the more extensive the deletion, the more severe the phenotype. Surprisingly, while elongation could be almost quantitatively blocked with large mutations, tetramer formation and higher ordered lateral interactions still occurred. Collectively, our in vitro data (a) provide a molecular basis for the dominance of our mutants in vivo, (b) offer new insights as to why different mutants may generate different phenotypes in vivo, and (c) delineate the limit sequences necessary for K14 to both incorporate properly into a preexisting keratin filament network in vivo and assemble efficiently into 10-nm keratin filaments in vitro.
为了研究对角蛋白组装成10纳米细丝很重要的序列，我们采用了一种联合方法：(a) 在体内将突变角蛋白cDNA转染到上皮细胞中，以及(b) 对突变型和野生型角蛋白进行体外组装。缺失非螺旋羧基末端和氨基末端结构域的角蛋白K14突变体不仅在体内能毫无干扰地整合到内源性角蛋白细丝网络中，而且在体外还能与K5形成10纳米细丝。令人惊讶的是，缺失所有中间丝蛋白共有的、位于α螺旋杆状结构域羧基末端的高度保守的LLEGE序列的角蛋白突变体，也能组装成细丝，只是效率略有降低。即使是缺失约10%杆状结构域的羧基端K14突变体也能组装成细丝，尽管在这种情况下细丝会明显聚集。尽管这些突变体在体外有形成细丝的能力，但它们在体内常常扰乱角蛋白细丝的组织。相比之下，杆状结构域氨基末端的小片段缺失在体外和体内都更严重地破坏了细丝组装过程，尤其是限制了伸长。对于羧基端和氨基端杆状结构域的缺失，缺失越广泛，表型越严重。令人惊讶的是，虽然大的突变几乎能定量地阻断伸长，但四聚体形成和更高阶的侧向相互作用仍然会发生。总的来说，我们的体外数据：(a) 为我们的突变体在体内的显性提供了分子基础；(b) 对于不同突变体在体内可能产生不同表型的原因提供了新的见解；(c) 描绘了K14在体内正确整合到预先存在的角蛋白细丝网络中以及在体外有效组装成10纳米角蛋白细丝所需的极限序列。
