Coulombe P A, Fuchs E
Howard Hughes Medical Institute, Department of Molecular Genetics, University of Chicago, IL 60637.
J Cell Biol. 1990 Jul;111(1):153-69. doi: 10.1083/jcb.111.1.153.
Because of extraordinarily tight coiled-coil associations of type I and type II keratins, the composition and structure of keratin subunits has been difficult to determine. We report here the use of novel genetic and biochemical methods to explore the early stages of keratin filament assembly. Using bacterially expressed humans K5 and K14, we show that remarkably, these keratins behave as 1:1 complexes even in 9 M urea and in the presence of a reducing agent. Gel filtration chromatography and chemical cross-linking were used to identify heterodimers and heterotetramers as the most stable building blocks of keratin filament assembly. EM suggested that the dimer consists of a coiled-coil of K5 and K14 aligned in register and in parallel fashion, and the tetramer consists of two dimers in antiparallel fashion, without polarity. In 4 M urea, both end-to-end and lateral packing of tetramers occurred, leading to a variety of larger heteromeric complexes. The coexistence of multiple, higher-ordered associations under strongly denaturing conditions suggests that there may not be a serial sequence of events leading to the assembly of keratin intermediate filaments, but rather a number of associations may take place in parallel.
由于I型和II型角蛋白的卷曲螺旋结合异常紧密，角蛋白亚基的组成和结构一直难以确定。我们在此报告使用新的遗传和生化方法来探索角蛋白丝组装的早期阶段。利用细菌表达的人K5和K14，我们发现，值得注意的是，即使在9M尿素和存在还原剂的情况下，这些角蛋白也表现为1:1复合物。凝胶过滤色谱法和化学交联法被用于鉴定异二聚体和异四聚体是角蛋白丝组装中最稳定的构建块。电子显微镜显示，二聚体由K5和K14的卷曲螺旋以对齐和平行方式排列组成，四聚体由两个以反平行方式排列的二聚体组成，没有极性。在4M尿素中，四聚体发生了端对端和侧向堆积，导致形成各种更大的异聚复合物。在强变性条件下多种高阶结合的共存表明，可能不存在导致角蛋白中间丝组装的一系列连续事件，而是许多结合可能并行发生。
