Cordes V C, Hase M E, Müller L
Medical Nobel Institute, Karolinska Institutet, Stockholm, S-17177, Sweden.
Exp Cell Res. 1998 Nov 25;245(1):43-56. doi: 10.1006/excr.1998.4246.
Tpr is a 267-kDa protein of unknown function recently identified as a constitutive component of the nuclear pore complex (NPC2)-attached intranuclear filaments. Secondary structure predictions suggest that the protein is divided into a large, coiled-coil forming aminoterminal domain and a shorter, highly acidic carboxyterminal domain. To identify which of Tpr's molecular segments determine its specific intranuclear localization, we have constructed expression vectors encoding various Tpr deletion mutants as well as chimeric combinations of Tpr sequences with the soluble cytoplasmic protein pyruvate kinase. Following transfection of cultured mammalian cells, we have identified a short region within Tpr's carboxyterminal domain that is essential and sufficient to mediate nuclear import of Tpr and which can also confer nuclear accumulation of pyruvate kinase. Tpr deletion mutants that contain this nuclear targeting segment, but lack the aminoterminal domain, appear evenly dispersed throughout the nucleus without any noticeable association to the NPC. In contrast, the aminoterminal domain lacking the carboxyterminal region remains located within the cytoplasm, forming aggregate-like structures not associated with the nuclear envelope. However, when tagged to Tpr's short nuclear targeting segment or to the nuclear localization signal of the SV40 large T protein, the aminoterminal domain is imported into the nucleus, where it then associates with the NPC. This association is mediated by shorter molecular segments within the aminoterminal domain which contain clusters of heptad repeats, whereas other regions are dispensable. This assignment of different topogenetic properties to distinct molecular segments of Tpr will now allow the design of future experiments to study the protein's structural properties further and determine its actual function.
Tpr是一种分子量为267 kDa、功能未知的蛋白质，最近被鉴定为附着于核孔复合体（NPC2）的核内细丝的组成成分。二级结构预测表明，该蛋白质分为一个形成卷曲螺旋的大的氨基末端结构域和一个较短的、高度酸性的羧基末端结构域。为了确定Tpr的哪些分子片段决定其特定的核内定位，我们构建了编码各种Tpr缺失突变体以及Tpr序列与可溶性细胞质蛋白丙酮酸激酶的嵌合组合的表达载体。在转染培养的哺乳动物细胞后，我们在Tpr的羧基末端结构域内鉴定出一个短区域，该区域对于介导Tpr的核输入是必不可少且足够的，并且还可以赋予丙酮酸激酶核内积累。含有该核靶向片段但缺乏氨基末端结构域的Tpr缺失突变体似乎均匀地分散在整个细胞核中，与NPC没有任何明显的关联。相反，缺乏羧基末端区域的氨基末端结构域仍位于细胞质中，形成与核膜无关的聚集体样结构。然而，当与Tpr的短核靶向片段或SV40大T蛋白的核定位信号标记时，氨基末端结构域被导入细胞核，然后在那里与NPC结合。这种结合是由氨基末端结构域内较短的分子片段介导的，这些片段包含七肽重复序列簇，而其他区域是可有可无的。将不同的拓扑发生特性赋予Tpr的不同分子片段，现在将允许设计未来的实验，以进一步研究该蛋白质的结构特性并确定其实际功能。
