Molecular segments of protein Tpr that confer nuclear targeting and association with the nuclear pore complex
Molecular segments of protein Tpr that confer nuclear targeting and association with the nuclear pore complex
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DOI：
10.1006/excr.1998.4246
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发表时间：
1998-11-25
影响因子：
3.7
通讯作者：
Müller, L
中科院分区：
文献类型：
--
作者：
Cordes, VC;Hase, ME;Müller, L
TPR是一种功能未知的267 kDa蛋白质，最近被鉴定为核孔复合体(NPC2)连接的核内细丝的组成成分。二级结构预测表明，该蛋白分为一个大的螺旋卷曲形成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在核内的特异性定位，我们构建了编码各种Tpr缺失突变体的表达载体，以及Tpr序列与可溶性细胞质蛋白丙酮酸激酶的嵌合组合。在对培养的哺乳动物细胞进行转染后，我们在Tpr的羧基末端结构域中发现了一个短小的区域，该区域对介导Tpr的核输入是必要的，也是充分的，它还可以提供丙酮酸激酶的核积聚。含有这个核靶向片段但缺乏氨基末端结构域的TPR缺失突变体，均匀分布在整个细胞核中，与NPC没有任何明显的联系。相反，缺少羧基末端区域的氨基末端结构域仍然位于细胞质内，形成与核膜无关的集合体结构。然而，当标记到Tpr的短核靶向片段或SV40大T蛋白的核定位信号时，氨基末端结构域被输入到核中，在那里它与NPC结合。这种联系是由氨基末端结构域中较短的分子片段介导的，这些分子片段包含七肽重复序列簇，而其他区域是必不可少的。将不同的拓扑发生特性分配给Tpr的不同分子片段，现在将允许设计未来的实验，以进一步研究蛋白质的结构特性，并确定其实际功能。(C)1998年学术出版社。
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. (C) 1998 Academic Press.