Molecular mechanisms of protein translocation through nuclear pores

大多数亲核蛋白通过选择性调节机制通过核孔迅速积聚到细胞核中。亲核蛋白的选择性核输入是由被称为核定位信号(NLSS)的短氨基酸序列控制的。亲核蛋白的转运过程被认为至少包括两个步骤：依赖于NLS结合到核孔的细胞质表面，然后通过核孔复合体转运。洋地黄素通透性的无细胞转运系统有助于识别与核进口相关的可溶性因子。利用该体外系统，我们获得了以下数据：1.我们发现，在活跃的核运输过程中，亲核蛋白与细胞质因子形成稳定的复合体，靶向核孔。我们将其称为核孔靶向复合体(PTAC)。2.我们鉴定了PTAC的两个基本成分，并分离了它们的cDNA克隆。根据它们的计算分子质量，我们将它们命名为PTAC58和PTAC97.3。我们发现PTAC58具有特异的NLS结合活性。部分胞质注射的抗PTAC58抗体迅速迁移到细胞核，表明这种蛋白在核膜上的动态移动。4.研究表明，PTAC97和PTAC58的摩尔比为1：1。5.PTAC58和PTAC97的复合体定位于核孔，这取决于亲核物质的存在。这些结果表明，核导入的第一步是通过PTAC58与PTAC97结合的亲核分子形成靶向复合体来实现的。

Most karyophilic proteins accumulate rapidly into the nucleus through nuclear pores by selective mediated mechanisms. The selective nuclear import of karyophilic proteins is directed by short amino acid sequences termed nuclear localization signals (NLSs). The process of the mediated import of karyophilic proteins has been considered to involve at least two steps ; NLS-dependent binding to the cytoplasmic face of nuclear pores, followed by translocation through the nuclear pore complex.The digitonin-permeabilized cell-free transport system facilitates the identification of soluble factors involved in nuclear import. Using this in vitro system, we obtained the following data.1.We found that a karyophilic protein forms a stable complex with cytoplasmic factors to target nuclear pores in active nuclear transport process. We termed this as nuclear pore-targeting complex (PTAC).2.We identified two essential components of PTAC and isolated their cDNA clones. Based on their calculated molecular masses, we named them as PTAC58 and PTAC97.3.PTAC58 was found to have specific NLS-binding activity. A portion of cytoplasmically injected antibodies against PTAC58 migrated rapidly into the nucleus, indicating dynamic movement of this protein across the nuclear envelope.4.It was shown that PTAC97 is associated with PTAC58 in a 1 : 1 molar ratio.5.A complex of PTAC58 and PTAC97 targets nuclear pores, depending on the presence of a karyophile.These results indicate that the first step in nuclear import occurs through the targeting-complex formation of a karyophile with PTAC58 bound to PTAC97.