Cordes V C, Rackwitz H R, Reidenbach S
Division of Cell Biology, German Cancer Research Center, Heidelberg, Germany.
Exp Cell Res. 1997 Dec 15;237(2):419-33. doi: 10.1006/excr.1997.3806.
Nuclear pore complexes are constitutive structures of the nuclear envelope in eukaryotic cells and represent the sites where transport of molecules between nucleus and cytoplasm takes place. However, pore complexes of similar structure, but with largely unknown functional properties, are long known to occur also in certain cytoplasmic cisternae that have been termed annulate lamellae (AL). To analyze the capability of the AL pore complex to interact with the soluble mediators of nuclear protein import and their karyophilic protein substrates, we have performed a microinjection study in stage VI oocytes of Xenopus laevis. In these cells AL are especially abundant and can easily be identified by light and electron microscopy. Following injection into the cytoplasm, fluorochrome-labeled mediators of two different nuclear import pathways, importin beta and transportin, not only associate with the nuclear envelope but also with AL. Likewise, nuclear localization signals (NLS) of the basic and M9 type, but not nuclear export signals, confer targeting and transient binding of fluorochrome-labeled proteins to cytoplasmic AL. Mutation or deletion of the NLS signals prevents these interactions. Furthermore, binding to AL is abolished by dominant negative inhibitors of nuclear protein import. Microinjections of gold-coupled NLS-bearing proteins reveal specific gold decoration at distinct sites within the AL pore complex. These include such at the peripheral pore complex-attached fibrils and at the central "transporter" and closely resemble those of "transport intermediates" found in electron microscopic studies of the nuclear pore complex (NPC). These data demonstrate that AL can represent distinct sites within the cytoplasm of transient accumulation of nuclear proteins and that the AL pore complex shares functional binding properties with the NPC.
核孔复合体是真核细胞中核膜的组成结构，代表着细胞核与细胞质之间分子运输发生的位点。然而，结构相似但功能特性大多未知的孔复合体，长期以来已知也存在于某些被称为环孔片层（AL）的细胞质池中。为了分析AL孔复合体与核蛋白输入的可溶性介质及其亲核蛋白底物相互作用的能力，我们在非洲爪蟾的VI期卵母细胞中进行了显微注射研究。在这些细胞中，AL特别丰富，并且可以通过光学显微镜和电子显微镜轻松识别。将两种不同核输入途径的荧光染料标记介质——输入蛋白β和运输蛋白注射到细胞质后，它们不仅与核膜结合，还与AL结合。同样，碱性和M9型的核定位信号（NLS），而非核输出信号，使荧光染料标记的蛋白质靶向并短暂结合到细胞质AL上。NLS信号的突变或缺失会阻止这些相互作用。此外，核蛋白输入的显性负性抑制剂可消除与AL的结合。对金偶联的携带NLS的蛋白质进行显微注射，揭示了AL孔复合体内不同位点的特异性金标记。这些位点包括外周孔复合体附着的纤维以及中央“转运体”处的位点，与在核孔复合体（NPC）的电子显微镜研究中发现的“运输中间体”的位点非常相似。这些数据表明，AL可以代表细胞质中核蛋白短暂积累的不同位点，并且AL孔复合体与NPC具有共同的功能结合特性。
