Cordes V C, Reidenbach S, Rackwitz H R, Franke W W
Division of Cell Biology, German Cancer Research Center, Heidelberg, Federal Republic of Germany.
J Cell Biol. 1997 Feb 10;136(3):515-29. doi: 10.1083/jcb.136.3.515.
Using a monoclonal antibody, mAb 203-37, we have identified a polypeptide of M(r) approximately 270 kD (p270) as a general constituent of the intranuclear filaments attached to the nucleoplasmic annulus of the nuclear pore complex (NPC) in diverse kinds of vertebrate cells. Using cDNA cloning and immunobiochemistry, we show that human protein p270 has a predicted molecular mass of 267 kD and is essentially identical to the coiled-coil dominated protein Tpr reported by others to be located on the outer, i.e., cytoplasmic surface of NPCs (Byrd, D.A., D.J. Sweet, N. Pante, K.N. Konstantinov, T. Guan, A.C.S. Saphire, P.J. Mitchell, C.S. Cooper, U. Aebi, and L. Gerace. 1994. J. Cell Biol. 127: 1515-1526). To clarify this controversial localization, we have performed immunoelectron microscopy in diverse kinds of mammalian and amphibian cells with a series of antibodies raised against different epitopes of human and Xenopus laevis p270/Tpr. In these experiments, the protein has been consistently and exclusively detected in the NPC-attached intranuclear filaments, and p270/Tpr-containing filament bundles have been traced into the nuclear interior for up to 350 nm. No reaction has been noted at the cytoplasmic side of NPCs with any of the p270/Tpr antibodies, whereas control antibodies such as those against protein RanBP2/Nup358 specifically decorate the cytoplasmic annulus of NPCs. Pore complexes of cytoplasmic annulate lamellae in various mammalian and amphibian cells are also devoid of immunodetectable protein p270/Tpr. We conclude that this coiled-coil protein is a general and ubiquitous component of the intranuclear NPC-attached filaments and discuss its possible functions.
利用单克隆抗体mAb 203 - 37，我们鉴定出一种分子量约为270 kD的多肽（p270），它是多种脊椎动物细胞核孔复合体（NPC）核质环上附着的核内细丝的普遍组成成分。通过cDNA克隆和免疫生物化学方法，我们发现人类蛋白p270的预测分子量为267 kD，并且与其他人报道的位于NPC外表面（即细胞质表面）的以卷曲螺旋为主的蛋白Tpr基本相同（Byrd, D.A., D.J. Sweet, N. Pante, K.N. Konstantinov, T. Guan, A.C.S. Saphire, P.J. Mitchell, C.S. Cooper, U. Aebi, and L. Gerace. 1994. J. Cell Biol. 127: 1515 - 1526）。为了阐明这一有争议的定位，我们用一系列针对人类和非洲爪蟾p270/Tpr不同表位产生的抗体，在多种哺乳动物和两栖动物细胞中进行了免疫电子显微镜实验。在这些实验中，该蛋白始终且仅在附着于NPC的核内细丝中被检测到，并且含有p270/Tpr的细丝束已被追踪到核内达350 nm。用任何一种p270/Tpr抗体在NPC的细胞质侧均未观察到反应，而对照抗体如针对RanBP2/Nup358蛋白的抗体则特异性地标记NPC的细胞质环。各种哺乳动物和两栖动物细胞中细胞质环状片层的孔复合体也没有可免疫检测到的蛋白p270/Tpr。我们得出结论，这种卷曲螺旋蛋白是附着于核内NPC的细丝的普遍且广泛存在的成分，并讨论了其可能的功能。
