Identification of protein p270/Tpr as a constitutive component of the nuclear pore complex-attached intranuclear filaments.
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.
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Division of Cell Biology, German Cancer Research Center, Heidelberg, Federal Republic of Germany.
Reidenbach
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Rackwitz
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Franke
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D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D018938:Caco-2 Cells; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004285:Dogs; D000939:Epitopes / Q000276:immunology; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009685:Nuclear Envelope / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013552:Swine; D014407:Tumor Cells, Cultured; D014982:Xenopus laevis / Q000378:metabolism
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D000939:Epitopes; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; C073597:TPR protein, human