Identification of Protein p270/Tpr as a Constitutive Component of the Nuclear Pore Complex-attached Intranuclear Filaments

doi:10.1083/jcb.136.3.515

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© The Rockefeller University Press, 0021-9525/1997//515 $5.00
The Journal of Cell Biology, Volume 136, Number 3, , 1997 515-529

Article

Identification of Protein p270/Tpr as a Constitutive Component of the Nuclear Pore Complex–attached Intranuclear Filaments

Volker C. Cordes, Sonja Reidenbach, Hans-Richard Rackwitz, and Werner W. Franke

Division of Cell Biology, German Cancer Research Center, D-69120 Heidelberg, Federal Republic of Germany

Using a monoclonal antibody, mAb 203-37, we have identifieda polypeptide of M_r $~$ 270 kD (p270) as a general constituentof the intranuclear filaments attached to the nucleoplasmicannulus of the nuclear pore complex (NPC) in diverse kindsof vertebrate cells. Using cDNA cloning and immunobiochemistry, we show that human protein p270 has a predicted molecular massof 267 kD and is essentially identical to the coiled-coil dominatedprotein 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 kindsof mammalian and amphibian cells with a series of antibodiesraised against different epitopes of human and Xenopus laevisp270/Tpr. In these experiments, the protein has been consistentlyand exclusively detected in the NPC-attached intranuclear filaments, and p270/Tpr-containing filament bundles have been tracedinto the nuclear interior for up to 350 nm. No reaction hasbeen noted at the cytoplasmic side of NPCs with any of thep270/Tpr antibodies, whereas control antibodies such as thoseagainst protein RanBP2/ Nup358 specifically decorate the cytoplasmicannulus of NPCs. Pore complexes of cytoplasmic annulate lamellaein various mammalian and amphibian cells are also devoid ofimmunodetectable protein p270/Tpr. We conclude that this coiled-coilprotein is a general and ubiquitous component of the intranuclearNPC- attached filaments and discuss its possible functions.

Abbreviations used in this paper: aa, amino acid; CMV, cytomegalovirus; ECL, enhanced chemiluminescence; KLH, keyhole limpet hemocyanin; NPC, nuclear pore complex; TBST, TBS containing 0.05% Tween-20.

The human fetal brain p270/Tpr sequence and the partial Xenopuskidney sequence have been submitted to the GenBank and are availableunder accession numbers U69668 and U69669, respectively.

Address all correspondence to Volker C. Cordes and Werner W.Franke, Division of Cell Biology, German Cancer Research Center,Im Neuenheimer Feld 280, D-69120 Heidelberg, FRG. Tel.: (49)6221-423400. Fax: (49) 6221-423404.

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