The nucleoporin Nup98 associates with the intranuclear filamentous protein network of TPR.
The Nup98 gene codes for several alternatively spliced protein precursors. Two in vitro translated and autoproteolytically cleaved precursors yielded heterodimers of Nup98-6kDa peptide and Nup98-Nup96. TPR (translocated promoter region) is a protein that forms filamentous structures extending from nuclear pore complexes (NPCs) to intranuclear sites. We found that in vitro translated TPR bound to in vitro translated Nup98 and, via Nup98, to Nup96. Double-immunofluorescence microscopy with antibodies to TPR and Nup98 showed colocalization. In confocal sections the nucleolus itself was only weakly stained but there was intensive perinucleolar staining. Striking spike-like structures emanated from this perinucleolar ring and attenuated into thinner structures as they extended to the nuclear periphery. This characteristic staining pattern of the TPR network was considerably enhanced when a myc-tagged pyruvate kinase-6kDa fusion protein was overexpressed in HeLa cells. Double-immunoelectron microscopy of these cells using anti-myc and anti-TPR antibodies and secondary gold-coupled antibodies yielded row-like arrangements of gold particles. Taken together, the immunolocalization data support previous electron microscopical data, suggesting that TPR forms filaments that extend from the NPC to the nucleolus. We discuss the possible implications of the association of Nup98 with this intranuclear TPR network for an intranuclear phase of transport.
Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021, USA.
B M
BM
Fontoura
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S
Dales
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G
Blobel
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Zhong
D002467:Cell Nucleus / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D010455:Peptides; D011498:Protein Precursors; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C073597:TPR protein, human; C119239:nuclear pore complex protein 96; C093350:nuclear pore complex protein 98; D010450:Endopeptidases