The nucleoporin Nup98 associates with the intranuclear filamentous proteinnetwork 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 promo ter region) is a protein that forms filamentous structures extending from n uclear 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 show ed colocalization. In confocal sections the nucleolus itself was only weakl y stained but there was intensive perinucleolar staining, Striking spike-li ke structures emanated from this perinucleolar ring and attenuated into thi nner structures as they extended to the nuclear periphery. This characteris tic staining pattern of the TPR network was considerably enhanced when a my c-tagged pyruvate kinase-6kDa fusion protein was overexpressed in HeLa cell s. Double-immunoelectron microscopy of these cells using anti-myc and anti- TPR antibodies and secondary gold-coupled antibodies yielded row-like arran gements of gold particles. Taken together, the immunolocalization data supp ort previous electron microscopical data, suggesting that TPR forms filamen ts that extend from the NPC to the nucleolus. We discuss the possible impli cations of the association of Nup98 with this intranuclear TPR network for an intranuclear phase of transport.