TPR, A LARGE COILED-COIL PROTEIN WHOSE AMINO-TERMINUS IS INVOLVED IN ACTIVATION OF ONCOGENIC KINASES, IS LOCALIZED TO THE CYTOPLASMIC SURFACE OF THE NUCLEAR-PORE COMPLEX

From a panel of monoclonal antibodies raised against fractions of rat liver nuclear envelopes (NEs), we have identified an antibody, RL30, w hich reacts with novel nuclear pore complex (NPC) antigens that are no t O-glycosylated. By immunofluorescence staining of cultured cells, RL 30 reacts exclusively with the NE in a punctate pattern that largely c oincides with that of identified NPC proteins. RL30 labels only the cy toplasmic surface of the NPC in immunogold electron microscopy, predom inantly in peripheral regions nearby the cytoplasmic ring. In immunobl ots of isolated rat liver NEs and cultured rat cells, RL30 recognizes a 265-kD band, as well as a series of 175-265-kD bands in rat liver NE s that are likely to be proteolytic products of p265. Sequencing of pe ptides from the 175- and 265-kD RL30 antigens of rat liver revealed th at they are both closely related to human Tpr, a protein whose amino-t erminal 150-250 amino acids appear in oncogenic fusions with the kinas e domains of the met, trk, and raf protooncogenes. We found that in vi tro translation of human Tpr mRNA yields a major 265-kD band. Consider ed together, these data indicate that the 265-kD RL30 antigen in the N PC is the rat homologue of Tpr. Interestingly, Tpr contains an excepti onally long predicted coiled coil domain (similar to 1600 amino acids) . The localization and predicted structure of Tpr suggest that it is a component of the cytoplasmic fibrils of the NPC implicated in nuclear protein import. Immunofluorescence microscopy shows that during NPC r eassembly at the end of mitosis, Tpr becomes concentrated at the NE si gnificantly later than O-linked glycoproteins, including p62. This ind icates that reassembly of the NPC after mitosis is a stepwise process, and that the Tpr-containing peripheral structures are assembled later than p62.