The mechanism by which proteins are transported to the nucleus in eucaryotic cells will be examined. Elucidation of the mechanism(s) governing nuclear transport and nucleocytoplasmic interactions has broad implications to understanding the regulation of gene expression, oncogenic transformation, and the control of early embryonic development. The simiam virus 40 (SV40) large tumor (T) antigen offers an exceptional model for the analysis of nuclear transport; the T antigen polypeptide is well characterized, the role of T antigen in replication and oncogenic transformation is well established, the nuclear transport signal of T antigen is known, and the T antigen polypeptide is localized at both the nucleus and the cell surface. A system for the analysis of nuclear transport has been developed which utilizes microinjection of mammalian cells with synthetic peptide nuclear transport signals crosslinked to carrier proteins. A peptide homologous to the T antigen nuclear transport signal induces the nuclear transport of carrier proteins, but no transport occur when proteins are coupled to a synthetic peptide homologous to a nuclear-transport defective T antigen. The first specific aim will be to examine modified T antigen nuclear transport signal peptides, as well as signal peptides from other proteins for induction of nuclear transport. The flexibility of the nuclear transport machinery will be evaluated by measuring the kinetics of transport for modified peptides, and competitive microinjection experiments will determine whether different cellular factors are involved in recognition of dissimilar transport signals from other proteins. Second, the cellular protein that binds to the T antigen transport signal will be isolated and characterized. The signal peptide and anti-idiotypic antibodies that mimic the conformation of the signal peptide will be used to isolate cellular transport factors. The third specific aim will be to isolate and sequence the gene encoding the cellular factor that binds the T antigen signal sequence. The cloned gene will be used to analyze the conservation of the gene during eucaryotic evolution and to construct expression vectors. Fourth, a detailed immunological characterization of the ligand-receptor (signal sequence-transport factor) interaction will be performed. Finally, an attempt will be made to reconstitute an in vitro nuclear transport system using isolated nuclear transport factors.
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