The long term objective of this work is to understand the mechanisms by which large proteins are transported into the nucleus across the nuclear pore complex. Since import of most nuclear proteins occurs by selective mediated processes, the pore complex is likely to have an important role in controlling cell physiology. In this proposal, the specific aims are focused on detailed characterization of three proteins associated with the pore complex that have putative roles in protein import: the transport receptor for the nuclear location signal of the SV40 T antigen, the pore complex component that interacts with this transport receptor, and a major glycoprotein of the pore complex that contains O-linked N- acetylglucosamine. Monoclonal and domain-specific polyclonal antibodies will be prepared to the three proteins, and cDNA clones for the first two proteins will be isolated and sequenced. With these reagents, biochemical properties and localization of these proteins will be analyzed. Subsequently, the functions of these components will be investigated in cultures cells by microinjecting antibodies and overexpressing domains and deletion mutants of the proteins, to describe effects on mediated proteins import and on diffusional permeability of the pore complex. To complement this in vivo work, biochemical properties of nuclear proteins import assays. Using this system, antibodies to the three proteins considered above together with biochemical inhibitors will be used to begin dissecting the pathway for protein transport across the pore complex and to define intermediates in this process. Collectively, these studies will provide a paradigm for approaching study of RNA export from the nucleus, which probably shares many features with protein import.
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