This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The nuclear pore complex (NPC) regulates the vital flow of proteins and RNA between the cytoplasm and the nucleoplasm of eukaryotic cells. It is a supramolecular sieve that spans both membranes of the nuclear envelope to create a forty-nanometer aqueous channel that connects the cytoplasm and the nucleoplasm of cells. Small metabolites diffuse freely through the NPC, but the flow of large proteins and RNA is selective and requires specific transport signals in the cargo. The signals are recognized by mobile receptors termed karyopherins (also called importins, exportins and transportins), which interact with proteins of the NPC (nucleoporins; Nups) to translocate across and shuttle cargo between the cytoplasm and the nucleoplasm. We are obtaining a map of protein interactions between the 54 components of the nucleocytoplasmic transport machinery in S. cerevisiae using a GST based affinity-capture assay coupled to mass spectrometric identification of captured proteins. The approach has allowed the identification of dozens of proteins in yeast extracts that interact specifically with isolated components of the transport machinery (Nups, Kaps, Ran and its effectors). We are currently examining how the network of interactions is modulated under varying cellular conditions, such as different cell densities or temperature, and as cells progress through different stages of the cell cycle. The approach is providing a global picture of the molecular dynamics underlying nucleocytoplasmic transport, as well as a detailed map of the many interactions that each protein component of the machinery makes.
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