The nuclear transport machinery integrates cytoplasmic and nuclear functions through multiple import and export. These pathways rely on targeting signals within cargo, recognition by soluble receptors, and physical translocation of cargo/receptor complexes through the nuclear pore. Our long-term goal is to define the mechanisms responsible for nuclear protein export in mammalian cells. Our approach is to use functional assays to purify and characterize the soluble export receptors and their regulators. We developed an assay that reconstitute nuclear export mediated by the leucine-rich nuclear export signal (NES) in protein kinase inhibitor (PKI). We have used the PKI assay to purify a new cytosolic export factor, and we have shown that this biochemical, cellular, and molecular approaches. We will determine the molecular identity of the new export using biochemical, cellular, and molecular approaches. We will determine the molecular identity of the new export factor and whether it requires co-factors for its nuclear export function. We will examine its subcellular distribution and determine if it shuttles between the nucleus and cytoplasm. A structure-function analyses of the export factor will reveal whether it physically contacts the NES and whether the Ran GTPase regulates this interaction. We will determine how the export factor interacts with the nuclear pore complex and describe its mechanisms for recycling. The in vivo function of the export factor will be addressed by examining its ability to mediate export of two medically-relevant substrates: the glucocorticoid receptor, and the HIV-1 protein rev complexes with RNA. These macromolecules are likely substrates of the new export factor because it mediates the nuclear export in vitro. Our hypothesis is that mammalian cells use multiple export pathways that feature distinct NES receptors. Elucidating the biochemistry and molecular details of these pathways is of general interest because the same machinery is used to sort proteins and RNAS of both cellular and viral origin.
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