| The nuclear pore complex (NPC) mediates bidirectional macromolecular transport between the nucleus and cytoplasm of the eukaryotic cell. Transport substrates are recognized in the compartment of origin by soluble receptors that move through the NPC and release their cargo in the compartment of destination. Different receptors have been identified that bind to different types of importand export cargoes, explaining the existence of independent transport pathways. However, genetic analysis in yeast and biochemical data in vertebrates indicate that an additional level of specificity may be imposed by the nuclear pore complex. To study this, I propose to use in vitro nuclear reconstitution and assay the effect of depletion of specific nuclear pore components (nucleoporins) on different nuclear cytoplasmic transport pathways and NPC ultrastructure. Nucleoporins will be targeted that show specificity in nuclear transport receptors in vitro and those that are known to interact with trnsport receptors per se. I propose to test if transport and ultrastructural defects can be reversed by adding back recombinant nucleoporins. This opens the way to precisely delineate the functional interaction domains for different transport receptors and create part of a first functional map of the vertebrate NPC. |
