The transport of large molecules between the nucleus and cytoplasm of eukaryotic cells occurs through large, multi-protein structures termed nuclear pore complexes (NPCs). Molecules being transported are targeted to and carried across the NPC while associated with members of a protein family termed the """"""""importins"""""""" or """"""""karyopherins"""""""" (Kaps). Different Kaps are responsible for the transport of distinct substrate molecules. While it is clear that the Kaps associate with proteins in the NPC during transport, it remains unclear exactly which Kaps bind which NPC proteins. In addition, the role this binding has in the physical translocation of the Kaps and their substrates across the NPC in unclear. In this proposal, the budding yeast Saccharomyces cerevisiae is used to examine the physical and functional interactions between the nuclear pore complex protein Nupip and all fourteen yeast karyopherins. In order to identify which Kaps have their kinetics of transport altered by a loss of Nupip, changes in the subcellular localization of Kaps expressed in NUP1 mutant cells will be examined by fluorescence microscopy using Kap::GFP fusions. Physical interactions between Nupip and each of the Kaps will also be explored using recombinant Nupip proteins and affinity column chromatography, as well as fluorescence light microscopy. Finally, a genetic screen used to identify mutants that suppress Nupi-mediated transport defects will be completed, and the resulting mutants examined for interactions with proteins in the karyopherin family. Importantly, original experiments examining Nupi-Kap interactions will be incorporated into an upper-level undergraduate Cell Biology laboratory course, as well as an intensive undergraduate research program, thus exposing large numbers of undergraduate students to the process of designing, performing, analyzing, and publishing basic research in molecular cell biology.
