Examination of Nup1 Mediated Nucleocytoplasmic Transport

描述（由申请人提供） 大分子在细胞核和细胞质之间的运输 真核细胞通过称为核的大的多蛋白质结构发生 孔复合物（NPC）。分子被运送到 在NPC中，与蛋白质家族成员相关的蛋白质称为 “输入蛋白”或“核转运蛋白”（Kaps）。不同的卡普负责 不同底物分子的运输。很明显卡普一家 在运输过程中与NPC中的蛋白质相关联， 哪些Kaps结合哪些NPC蛋白。此外，这种绑定在 Kaps及其底物在NPC中的物理易位， 不清楚在这个提议中，使用了芽殖酵母酿酒酵母（Saccharomyces cerevisiae 研究核孔之间的物理和功能相互作用， 复合蛋白Nupip和所有14种酵母核转运蛋白。以便识别 Kaps的运输动力学因Nupip的损失而改变， 在NUP1突变细胞中表达的Kaps的亚细胞定位中， 使用Kap：：GFP融合物通过荧光显微镜检查。物理 Nupip和每个Kap之间的相互作用也将使用 重组Nupip蛋白和亲和柱层析，以及 荧光显微镜。最后，一种基因筛选方法， 将完成抑制Nupi介导的转运缺陷的突变体， 所产生的突变体检查与蛋白质的相互作用， 核粒蛋白家族。重要的是，研究努皮卡普的原始实验 互动将被纳入一个高层次的本科细胞 生物学实验室课程，以及密集的本科研究 计划，从而暴露了大量的本科生的过程中， 设计、执行、分析和发表分子生物学基础研究的 细胞生物学

DESCRIPTION (provided by applicant) 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.