Influence of glycosylation on nuclear pore function

描述(申请人提供)：真核细胞的核与细胞质之间由双膜核膜隔开。核孔复合体(NPC)穿透核膜，并提供水通道，分子可通过该通道在核间和细胞质间移动。每个NPC由大约30种不同的称为核孔蛋白(Nups)的蛋白质组成，每个NPC有8-32个拷贝。这些核蛋白中有三个是跨越核膜的跨膜蛋白，将NPC与核膜连接起来。所有这三种跨膜核蛋白都可以通过糖基化来修饰。我们已经产生了糖基化改变NPC活性的证据，很可能是通过改变这些核苷酸中至少一个的功能，Pom152。在这项建议中，发芽酵母酿酒酵母被用来研究糖基化在影响NPC功能中的作用。将使用大规模、自动化的遗传筛查来识别编码影响NPC活性和糖基化的蛋白质的基因。酵母突变体库将首先筛选与核孔蛋白Nup1和Pom152突变的合成致死性或抑制者相互作用。与这些核蛋白相互作用的突变体随后将被测试表明在糖基化中发挥作用的表型，包括潮霉素敏感性和钒酸盐抗性。每个突变体随后将被检测鼻咽癌功能的变化，包括核运输动力学的变化，鼻咽癌分布的异常，以及鼻咽癌结构的改变。最后，由这些突变体的子集编码的蛋白质将被测试与Pom152糖基化区域的物理相互作用。重要的是，核运输的原创性实验将被纳入生物实验室入门课程、细胞生物学高级本科课程和本科生密集研究课程，从而使大量本科生接触到分子细胞生物学基础研究的设计、执行、分析和出版过程。

DESCRIPTION (provided by applicant): The nucleus of a eukaryotic cell is separated from the cytoplasm by the double-membrane nuclear envelope. Nuclear pore complexes (NPCs) perforate the nuclear envelope and provide aqueous channels through which molecules can move between the nuclear and cytoplasmic compartments. Each NPC is comprised of approximately 30 different proteins termed nucleoporins (Nups), each present in 8 - 32 copies per NPC. Three of these Nups are transmembrane proteins that span the nuclear membrane, linking the NPC to the nuclear envelope. All three of these transmembrane Nups may be modified by glycosylation. We have generated evidence that glycosylation alters NPC activity, most likely by altering the function of at least one of these Nups, Pom152. In this proposal, the budding yeast Saccharomyces cervisiae is used to examine the role of glycosylation in influencing NPC function. Large-scale, automated genetic screens will be employed to identify genes encoding proteins that influence both NPC activity and glycosylation. A library of yeast mutants will first be screened for synthetic lethal or suppressor interactions with mutations in the nucleoporins Nup1 and Pom152. Mutants that exhibit interactions with these Nups will then be tested for phenotypes suggesting a role in glycosylation, including hygromycin sensitivity and vanadate resistance. Each mutant will subsequently be assayed for changes in NPC function, including alterations in nuclear transport kinetics, aberrant NPC distribution, and altered NPC structure. Finally, the proteins encoded by a subset of these mutants will be tested for physical interactions with the glycosylated region of Pom152. Importantly, original experiments examining nuclear transport will be incorporated into an introductory biology lab course, an upper-level undergraduate cell biology course, and 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.