Influence of glycosylation on nuclear pore function

描述（由申请人提供）：真核细胞的细胞核与细胞质由双膜核膜分隔。核孔复合物（NPC）包裹着核膜，并提供水通道，分子可以通过该水通道在核和细胞质区室之间移动。每个NPC由大约30种不同的蛋白质组成，称为核孔蛋白（Nups），每个NPC以8 - 32个拷贝存在。其中三种Nup是跨核膜的跨膜蛋白，将NPC连接到核膜。 所有这三种跨膜Nup都可以通过糖基化修饰。我们已经产生了糖基化改变NPC活性的证据，最有可能是通过改变这些Nups中至少一个的功能，Pom 152。在这个提议中，芽殖酵母酿酒酵母被用来检查糖基化在影响NPC功能中的作用。将采用大规模的自动化遗传筛选来鉴定编码影响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Nup 1和Pom 152中突变的合成致死或抑制相互作用。然后将测试表现出与这些Nup相互作用的突变体的表型，表明其在糖基化中的作用，包括潮霉素敏感性和钒酸盐抗性。随后将测定每种突变体的NPC功能变化，包括核转运动力学的改变、异常NPC分布和改变的NPC结构。最后，将测试由这些突变体的子集编码的蛋白质与Pom 152的糖基化区域的物理相互作用。重要的是，检查核运输的原始实验将被纳入生物学实验室入门课程，高级本科细胞生物学课程和密集的本科研究计划，从而使大量本科生接触到设计，执行，分析和出版分子细胞生物学基础研究的过程。

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.