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.
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