Transport across the nuclear envelope separating the nucleus and cytoplasm of eukaryotic cells occurs through nuclear pore complexes (NPCs). The mechanisms by which regulated protein transport occurs across the NPCs are poorly understood. In particular, while much research has been done on nuclear protein import, relatively little work has examined the export of proteins from the nucleus to the cytoplasm. We have developed a kinetic assay to investigate the relative rate of transport out of the nucleus in wild type yeast and in cells lacking specific NPC components (Nups) and have used this assay to identify a subset of Nups necessary for efficient nuclear protein export. We will investigate which Nups and Nup domains are required for efficient translocation of substrate across the NPC and determine if different subsets of Nups are required for translocation of protein cargos in different directions. Four specific aims will be used to investigate the role of specific Nups in nucleocytoplasmic protein transport. In Specific Aim 1, we will identify the Nups necessary for efficient protein export from the nucleus. Specific Aim 2 will investigate the function of Nup subdomains in translocation by generating mutations in specific Nup regions. We will also swap domains between Nups to determine if the context of specific Nup domains within the NPC is important for transport activity. In Specific Aim 3, we will examine whether distinct Nups and Nup domains are essential for protein export and import mediated by different transport factors. Using regulated nuclear export and small molecule-mediated protein dimerization, we will examine the kinetics of nuclear import and export of distinct substrates by different transport proteins in cells lacking specific Nups and Nup domains. Finally, Specific Aim 4 will incorporate undergraduate students into the experiments described in Aims 1 - 3, through both the development of a new investigative lab component for an intermediate-level lab course and the support of independent research projects during the summer and academic year. This research utilizes a novel nuclear protein export assay to provide the first comprehensive kinetic analysis of Nup function in nuclear protein export. The resulting data will provide insights into the poorly understood mechanism for protein translocation across the NPC. Importantly, we will identify whether distinct Nups are necessary for export by different transport factors and if different subsets of Nups mediate nuclear protein import and export. PUBLIC HEALTH RELEVANCE: In order to function properly, cells must carefully regulate the movement of proteins into and out of the nucleus. Failure to do so results in changes in cell function, including alterations in gene expression, uncontrolled cell growth, and premature cell death. The research described in this proposal will provide insights into how cells regulate the movement of molecules between the cytoplasm and nucleus and how that regulation impacts cellular activity.