Molecular Mechanisms of Nuclear Pore Complex Assembly

Texas A&M Research Foundation, College Station TX

Investigators

Abstract

Intellectual Merit Nuclear pore complexes (NPCs) are specialized structures that are located in the nuclear envelope of eukaryotic cells to allow for the bi-directional transport of metabolites, proteins, and RNAs between the nucleus and cytoplasm. As the only way into or out of the nucleus, NPCs are essential in linking gene expression to cellular physiology. The complexity of the NPC predicts that its assembly involves a number of discrete molecular events, and intermediates have been isolated. However, the molecular mechanisms underlying the assembly of the nuclear pore complex have not been elucidated. Using a combination of cell biological, biochemical, and genetic strategies in the brewers' yeast, Saccharomyces cerevisiae, this project focuses on the function of specific cellular proteins and a novel, vesicular intermediate during an early stage of the assembly pathway. Dr. Ryan hypothesizes that Ran, a small GTPase, and Kap95, a karyopherin/importin, two proteins whose functions in regulating traffic through nuclear pores have already been established, also play a role in regulating the fusion of these vesicles to the outer nuclear envelope where they nucleate new NPC formation. Aim I is to determine the functional significance of vesicles during interphase NPC assembly. This will be accomplished by biochemical purification of the vesicles that accumulate in Ran cycle mutants and determining their protein composition. The contribution of these factors to NPC assembly will be addressed using a number of microscopy, genetic, and biochemical based approaches. In Aim II, Dr. Ryan will take advantage of her large collection of NPC assembly mutants to identify additional components and stages in the assembly pathway. Taken together, these studies will provide crucial advances in understanding the mechanism of NPC assembly at the molecular level. Correct NPC assembly is critical in establishing and maintaining the unique cytoplasmic and nuclear environments. Changes in NPC composition are seen during development and cellular differentiation suggesting that NPCs are not simply static passageways between the two compartments but instead provide an additional level of regulation. Recent studies indicate that the NPC also has an active role in regulating gene expression. In addition to allowing transcriptional regulators in and mRNAs out of the nucleus, NPCs have been found to regulate transcription directly, by acting as transcriptional activators or influencing chromatin structure. Thus, determining how NPCs are assembled is pivotal in understanding the many ways NPCs link gene expression to cellular physiology in all eukaryotic cells. NPC biogenesis requires a number of basic cellular processes including protein trafficking, vesicle targeting, and membrane fusion. Thus, these studies on how these events are coordinated to form functional NPCs will address an array of fundamental biological questions and impact diverse fields. Broader Impacts The undergraduate and graduate students supported by this grant will receive training in cell biology, biochemistry, molecular biology, and genetics. All students will present their research in lab/group meetings, and symposia at the department and University levels. In addition, senior students will have the opportunity to attend national meetings to present their work to a larger audience and to make contacts to advance the next stage of their scientific career.