Nuclear pore complexes (NPCs) form the site for entry and exit from the nucleus. The most outstanding issue in the nuclear transport field involves delineating how cargo crosses the aqueous NPC channel. Translocation thought to be is based on physical interactions between soluble shuttling transport factors and the NPC. How these interactions result in movement across the NPC is unknown. The long-range goal of this project is to understand the order of events at the NPC during nuclear transport.
Our specific aims will analyze the mechanism and regulation of transport factor-NPC interaction during protein and mRNA transport.
The first aim builds on our recent discovery that mRNA export requires a nuclear enzymatic pathway that converts soluble inositol 1,4,5-trisphosphate to inositol hexakisphosphate (IP6). We hypothesize that IP6 production influences events at the NPC. IP6 binding, two-hybrid and genetic strategies will be used to identify IP6 targets in S. cerevisiae and pinpoint the step in the mRNA export pathway that requires IP6 production. In the second aim, we will test specific hypotheses for the role of the essential mRNA export factor Gle1 in yeast and human cells. IP6 production is required for Gle1 function. We will investigate whether Gle1 shuttles between the nucleus and cytoplasm, and determine the network of protein-protein interactions that mediate Gle1 shuttling, NPC localization, and mRNA translocation. Finally, we will initiate new studies to test models for the NPC translocation mechanism. Yeast mutant strains will be identified that harbor a minimal repertoire of NPC transport factor binding sites, or that harbor a transport factor defective for NPC interaction. By determining the rates of nuclear import /export and the transport arrest points at the NPC, we aim to reveal how binding at the NPC results in translocation.Knowledge of how the nuclear accessibility of molecules can be selectively targeted or inhibited will be essential for designing therapeutic strategies, and understanding viral proliferation/pathogenesis. Transport factors and nucleoporins are both targets for viral inhibition of cellular function and mediators of viral RNA export. Defects in inositol signaling pathways are also associated with disease states including cancer cell growth, inflammation, and neurotransmission. Thus, these studies have direct health relatedness.
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