Project Summary Trafficking of macromolecules between the nucleus and the cytoplasm is an essential cellular function intimately linked to many degenerative processes that lead to disease and is emerging as a promising target for therapeutic intervention. Over the last 30 years, dramatic progress in cell, molecular and structural biology has been instrumental in elucidating the `design principles' of nuclear transport. Critical soluble factors trafficking cargos through the Nuclear Pore Complex (NPC) have been identified. The organization, architecture and assembly of the yeast NPC has been revealed. The physical principles governing passage of macromolecules through the NPC have been deciphered. In stark contrast, far less is known about the regulation of nuclear transport and the mechanisms of `fine-tuning' that result in spatiotemporally regulated availability of critical signaling molecules in the cell nucleus. In this proposal, by integrating biophysical, biochemical and functional techniques, we seek to understand how isoforms of the import adaptor importin α regulate nuclear import of key factors of the NF-κB and Ran signaling pathways. Specifically, we propose to: 1.) Determine the mechanisms of importin α isoform selectivity for NF-κB heterodimers under physiological conditions and in disease states; 2.) Decipher how importin α3 modulates the quaternary structure and transport properties of the RCC1 import complex. Overall, our work will shed light on a poorly understood chapter of nuclear transport, with the goal of exploiting nature's complex regulation to identify novel pharmacological targets.