Biochemical Dissections of Importin Binding Modalities: Import Regulation Through Cargo Selection and Competition

Active nuclear transport depends on karyopherins, a class of chaperone proteins that recognize cargo in the cytoplasm, escort it through the hydrophobic nuclear pore, and release it in the nucleus. How karyopherins achieve specificity across hundreds of structurally diverse cargos is incompletely understood. This thesis dissects the binding specificity determinants of Importin 9 (IPO9), an essential karyopherin whose cargo set and binding mechanisms had not been systematically characterized, and in doing so challenges two established paradigms of nuclear import. In the first part of this thesis, I develop a direct pulldown approach using ectopically expressed FLAG-tagged IPO9 from cytoplasmic extracts, followed by mass spectrometry, that recapitulates over half of all previously identified IPO9 cargos, outperforming existing high-throughput methods. Using this platform, I show that IPO9 cargo specificity is not mediated by classical nuclear localization signals but by extended disordered loop features on the importin itself. Perturbation of the H7, H8, and H18-19 loops reveals that each governs a distinct subset of cargo interactions, and that disrupting them does not simply reduce cargo binding but reveals a secondary class of cargos normally excluded under physiological conditions. I further find that IPO9 cargos are variably sensitive to RanGTP addition, with a substantial fraction requiring additional nuclear factors for release. This is, to my knowledge, the first high-throughput demonstration that RanGTP-mediated release is not universal for an importin's cargo set. In the second part of this thesis, I perform a detailed biophysical dissection of the interaction between IPO9 and the cytoskeletal protein actin. Nuclear actin is required for transcription, chromatin remodeling, 3D genome organization, and the DNA damage response, making its regulated import consequential for broad nuclear function. The prevailing model held that IPO9 binding to actin depended on the actin depolymerization factor cofilin as an obligate adaptor. Using surface plasmon resonance, I demonstrate that IPO9 binds directly to monomeric actin with mid-nanomolar affinity in the complete absence of cofilin, and that cofilin binding is in fact inhibitory to this interaction. Profilin and DNase I are similarly inhibitory in competition experiments, indicating that IPO9 contacts multiple surfaces of the actin monomer simultaneously. These findings overturn the prior model and reframe actin import as a competitive process governed by the full complement of cytoplasmic actin binding proteins, linking nuclear actin homeostasis directly to cytoplasmic actin dynamics and cell state. In the final chapter, I situate these findings within the broader karyopherin literature and extend the structural analysis across all twenty mammalian karyopherins. Annotation of HEAT repeat lengths, inter-HEAT loop positions, 3D geometry, and AlphaMissense pathogenicity scores reveals that karyopherins are exceptionally sensitive to missense mutations, with hotspots mapping to residues critical for HEAT repeat folding and cargo contact. Together, these findings reframe cargo selectivity, competitive regulation, and nuclear release as deeply interconnected properties of the karyopherin:cargo system and provide a mechanistic foundation for a markedly more complex model of nuclear transport in cells.