Abstract of grant R35GM141461: Twenty homologous Karyopherin- (Kap) proteins mediate the majority of protein transport between the nucleus and the cytoplasm. The Chook Lab is a leader in understanding the mechanisms by which Kaps control macromolecular traffic. We achieve this understanding through a combination of structural, biochemical, cell biological and bioinformatic analyses of Kap-ligand interactions. Our studies of the nuclear import receptor Kapβ2 led to discovery of the proline-tyrosine nuclear localization signal (PY-NLS), which it recognizes. This study led to a new concept—that many nuclear targeting signals are defined not merely by sequence but by a collection of physical features—which explained how Kapβ2 can recognize hundreds of different sequences, and enabled us to design the first nuclear import inhibitor. Although two additional NLS types have recently been determined, cargo recognition by most importins, including Importins-4, -7, -8, -9 and -11 is not understood even though they account for 50% of nuclear import. Few of their cargos are known and the NLS classes that they recognize are unknown. The missing knowledge obscures our understanding of how a significant portion of the proteome lacking recognizable NLSs are trafficked into the nucleus. We aim to discover new cargos, classes of NLSs and pathway-specific inhibitors for all importins. We will combine this knowledge with bioinformatic analyses and experiments to define the full cargo repertoires of each importin, and thus map both the traffic and cellular processes that they each control. We showed that in addition to mediating nuclear import, importins also act as chaperones. We will learn how Kap2 chaperones RNA-binding proteins, preventing their aggregation and understand the functional significance of the Importin-9 histone-chaperone function in histone storage and nucleosome assembly. In our studies of nuclear export, we were the first to reveal how CRM1 recognizes the only known nuclear export signal (NES). This work was foundational for development of the anti-cancer drug Selinexor, and the physical/chemical mechanisms of inhibition that we later revealed contributed to FDA-approval of the drug. We also revealed the sequence and structural degeneracy of the NES, explaining how CRM1 is able to recognize hundreds of diverse NES sequences. CRM1 also exports thousands of mRNAs, but little is known about the mechanism. We will study CRM1-mRNA export complexes to shed light on this critical step of gene expression. Finally, we will expand study to the exportin Msn5, which binds intrinsically disordered and phosphorylated segments of multiple cargos. This work will reveal a second, new class of NES.