Background: Nup214 interacts with the nuclear export receptor CRM1 and promotes export of certain cargos. Results: Several FG motives in the C-terminal region participate in CRM1 binding. Conclusion: CRM1, like other transport receptors, makes multiple contacts to nucleoporins. Significance: Elucidation of the details of nucleoporin-receptor interactions is essential for our understanding of the transition process of transport complexes through the nuclear pore.

Nuclear pore complexes (NPCs) are giant units of the nuclear envelope, mediating directional transport into and out of the nucleus. Nucleoporins are the main components of the NPC, and one third of them contain phenylalanine-glycine (FG) repeats. They are believed to serve as binding sites for transport receptors of the karyopherin family. CRM1 is the main export receptor in eukaryotic cells. It has a ring-like structure that is composed of HEAT (Huntingtin Elongation Factor A Subunit TOR) repeats (HRs) and exports various cargo proteins containing a leucine-rich nuclear export signal (NES). In the nucleus, the export complex assembles together with the small GTPase Ran in its GTPbound state. On its way through the nuclear pore, the trimeric export complex interacts with several nucleoporins, among them Nup214. Nup214 is located at the cytoplasmic side of the NPC and its FGrich C-terminus interacts with CRM1 in a RanGTP-dependent manner, providing a terminal docking site for CRM1-RanGTP-cargo complexes in the late steps of nuclear export. This interaction also indicates a specific role for Nup214 in nuclear export, at least for some cargos. Whereas the CRM1binding region in Nup214 is known, the CRM1-binding sites for Nup214 have not been identified so far. In this work, we analyzed the localization and the export potential of CRM1-fragments, mutants and chimeras, derived from H. sapiens and C. elegans CRM1. Whereas CRM1-fragments did not associate with the nuclear envelope and were not able to mediate export in mammalian cells, we found one CRM1-chimera which was located at the nuclear envelope and was able to transport NES-cargos through the nuclear pore. From that we conclude that association of CRM1 with the nuclear pore correlates with the ability to promote nuclear export. By comparing several chimeras with respect to their localization and functionality, we hypothesize that Nup214 binding occurs between HRs 9-11 of CRM1.Transport receptors bind preferentially to FGs in nucleoporins. For CRM1 this had not been analyzed in detail. Here we show that CRM1 specifically binds to the FGs in Nup214. Moreover, we could narrow down the Nup214 region for CRM1-binding. By analyzing short Nup214-fragments and mutants, we identified a prominent FG-motif in Nup214, comprising 20 amino acid residues. This motif interacts with CRM1 in a RanGTP-dependent manner, with enhanced binding in the presence of an NES-cargo. Two other Nup214 regions downstream of this motif contribute to the stability of this interaction.