Nuclear import of proteins containing classical nuclear localization signals (NLS) is mediated by the importin‐α:β complex that binds cargo in the cytoplasm and facilitates its passage through nuclear pores, after which nuclear RanGTP dissociates the import complex and the importins are recycled. In vertebrates, import is stimulated by nucleoporin Nup50, which has been proposed to accompany the import complex through nuclear pores. However, we show here that the Nup50 N‐terminal domain actively displaces NLSs from importin‐α, which would be more consistent with Nup50 functioning to coordinate import complex disassembly and importin recycling. The crystal structure of the importin‐α:Nup50 complex shows that Nup50 binds at two sites on importin‐α. One site overlaps the secondary NLS‐binding site, whereas the second extends along the importin‐α C‐terminus. Mutagenesis indicates that interaction at both sites is required for Nup50 to displace NLSs. The Cse1p:Kap60p:RanGTP complex structure suggests how Nup50 is then displaced on formation of the importin‐α export complex. These results provide a rationale for understanding the series of interactions that orchestrate the terminal steps of nuclear protein import.

Interactions with nucleoporins containing FxFG‐repeat cores are crucial for the nuclear import of RanGDP mediated by nuclear transport factor 2 (NTF2). We describe here the 1.9 Å resolution crystal structure of yeast NTF2‐N77Y bound to a FxFG‐nucleoporin core, which provides a basis for understanding this interaction and its role in nuclear trafficking. The two identical FxFG binding sites on the dimeric molecule are formed by residues from each chain of NTF2. Engineered mutants at the interaction interface reduce the binding of NTF2 to nuclear pores and cause reduced growth rates and Ran mislocalization when substituted for the wild‐type protein in yeast. Comparison with the crystal structure of FG‐nucleoporin cores bound to importin‐β and TAP/p15 identified a number of common features of their binding sites. The structure of the binding interfaces on these transport factors provides a rationale for the specificity of their interactions with nucleoporins that, combined with their weak binding constants, facilitates rapid translocation through NPCs during nuclear trafficking.

The yeast Sac3:Cdc31:Sus1:Thp1 (TREX-2) complex facilitates the repositioning and association of actively transcribing genes with nuclear pores (NPCs)-"gene gating"-that is central to integrating transcription, processing, and mRNA nuclear export. We present here the crystal structure of Sus1 and Cdc31 bound to a central region of Sac3 (the CID domain) that is crucial for its function. Sac3(CID) forms a long, gently undulating alpha helix around which one Cdc31 and two Sus1 chains are wrapped. Sus1 has an articulated helical hairpin fold that facilitates its wrapping around Sac3. In vivo studies using engineered mutations that selectively disrupted binding of individual chains to Sac3 indicated that Sus1 and Cdc31 function synergistically to promote NPC association of TREX-2 and mRNA nuclear export. These data indicate Sac3(CID) provides a scaffold within TREX-2 to integrate interactions between protein complexes to facilitate the coupling of transcription and mRNA export during gene expression.