Structural and functional investigation of cargo recognition by exportins

Abstract

Nucleocytoplasmic exchange is essential for eukaryotic cells. It proceeds through nuclear pore complexes and is largely mediated by Importin β-like nuclear transport receptors (Impβ-like NTRs). According to the direction of the transport, Impβ-like NTRs are classified as importins or exportins. Exportin 4 (Xpo4) is a bidirectional receptor that can function both as importin and exportin. Xpo4 mediates export of the eukaryotic translation initiation factor 5A (eIF5A) and Smad3 as well as import of transcription factors such as Sox2 and SRY. So far, it has been unclear how Xpo4 facilitates transport of structurally diverse cargoes to opposing compartments. In order to understand the mechanisms of Xpo4-dependent bidirectional transport, in my PhD work, I aimed at crystallizing the cargo-bound and unbound complexes of Xpo4. Here I present the crystal structure of the RanGTP•Xpo4•eIF5A export complex. The structure shows that Xpo4 recognizes not just a linear peptide, but the two folded domains of eIF5A at the same time. eIF5A contains hypusine, a unique amino acid with two positive charges, that is essential for cell viability and for eIF5A function in translation. The hypusine docks into a deep, acidic pocket of Xpo4. The interactions at the acidic pocket are essential for export complex formation and eIF5A export by Xpo4. Therefore, hypusine is a critical element of eIF5A’s complex export signature. The structure also reveals that Ran promotes eIF5A binding through conformational changes in Xpo4, including the stabilization of a conserved acidic loop. Similar acidic loops in unidirectional importins have critical function in cargo assembly and disassembly processes. Therefore, this detail also gives mechanistic hints about how Xpo4 can act as a bidirectional transport receptor. In a parallel project, I aspired to identify novel transport substrates for exportin 7 (Xpo7). To address this, I employed an Xpo7 affinity chromatography to enrich Xpo7 interaction partners from a cytoplasmic extract. This revealed not only further Xpo7 export substrates but also several potential import cargoes. The results suggest that in addition to its characterized role in nuclear export, Xpo7 could function as a nuclear import receptor. Therefore, similar to Xpo4, Xpo7 can be another bidirectional nuclear transport receptor.