Cleavage of affinity tags by specific proteases can be exploited for highly selective affinity chromatography. The SUMO/SENP1 system is the most efficient for such application but fails in eukaryotic expression because it cross-reacts with endogenous proteases. Using a novel selection system, we have evolved the SUMOEu/SENP1Eu pair to orthogonality with the yeast and animal enzymes. SUMOEu fusions therefore remain stable in eukaryotic cells. Likewise, overexpressing a SENP1Eu protease is nontoxic in yeast. We have used the SUMOEu system in an affinity-capture-proteolytic-release approach to identify interactors of the yeast importin Pdr6/Kap122. This revealed not only further nuclear import substrates such as Ubc9, but also Pil1, Lsp1, eIF5A, and eEF2 as RanGTP-dependent binders and thus as export cargoes. We confirmed that Pdr6 functions as an exportin in vivo and depletes eIF5A and eEF2 from cell nuclei. Thus, Pdr6 is a bidirectional nuclear transport receptor (i.e., a biportin) that shuttles distinct sets of cargoes in opposite directions.

Importins ferry proteins into nuclei while exportins carry cargoes to the cytoplasm. In the accompanying paper in this issue (Vera Rodriguez et al. 2019. J. Cell Biol. https://doi.org/10.1083/jcb.201812091), we discovered that Pdr6 is a biportin that imports, e.g., the SUMO E2 ligase Ubc9 while depleting the translation factor eIF5A from the nuclear compartment. In this paper, we report the structures of key transport intermediates, namely, of the Ubc9•Pdr6 import complex, of the RanGTP•Pdr6 heterodimer, and of the trimeric RanGTP•Pdr6•eIF5A export complex. These revealed nonlinear transport signals, chaperone-like interactions, and how the RanGTPase system drives Pdr6 to transport Ubc9 and eIF5A in opposite directions. The structures also provide unexpected insights into the evolution of transport selectivity. Specifically, they show that recognition of Ubc9 by Pdr6 differs fundamentally from that of the human Ubc9-importer Importin 13. Likewise, Pdr6 recognizes eIF5A in a nonhomologous manner compared with the mammalian eIF5A-exporter Exportin 4. This suggests that the import of Ubc9 and active nuclear exclusion of eIF5A evolved in different eukaryotic lineages more than once and independently from each other.

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