Engineered SUMO/protease system identifies Pdr6 as a bidirectional nuclear transport receptor.

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.
Mesh Terms:
Active Transport, Cell Nucleus, Amino Acid Sequence, Animals, Cell Nucleus, Cysteine Endopeptidases, HEK293 Cells, Humans, Peptide Initiation Factors, Protein Binding, RNA-Binding Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Sequence Homology, Small Ubiquitin-Related Modifier Proteins, Ubiquitin-Conjugating Enzymes, beta Karyopherins
J Cell Biol
Date: Dec. 03, 2018
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