Abstract
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.
© 2019 Vera Rodriguez et al.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Active Transport, Cell Nucleus
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Amino Acid Sequence
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Animals
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Cell Nucleus / metabolism*
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Cysteine Endopeptidases / genetics
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Cysteine Endopeptidases / metabolism
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Eukaryotic Translation Initiation Factor 5A
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HEK293 Cells
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Humans
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Peptide Initiation Factors / genetics
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Peptide Initiation Factors / metabolism
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Protein Binding
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RNA-Binding Proteins / genetics
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RNA-Binding Proteins / metabolism
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / growth & development
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Saccharomyces cerevisiae / metabolism*
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism*
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Sequence Homology
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Small Ubiquitin-Related Modifier Proteins / genetics
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Small Ubiquitin-Related Modifier Proteins / metabolism*
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Ubiquitin-Conjugating Enzymes / genetics
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Ubiquitin-Conjugating Enzymes / metabolism
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beta Karyopherins / genetics
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beta Karyopherins / metabolism*
Substances
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KAP122 protein, S cerevisiae
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Peptide Initiation Factors
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RNA-Binding Proteins
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Saccharomyces cerevisiae Proteins
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Small Ubiquitin-Related Modifier Proteins
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beta Karyopherins
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Ubiquitin-Conjugating Enzymes
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SENP1 protein, human
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Cysteine Endopeptidases