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

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 SUMO^Eu/SENP1^Eu pair to orthogonality with the yeast and animal enzymes. SUMO^Eu fusions therefore remain stable in eukaryotic cells. Likewise, overexpressing a SENP1^Eu protease is nontoxic in yeast. We have used the SUMO^Eu 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,

SUBMITTER: Vera Rodriguez A 

PROVIDER: S-EPMC6548132 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature