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

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Affinity chromatography relies on specific protease cleavage of affinity tags.
• The SUMO/SENP1 system is efficient but unsuitable for eukaryotic expression due to cross-reactivity with endogenous proteases.

Purpose of the Study:

• To evolve an orthogonal SUMO/SENP1 system (SUMO^Eu/SENP1^Eu) for stable eukaryotic expression and selective protein purification.
• To identify novel interactors and functions of the yeast importin Pdr6/Kap122 using the developed system.

Main Methods:

• Development of a novel selection system to evolve orthogonal SUMO^Eu/SENP1^Eu protease pairs.
• Application of an affinity-capture-proteolytic-release strategy to identify Pdr6/Kap122 interactors.
• Confirmation of Pdr6 function in vivo using techniques like depletion assays.

Main Results:

• The SUMO^Eu/SENP1^Eu system demonstrated orthogonality with yeast and animal proteases, ensuring SUMO^Eu fusion stability in eukaryotic cells.
• Identification of Ubc9, Pil1, Lsp1, eIF5A, and eEF2 as novel nuclear import substrates and RanGTP-dependent export cargoes of Pdr6.
• In vivo evidence confirmed Pdr6's role as an exportin, depleting eIF5A and eEF2 from cell nuclei.

Conclusions:

• The evolved SUMO^Eu/SENP1^Eu system is a valuable tool for selective protein purification in eukaryotic systems.
• Pdr6 functions as a bidirectional nuclear transport receptor (biportin), mediating the transport of distinct cargoes in both import and export pathways.