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 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.

Structural basis for the nuclear import and export functions of the biportin Pdr6/Kap122

“…Pdr6 has so far been a rather poorly studied shuttling NTR with just two import cargoes, subunits of TFIIA and a Wtm1 ribonucleotide reductase complex, being known. In the accompanying paper (Vera Rodriguez et al, 2019), we reevaluated its cargo spectrum and identified not only Ubc9 as a novel import cargo but also export cargoes, namely, the translation elongation factors eIF5A and eEF2, as well as the membrane trafficking components Pil1 and Lsp1. This defines Pdr6 as a bidirectional NTR or a biportin.…”

“…Recombinant mouse Xpo4, human RanQ69L (5–180), human eIF5A, and S. cerevisiae eIF5A variants, as well as human Imp13, were expressed and purified as previously described (Aksu et al, 2016; Vera Rodriguez et al, 2019).…”

WNK1 is an assembly factor for the human ER membrane protein complex

“…The coding sequence of full length human WNK1 was derived from a commercial expression vector (#RC218208, Origene, USA). Expression constructs needed to generate biotinylated anti-GFP nanobody for native purification from human cells are available from Addgene (#149336, #149334, #149333) (Pleiner et al, 2015;Pleiner et al, 2020;Vera Rodriguez et al, 2019). Expression constructs for the bdNEDP1, bdSENP1 and SENP EuB proteases were kind gifts of Dirk Görlich and are available from Addgene (#104129, #104962, #149333) (Frey and Görlich, 2014;Pleiner et al, 2018;Vera Rodriguez et al, 2019).…”

“…The expression of Biotin ligase BirA from pTP264 (Addgene #149334) was carried out as described before (Pleiner et al, 2020). The engineered SUMO Eu1 module is resistant to cleavage by endogenous eukaryotic deSUMOylases and thus allows stable isolation of nanobody-target complexes from eukaryotic cell lysates, followed by proteolytic release on ice in physiological buffer using the cognate SENP EuB protease (Vera Rodriguez et al, 2019). The expression of SENP EuB protease was carried out as described before (Pleiner et al, 2020;Vera Rodriguez et al, 2019).…”

“…After 1 hour of binding head-over-tail at 4˚C, the beads were washed three times with wash buffer (solubilization buffer with 0.1% [w/v] LMNG). Finally, the anti-GFP nanobody along with all bound proteins was released under native conditions and in minimal volume by cleavage with 250 nM SENP EuB protease (Vera Rodriguez et al, 2019) in wash buffer for 20 min at 4˚C. The eluate was then analyzed by SDS-PAGE and Western blotting or Sypro Ruby staining (Bio-Rad Laboratories, USA).…”