Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
J Cell Biol. 2019 Jun 3;218(6):2006-2020. doi: 10.1083/jcb.201812091. Epub 2019 Apr 25.
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/SENP1 pair to orthogonality with the yeast and animal enzymes. SUMO fusions therefore remain stable in eukaryotic cells. Likewise, overexpressing a SENP1 protease is nontoxic in yeast. We have used the SUMO 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.
通过特定蛋白酶切割亲和标签可用于高度选择性亲和层析。SUMO/SENP1 系统是最有效的，但在真核表达中失败，因为它与内源性蛋白酶发生交叉反应。我们使用一种新的选择系统，使 SUMO/SENP1 对与酵母和动物酶正交。因此，SUMO 融合在真核细胞中保持稳定。同样，过表达 SENP1 蛋白酶在酵母中也没有毒性。我们在亲和捕获-蛋白水解-释放方法中使用 SUMO 系统来鉴定酵母输入蛋白 Pdr6/Kap122 的相互作用物。这不仅揭示了更多的核输入底物，如 Ubc9，还揭示了 Pil1、Lsp1、eIF5A 和 eEF2 作为 RanGTP 依赖性结合物，因此是输出货物。我们证实 Pdr6 在体内作为输出蛋白发挥作用，并从细胞核中耗尽 eIF5A 和 eEF2。因此，Pdr6 是一种双向核转运受体（即双向蛋白），可在相反方向上转运不同的货物。
