Kimura Makoto, Thakar Ketan, Karaca Samir, Imamoto Naoko, Kehlenbach Ralph H
Cellular Dynamics Laboratory, RIKEN, Wako, Saitama, Japan.
Department of Molecular Biology, Faculty of Medicine, Georg-August-University of Göttingen, Göttingen, Germany.
Methods Cell Biol. 2014;122:353-78. doi: 10.1016/B978-0-12-417160-2.00016-3.
Nucleocytoplasmic transport affects the subcellular localization of a large proportion of cellular proteins. Transported proteins interact with a set of ~20 transport receptors, importins and exportins, which mediate translocation through the nuclear pore complex. Here we describe two novel methods based on quantitative proteome analysis for the identification of cargo proteins that are transported by a specific importin or exportin. The first approach is based on SILAC (stable isotope labeling of amino acids in cells) using cells that have been treated or not with specific reagents, followed by subcellular fractionation. Applying this approach to cells treated with or without the selective CRM1 inhibitor leptomycin B, we identified substrates of CRM1, the major nuclear export receptor. In the second SILAC approach, digitonin-permeabilized cells are incubated with nuclear and cytosolic extracts in the absence or presence of particular import receptors of interest. Proteomic analysis of the permeabilized cells then yields proteins whose nuclear import depends specifically on the added import receptor. Using this system, we identified substrates of two representative import receptors, transportin and importin-α/β.
核质运输影响着很大一部分细胞蛋白质的亚细胞定位。被运输的蛋白质与一组约20种运输受体相互作用，即输入蛋白和输出蛋白，它们介导通过核孔复合体的转运。在此，我们描述了基于定量蛋白质组分析的两种新方法，用于鉴定由特定输入蛋白或输出蛋白运输的货物蛋白。第一种方法基于SILAC（细胞中氨基酸的稳定同位素标记），使用经过或未经过特定试剂处理的细胞，随后进行亚细胞分级分离。将这种方法应用于用选择性CRM1抑制剂莱普霉素B处理或未处理的细胞，我们鉴定出了主要的核输出受体CRM1的底物。在第二种SILAC方法中，将洋地黄皂苷通透的细胞在不存在或存在特定感兴趣的输入受体的情况下与核提取物和胞质提取物一起孵育。然后，对通透细胞进行蛋白质组分析，得到其核输入特别依赖于添加的输入受体的蛋白质。使用这个系统，我们鉴定出了两种代表性输入受体——运输蛋白和输入蛋白α/β的底物。
