Furuta Maiko, Kose Shingo, Kehlenbach Ralph H, Imamoto Naoko
Department of Molecular Genetics, National Institute of Genetics, The Graduate University for Advanced Studies Sokendai, Mishima, Shizuoka, Japan.
Cellular Dynamics Laboratory, RIKEN, Wako, Saitama, Japan.
Methods Cell Biol. 2014;122:331-52. doi: 10.1016/B978-0-12-417160-2.00015-1.
The regulation of nucleocytoplasmic transport is crucial not only for basic cellular activities but also for physiological adaptation to specific situation during the cell cycle, development, or stress. Although a wide variety of transport pathways have been identified in eukaryotic cells, the functional significance of their multiplicity remains unclear. The best-characterized nuclear transport receptors (NTRs) are the members of the importin β family (karyopherin, transportin) whose association with specific cargoes is regulated by the GTPase Ran. In this chapter, we first provide an overview of the various expression vectors used to purify recombinant NTRs. We then describe two sets of recent examples of using well-established digitonin-permeabilized cell-free transport systems in mammalian cells to mimic different cellular conditions in living cells: normal/heat-shock conditions and interphase/mitosis. In the former case, physiological regulation impacts different transport pathways in opposite ways. In the latter case, the importin β-Ran system is used at different cell-cycle stages but with the same biochemical principle to specify the nuclear localization and chromatin loading of a specific protein, respectively. This in vitro transport assay, when adapted to specific cellular conditions or particular substrates, should help to uncover specific transport pathways or transport factors function under different cellular conditions.
核质运输的调控不仅对基本细胞活动至关重要，而且对于细胞周期、发育或应激期间对特定情况的生理适应也很关键。尽管在真核细胞中已鉴定出多种运输途径，但其多样性的功能意义仍不清楚。特征最明确的核运输受体（NTRs）是输入蛋白β家族（核转运蛋白、运输蛋白）的成员，其与特定货物的结合受GTP酶Ran调控。在本章中，我们首先概述用于纯化重组NTRs的各种表达载体。然后，我们描述两组最近的例子，这些例子利用成熟的洋地黄皂苷通透化无细胞运输系统在哺乳动物细胞中模拟活细胞中的不同细胞状况：正常/热休克状况以及间期/有丝分裂。在前一种情况下，生理调控以相反的方式影响不同的运输途径。在后一种情况下，输入蛋白β-Ran系统在不同的细胞周期阶段使用，但基于相同的生化原理分别指定特定蛋白质的核定位和染色质负载。这种体外运输测定法，当适用于特定细胞状况或特定底物时，应有助于揭示不同细胞状况下的特定运输途径或运输因子功能。
