Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors.
Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors.
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核蛋白在透化的哺乳动物细胞中进口需要可溶性细胞质因子。
DOI：
10.1083/jcb.111.3.807
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发表时间：
1990-09
影响因子：
7.8
通讯作者：
Gerace, L
中科院分区：
文献类型：
--
作者：
Adam, S A;Marr, R S;Gerace, L
我们已经开发了一个涉及洋地黄素渗透性脊椎动物细胞的体外系统，以研究跨核被膜运输大分子的生化事件。虽然用洋地黄素处理培养细胞可以使质膜渗透到大分子中，但核膜结构保持不变，细胞核保持运输和积累含有SV40大T抗原核定位序列的蛋白质的能力。转运需要将外源胞浆添加到通透性细胞中，这表明核进口所需的可溶性细胞质因子(S)在洋地黄素治疗期间被释放。在这个重组的输入系统中，含有核定位信号的蛋白质迅速积累在细胞核中，在30分钟内它的浓度达到周围介质的30倍。核进口是功能核定位序列所特有的，需要ATP和胞浆，并与温度有关。此外，转运底物在细胞核内的积累完全被小麦胚凝集素抑制，麦胚凝集素与核孔复合体结合，抑制体内的转运。综上所述，这些结果表明，渗透的细胞系统复制了真正的核蛋白输入。在对该体系的初步生化剖析中，我们观察到，巯基烷基化试剂N-乙基马来酰亚胺既能灭活核蛋白进口所需的不溶通透性细胞组分中的胞浆因子(S)，也能灭活其成分(S)。由于这种通透性细胞模型简单、高效，并能有效地处理来自各种不同脊椎动物来源的细胞和胞质组分，因此它将被证明是研究核运输的生化途径的有力工具。
We have developed an in vitro system involving digitonin-permeabilized vertebrate cells to study biochemical events in the transport of macromolecules across the nuclear envelope. While treatment of cultured cells with digitonin permeabilizes the plasma membranes to macromolecules, the nuclear envelopes remain structurally intact and nuclei retain the ability to transport and accumulate proteins containing the SV40 large T antigen nuclear location sequence. Transport requires addition of exogenous cytosol to permeabilized cells, indicating the soluble cytoplasmic factor(s) required for nuclear import are released during digitonin treatment. In this reconstituted import system, a protein containing a nuclear location signal is rapidly accumulated in nuclei, where it reaches a 30-fold concentration compared to the surrounding medium within 30 min. Nuclear import is specific for a functional nuclear location sequence, requires ATP and cytosol, and is temperature dependent. Furthermore, accumulation of the transport substrate within nuclei is completely inhibited by wheat germ agglutinin, which binds to nuclear pore complexes and inhibits transport in vivo. Together, these results indicate that the permeabilized cell system reproduces authentic nuclear protein import. In a preliminary biochemical dissection of the system, we observe that the sulfhydryl alkylating reagent N- ethylmaleimide inactivates both cytosolic factor(s) and also component(s) in the insoluble permeabilized cell fraction required for nuclear protein import. Because this permeabilized cell model is simple, efficient, and works effectively with cells and cytosol fractions prepared from a variety of different vertebrate sources, it will prove powerful for investigating the biochemical pathway of nuclear transport.