Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores.
Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores.
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DOI：
10.1083/jcb.104.2.189
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发表时间：
1987-02
期刊：
影响因子：
--
通讯作者：
Forbes DJ
中科院分区：
文献类型：
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作者：
Finlay DR;Newmeyer DD;Price TM;Forbes DJ
蛋白质的选择性转运是维持细胞质与细胞核之间生化差异的主要机制。为了开始研究核转运的分子机制，我们使用了一种由非洲爪蟾卵提取物、大鼠肝细胞核以及一种荧光标记的核蛋白——核质蛋白组成的体外转运系统。利用这个系统，我们筛选了转运抑制剂。我们发现凝集素——麦胚凝集素（WGA）完全抑制荧光标记的核质蛋白的核转运。所测试的其他凝集素均不影响核转运。当存在N - 乙酰葡糖胺时，未观察到WGA的抑制作用，并且随后添加糖可使其抑制作用逆转。当通过电子显微镜检查与铁蛋白标记的WGA一起孵育的大鼠肝细胞核时，发现多个WGA分子结合在每个核孔的细胞质面。凝胶电泳和硝酸纤维素转移鉴定出一条主要的和几条次要的核蛋白条带可结合125I标记的WGA。其中含量最丰富的一种63 - 65 - kD糖蛋白是WGA对核蛋白转运的抑制作用位点的候选蛋白。WGA是首个被鉴定出的核蛋白转运抑制剂，并且直接与核孔相互作用。
Selective transport of proteins is a major mechanism by which biochemical differences are maintained between the cytoplasm and nucleus. To begin to investigate the molecular mechanism of nuclear transport, we used an in vitro transport system composed of a Xenopus egg extract, rat liver nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. With this system, we screened for inhibitors of transport. We found that the lectin, wheat germ agglutinin (WGA), completely inhibits the nuclear transport of fluorescently labeled nucleoplasmin. No other lectin tested affected nuclear transport. The inhibition by WGA was not seen when N-acetylglucosamine was present and was reversible by subsequent addition of sugar. When rat liver nuclei that had been incubated with ferritin-labeled WGA were examined by electron microscopy, multiple molecules of WGA were found bound to the cytoplasmic face of each nuclear pore. Gel electrophoresis and nitrocellulose transfer identified one major and several minor nuclear protein bands as binding 125I-labeled WGA. The most abundant protein of these, a 63-65-kD glycoprotein, is a candidate for the inhibitory site of action of WGA on nuclear protein transport. WGA is the first identified inhibitor of nuclear protein transport and interacts directly with the nuclear pore.