Dean D A, Kasamatsu H
Department of Biology, University of California, Los Angeles 90024.
J Biol Chem. 1994 Feb 18;269(7):4910-6.
Nuclear transport signal (NTS)-containing proteins are transported into the nucleus through the nuclear pore complex by a mechanism that is not well understood. To better characterize the mechanisms of transport, we have established an homologous in vitro system using an NTS-containing structural protein of simian virus 40 (SV40) and isolated nuclei from cultured cells of its natural host. Isolated nuclei accumulated either fluorescently labeled SV40 Vp3-NTS peptide-BSA conjugates (NTSwt-BSA), as assayed cytochemically, or 125I-NTSwt-BSA, as assayed by filtration, in a signal- and ATP-dependent manner. Nuclear accumulation required nuclear membrane integrity and was inhibited by the lectin wheat germ agglutinin but not concanavalin A. Unlike several other systems, this system is not dependent on cytoplasmic extracts for the transport of SV40 proteins. NTSwt-BSA was transported with an apparent Km of 0.8 microM and Vmax of 0.8 nmol/min/10(*) nuclei. Thin section autoradiography confirmed the transport. This system faithfully reproduced what occurs in vivo: nuclear import of the SV40 capsid protein Vp3 was dependent on the presence of its functional NTS. Full-length Vp3, expressed as a glutathione S-transferase fusion protein, and a deletion mutant which retains its NTS, Vp3 delta C13, were transported by the nuclei but Vp3 delta C35, which lacks the NTS, and an NTS mutant, Vp3(202E/204T), were not transported.
含有核转运信号（NTS）的蛋白质通过一种尚未完全了解的机制，经核孔复合体转运至细胞核内。为了更好地表征转运机制，我们利用猿猴病毒40（SV40）的一种含NTS的结构蛋白，建立了一种同源体外系统，并从其天然宿主的培养细胞中分离出细胞核。经细胞化学检测，分离出的细胞核以信号和ATP依赖的方式积累荧光标记的SV40 Vp3-NTS肽-BSA缀合物（NTSwt-BSA）；经过滤检测，积累125I-NTSwt-BSA。核积累需要核膜完整性，并受到凝集素麦胚凝集素的抑制，但不受伴刀豆球蛋白A的抑制。与其他几个系统不同，该系统在转运SV40蛋白时不依赖细胞质提取物。NTSwt-BSA的转运表观Km为0.8 microM，Vmax为0.8 nmol/min/10（*）个细胞核。超薄切片放射自显影证实了转运过程。该系统忠实地再现了体内发生的情况：SV40衣壳蛋白Vp3的核输入依赖于其功能性NTS的存在。全长Vp3以谷胱甘肽S-转移酶融合蛋白形式表达，保留其NTS的缺失突变体Vp3 delta C13被细胞核转运，但缺乏NTS的Vp3 delta C35和NTS突变体Vp3（202E/204T）未被转运。
