Finlay D R, Newmeyer D D, Hartl P M, Horecka J, Forbes D J
Department of Biology, University of California, San Diego, La Jolla 92014.
J Cell Sci Suppl. 1989;11:225-42. doi: 10.1242/jcs.1989.supplement_11.17.
In this paper, progress towards the goal of understanding communication between the nucleus and cytoplasm using an in vitro system is reviewed. To probe the mechanism of nuclear targeting, we developed an in vitro transport system and have begun to dissect the highly selective process of nuclear transport. The basic parameters of transport were defined using an easily isolated nuclear protein, nucleoplasmin. To study the interaction of nuclear targeting signals with the pore, an artificial nuclear transport substrate was constructed, which consists of human serum albumin coupled to the signal sequence of the SV40 T-antigen. A similar peptide-protein conjugate was made using a mutant signal sequence. These conjugates were fluorescently labeled and/or tagged with gold and tested for transport in the in vitro system. High levels of nuclear transport of the wild-type signal sequence-containing protein were observed, while no transport of the mutant signal sequence-containing protein was seen. Thus, the in vitro system correctly recognizes the single amino acid change between the wild-type and mutant signal sequences. We found that the observed nuclear transport was completely dependent on the presence of ATP. Using the in vitro system we identified a specific inhibitor of nuclear transport, the lectin wheat germ agglutinin (WGA), which we find binds directly to the nuclear pore. Probing blots of nuclear proteins with 125I-WGA identified a family of nuclear pore glycoproteins, including one major glycoprotein of 62K (K = 10(3)Mr) molecular weight. With the inhibitor and the in vitro assay, it has been possible to experimentally separate nuclear transport into two steps: (1) a step in which the signal sequence-bearing protein binds to the pore, followed by (2) a step in which the protein translocates through the pore. It is this second step which is the ATP-dependent step of transport, since pore binding but not translocation was seen to occur in the absence of ATP.
本文综述了利用体外系统在理解细胞核与细胞质之间通讯目标方面所取得的进展。为了探究核靶向机制，我们开发了一种体外转运系统，并开始剖析核转运这一高度选择性的过程。利用一种易于分离的核蛋白核质素确定了转运的基本参数。为了研究核靶向信号与核孔的相互作用，构建了一种人工核转运底物，它由与人血清白蛋白偶联的SV40 T抗原信号序列组成。使用突变信号序列制备了类似的肽 - 蛋白质缀合物。这些缀合物用荧光标记和/或用金标记，并在体外系统中进行转运测试。观察到含野生型信号序列的蛋白质有高水平的核转运，而含突变信号序列的蛋白质则未见转运。因此，体外系统能正确识别野生型和突变型信号序列之间的单个氨基酸变化。我们发现观察到的核转运完全依赖于ATP的存在。利用体外系统我们鉴定出一种核转运的特异性抑制剂，即凝集素麦胚凝集素（WGA），我们发现它直接与核孔结合。用125I - WGA探测核蛋白印迹鉴定出一族核孔糖蛋白，包括一种分子量为62K（K = 10(3)Mr）的主要糖蛋白。借助该抑制剂和体外测定法，已能够通过实验将核转运分为两个步骤：（1）携带信号序列的蛋白质与核孔结合的步骤，随后是（2）蛋白质穿过核孔的步骤。正是第二步是转运中依赖ATP的步骤，因为在没有ATP的情况下可见到与核孔的结合但未见转运。
