Yamasaki L, Kanda P, Lanford R E
Department of Virology and Immunology, Southwest Foundation for Biomedical Research, San Antonio, Texas.
Mol Cell Biol. 1989 Jul;9(7):3028-36. doi: 10.1128/mcb.9.7.3028-3036.1989.
The transport of proteins into the nucleus requires not only the presence of a nuclear transport signal on the targeted protein but also the signal recognition proteins and the nuclear pore translocation apparatus. Complicating the search for the signal recognition proteins is the fact that the nuclear transport signals identified share little obvious homology. In this study, synthetic peptides homologous to the nuclear transport signals from the simian virus 40 large T antigen, Xenopus oocyte nucleoplasmin, adenovirus E1A, and Saccharomyces cerevisiae MAT alpha 2 proteins were coupled to a UV-photoactivable cross-linker and iodinated for use in an in vitro cross-linking reaction with cellular lysates. Four proteins, p140, p100, p70, and p55, which specifically interacted with the nuclear transport signal peptides were identified. Unique patterns of reactivity were observed with closely related pairs of nuclear transport signal peptides. Competition experiments with labeled and unlabeled peptides demonstrated that heterologous signals were able to bind the same protein and suggested that diverse signals use a common transport pathway. The subcellular distribution of the four nuclear transport signal-binding proteins suggested that nuclear transport involves both cytoplasmic and nuclear receptors. The four proteins were not bound by wheat germ agglutinin and were not associated tightly with the nuclear pore complex.
蛋白质向细胞核的转运不仅需要目标蛋白上存在核转运信号，还需要信号识别蛋白和核孔转运装置。使寻找信号识别蛋白变得复杂的是，已鉴定出的核转运信号几乎没有明显的同源性。在这项研究中，与猿猴病毒40大T抗原、非洲爪蟾卵母细胞核质蛋白、腺病毒E1A和酿酒酵母MATα2蛋白的核转运信号同源的合成肽与紫外线光活化交联剂偶联并碘化，用于与细胞裂解物进行体外交联反应。鉴定出了四种与核转运信号肽特异性相互作用的蛋白质，即p140、p100、p70和p55。在密切相关的核转运信号肽对中观察到了独特的反应模式。用标记和未标记肽进行的竞争实验表明，异源信号能够结合相同的蛋白质，并表明不同的信号使用共同的转运途径。四种核转运信号结合蛋白的亚细胞分布表明，核转运涉及细胞质和细胞核受体。这四种蛋白质不与麦胚凝集素结合，也不与核孔复合体紧密相关。
