Kosugi Shunichi, Hasebe Masako, Matsumura Nobutaka, Takashima Hideaki, Miyamoto-Sato Etsuko, Tomita Masaru, Yanagawa Hiroshi
Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan and the Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan; Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan and the Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan.
Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan and the Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan.
J Biol Chem. 2009 Jan 2;284(1):478-485. doi: 10.1074/jbc.M807017200. Epub 2008 Nov 10.
The importin alpha/beta pathway mediates nuclear import of proteins containing the classical nuclear localization signals (NLSs). Although the consensus sequences of the classical NLSs have been defined, there are still many NLSs that do not match the consensus rule and many nonfunctional sequences that match the consensus. We report here six different NLS classes that specifically bind to distinct binding pockets of importin alpha. By screening of random peptide libraries using an mRNA display, we selected peptides bound by importin alpha and identified six classes of NLSs, including three novel classes. Two noncanonical classes (class 3 and class 4) specifically bound the minor binding pocket of importin alpha, whereas the classical monopartite NLSs (class 1 and class 2) bound to the major binding pocket. Using a newly developed universal green fluorescent protein expression system, we found that these NLS classes, including plant-specific class 5 NLSs and bipartite NLSs, fundamentally require the regions outside the core basic residues for their activity and have specific residues or patterns that confer the activities differently between yeast, plants, and mammals. Furthermore, amino acid replacement analyses revealed that the consensus basic patterns of the classical NLSs are not essential for activity, thereby generating more unconventional patterns, including redox-sensitive NLSs. These results explain the causes of the NLS diversity. The defined consensus patterns and properties of importin alpha-dependent NLSs provide useful information for identifying NLSs.
输入蛋白α/β途径介导含有经典核定位信号（NLSs）的蛋白质的核输入。尽管已经定义了经典NLSs的共有序列，但仍有许多NLSs不符合共有规则，也有许多符合共有序列的无功能序列。我们在此报告六种不同的NLS类别，它们特异性结合输入蛋白α的不同结合口袋。通过使用mRNA展示筛选随机肽库，我们选择了与输入蛋白α结合的肽，并鉴定出六类NLSs，包括三类新的类别。两类非经典类别（第3类和第4类）特异性结合输入蛋白α的次要结合口袋，而经典的单分型NLSs（第1类和第2类）则结合主要结合口袋。使用新开发的通用绿色荧光蛋白表达系统，我们发现这些NLS类别，包括植物特异性的第5类NLSs和双分型NLSs，其活性基本上需要核心碱性残基以外的区域，并且具有特定的残基或模式，这些残基或模式在酵母、植物和哺乳动物之间赋予不同的活性。此外，氨基酸替换分析表明，经典NLSs的共有碱性模式对活性并非必不可少，从而产生了更多非常规模式，包括氧化还原敏感的NLSs。这些结果解释了NLS多样性的原因。所定义的输入蛋白α依赖性NLSs的共有模式和特性为鉴定NLSs提供了有用的信息。
