Leung Sara W, Harreman Michelle T, Hodel Mary R, Hodel Alec E, Corbett Anita H
Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road NE, Atlanta, GA 30322, USA.
J Biol Chem. 2003 Oct 24;278(43):41947-53. doi: 10.1074/jbc.M307162200. Epub 2003 Aug 13.
Classical protein import, mediated by the binding of a classical nuclear localization signal (NLS) to the NLS receptor, karyopherin/importin alpha, is the most well studied nuclear transport process. Classical NLSs are either monopartite sequences that contain a single cluster of basic amino acids (Lys/Arg) or bipartite sequences that contain two clusters of basic residues separated by an unconserved linker region. We have created mutations in conserved residues in each of the three NLS-binding sites/regions in Saccharomyces cerevisiae karyopherin alpha (SRP1). For each mutant we have analyzed binding to both a monopartite and a bipartite NLS cargo in vitro. We have also expressed each karyopherin alpha mutant in vivo as the only cellular copy of the NLS receptor and examined the impact on cell growth and import of both monopartite and bipartite NLS-containing cargoes. Our results reveal the functional significance of specific residues within karyopherin alpha for NLS cargo binding. A karyopherin alpha variant with a mutation in the major NLS-binding site exhibits decreased binding to both monopartite and bipartite NLS cargoes, and this protein is not functional in vivo. However, we also find that a karyopherin alpha variant with a mutation in the minor NLS-binding site, which shows decreased binding only to bipartite NLS-containing cargoes, is also not functional in vivo. This suggests that the cell is dependent on the function of at least one bipartite NLS cargo that is imported into the nucleus by karyopherin alpha. Our experiments also reveal functional importance for the linker-binding region. This study provides insight into how changes in binding to cellular NLS sequences could impact cellular function. In addition, this work has led to the creation of conditional alleles of karyopherin alpha with well characterized defects in NLS binding that will be useful for identifying and characterizing novel NLS cargoes.
由经典核定位信号（NLS）与NLS受体核转运蛋白/输入蛋白α结合介导的经典蛋白质导入是研究最为深入的核运输过程。经典NLSs要么是包含单个碱性氨基酸簇（赖氨酸/精氨酸）的单分型序列，要么是包含两个由非保守连接区隔开的碱性残基簇的双分型序列。我们在酿酒酵母核转运蛋白α（SRP1）的三个NLS结合位点/区域中的每一个的保守残基处产生了突变。对于每个突变体，我们在体外分析了其与单分型和双分型NLS货物的结合情况。我们还在体内将每个核转运蛋白α突变体作为NLS受体的唯一细胞拷贝进行表达，并研究了其对细胞生长以及单分型和双分型含NLS货物导入的影响。我们的结果揭示了核转运蛋白α中特定残基对于NLS货物结合的功能重要性。在主要NLS结合位点发生突变的核转运蛋白α变体与单分型和双分型NLS货物的结合均减少，并且该蛋白在体内无功能。然而，我们还发现，在次要NLS结合位点发生突变的核转运蛋白α变体仅与双分型含NLS货物的结合减少，其在体内也无功能。这表明细胞依赖于至少一种由核转运蛋白α导入细胞核的双分型NLS货物的功能。我们的实验还揭示了连接区结合区域的功能重要性。这项研究深入了解了与细胞NLS序列结合的变化如何影响细胞功能。此外，这项工作导致产生了具有在NLS结合方面特征明确缺陷的核转运蛋白α条件等位基因，这将有助于鉴定和表征新型NLS货物。
