Dissection of a nuclear localization signal
Dissection of a nuclear localization signal
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DOI：
10.1074/jbc.m008522200
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发表时间：
2001-01-12
影响因子：
4.8
通讯作者：
Hodel, AE
中科院分区：
文献类型：
--
作者：
Hodel, MR;Corbett, AH;Hodel, AE
蛋白质输入真核细胞核的调节过程是由蛋白质输入受体识别的特定核定位信号（NLS）介导的。本研究旨在通过对变体 NLS 的定量分析来破译受体输入 α 识别 NLS 的能量细节。使用丙氨酸扫描方法确定两个单部分 NLS 序列中每个残基的相对重要性。这些测量产生了单部分 NLS 序列的能量定义，其中所需的赖氨酸残基后面是序列 K(K/R)X(K/R) 中的两个其他基本残基。此外，还测量了二分NLS中第二基本簇的能量贡献（类似于3 kcal/mol）以及抑制导入蛋白cu导入蛋白β结合结构域的能量（类似于3 kcal/mol）。这些数据允许基于肽对输入蛋白α-输入蛋白β复合物的亲和力生成能量规模的核定位序列。在此规模上，功能性 NLS 的结合常数类似于 10 nra，而非功能性 NLS 的亲和力弱 100 倍，为 1 muM。当前体外数据与体内功能之间的进一步关联将为蛋白质输入的综合定量模型提供基础。
The regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein import receptors. This study seeks to decipher the energetic details of NLS recognition by the receptor importin alpha through quantitative analysis of variant NLSs. The relative importance of each residue in two monopartite NLS sequences was determined using an alanine scanning approach. These measurements yield an energetic definition of a monopartite NLS sequence where a required lysine residue is followed by two other basic residues in the sequence K(K/R)X(K/R). In addition, the energetic contributions of the second basic cluster in a bipartite NLS (similar to3 kcal/mol) as well as the energy of inhibition of the importin cu importin beta -binding domain (similar to3 kcal/mol) were also measured. These data allow the generation of an energetic scale of nuclear localization sequences based on a peptide's affinity for the importin alpha -importin beta complex. On this scale, a functional NLS has a binding constant of similar to 10 nra, whereas a nonfunctional NLS has a 100-fold weaker affinity of 1 muM. Further correlation between the current in vitro data and in vivo function will provide the foundation for a comprehensive quantitative model of protein import.