Harreman Michelle T, Hodel Mary R, Fanara Patrizia, Hodel Alec E, Corbett Anita H
Department of Biochemistry, School of Medicine and the Graduate Program in Biochemistry, Cell and Developmental Biology, Emory University, Atlanta, Georgia 30322, USA.
J Biol Chem. 2003 Feb 21;278(8):5854-63. doi: 10.1074/jbc.M210951200. Epub 2002 Dec 16.
Proteins that contain a classical nuclear localization signal (NLS) are recognized in the cytoplasm by a heterodimeric import receptor composed of importin/karyopherin alpha and beta. The importin alpha subunit recognizes classical NLS sequences, and the importin beta subunit directs the complex to the nuclear pore. Recent work shows that the N-terminal importin beta binding (IBB) domain of importin alpha regulates NLS-cargo binding in the absence of importin beta in vitro. To analyze the in vivo functions of the IBB domain, we created a series of mutants in the Saccharomyces cerevisiae importin alpha protein. These mutants dissect the two functions of the N-terminal IBB domain, importin beta binding and auto-inhibition. One of these importin alpha mutations, A3, decreases auto-inhibitory function without impacting binding to importin beta or the importin alpha export receptor, Cse1p. We used this mutant to show that the auto-inhibitory function is essential in vivo and to provide evidence that this auto-inhibitory-defective importin alpha remains bound to NLS-cargo within the nucleus. We propose a model where the auto-inhibitory activity of importin alpha is required for NLS-cargo release and the subsequent Cse1p-dependent recycling of importin alpha to the cytoplasm.
含有经典核定位信号（NLS）的蛋白质在细胞质中被一种由输入蛋白/核转运蛋白α和β组成的异二聚体输入受体识别。输入蛋白α亚基识别经典的NLS序列，输入蛋白β亚基将复合物导向核孔。最近的研究表明，在体外缺乏输入蛋白β的情况下，输入蛋白α的N端输入蛋白β结合（IBB）结构域调节NLS-货物结合。为了分析IBB结构域在体内的功能，我们在酿酒酵母输入蛋白α蛋白中创建了一系列突变体。这些突变体剖析了N端IBB结构域的两种功能，即输入蛋白β结合和自抑制。其中一个输入蛋白α突变体A3降低了自抑制功能，而不影响与输入蛋白β或输入蛋白α输出受体Cse1p的结合。我们使用这个突变体表明自抑制功能在体内是必不可少的，并提供证据表明这种自抑制缺陷的输入蛋白α在细胞核内仍与NLS-货物结合。我们提出了一个模型，其中输入蛋白α的自抑制活性是NLS-货物释放以及随后输入蛋白α依赖Cse1p循环回到细胞质所必需的。
