Harreman Michelle T, Cohen Pamela E, Hodel Mary R, Truscott Glyn J, Corbett Anita H, Hodel Alec E
Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road NE, Atlanta, GA 30322, USA.
J Biol Chem. 2003 Jun 13;278(24):21361-9. doi: 10.1074/jbc.M301114200. Epub 2003 Apr 2.
Protein cargoes that contain a classic nuclear localization signal (NLS) are transported into the nucleus through binding to a heterodimeric receptor comprised of importin/karyopherin alpha and beta. An evolutionarily conserved auto-inhibitory sequence within the N-terminal importin beta binding (IBB) domain of importin alpha regulates NLS-cargo binding to the NLS binding pocket on importin alpha. In this study, we have used site-directed mutagenesis coupled with in vitro binding assays and in vivo analyses to investigate the intramolecular interaction of the N-terminal IBB domain and the NLS binding pocket of Saccharomyces cerevisiae importin alpha, Srp1p. We find that mutations within the IBB domain that decrease the binding affinity of the auto-inhibitory sequence for the NLS binding pocket impact importin alpha function in vivo. In addition, the severity of the in vivo phenotype is directly correlated to the reduction of auto-inhibition measured in vitro, suggesting that the in vivo phenotypes are directly related to the loss of auto-inhibitory function. We exploit a conditional auto-inhibitory mutant, srp1-55, to study the in vivo functional overlap between the N-terminal IBB domain of importin alpha and other factors implicated in NLS-cargo release, Cse1p and Nup2p. We propose that the N-terminal IBB domain of importin alpha and Cse1p function together in NLS-cargo release, whereas Nup2p contributes to cargo release/importin alpha recycling through a distinct mechanism.
含有经典核定位信号（NLS）的蛋白质货物通过与由输入蛋白/核转运蛋白α和β组成的异二聚体受体结合而被转运到细胞核中。输入蛋白α的N端输入蛋白β结合（IBB）结构域内进化保守的自抑制序列调节NLS货物与输入蛋白α上NLS结合口袋的结合。在本研究中，我们使用定点诱变结合体外结合试验和体内分析，来研究酿酒酵母输入蛋白α（Srp1p）的N端IBB结构域与NLS结合口袋的分子内相互作用。我们发现，IBB结构域内降低自抑制序列与NLS结合口袋结合亲和力的突变会影响体内输入蛋白α的功能。此外，体内表型的严重程度与体外测量的自抑制降低直接相关，这表明体内表型与自抑制功能的丧失直接相关。我们利用一个条件性自抑制突变体srp1-55，来研究输入蛋白α的N端IBB结构域与其他参与NLS货物释放的因子（Cse1p和Nup2p）之间的体内功能重叠。我们提出，输入蛋白α的N端IBB结构域和Cse1p在NLS货物释放中共同发挥作用，而Nup2p通过一种不同的机制促进货物释放/输入蛋白α循环利用。
