Fukumoto Masahiro, Sekimoto Toshihiro, Yoneda Yoshihiro
Department of Frontier Biosciences, Osaka University Graduate School of Frontier Biosciences, Suita, Osaka 565-0871, Japan.
Cell Struct Funct. 2011;36(1):57-67. doi: 10.1247/csf.10026. Epub 2011 Feb 5.
Many transport factors, such as importins and exportins, have been identified, and the molecular mechanisms underlying nucleocytoplasmic transport have been characterized. The specific molecules that are carried by each transport factor and the temporal profiles that characterize the movements of various proteins into or out of the nucleus, however, have yet to be elucidated. Here, we used a proteomic approach to identify molecules that are transported into the nuclei of adult mouse brain cells via importin α5. We identified 48 proteins in total, among which we chose seven to characterize more extensively: acidic (leucine-rich) nuclear phosphoprotein 32 family member A (Anp32a), far upstream element binding protein 1 (FUBP1), thyroid hormone receptor β1 (TRβ1), transaldolase 1, CDC42 effector protein 4 (CDC42-ep4), Coronin 1B, and brain-specific creatine kinase (CK-B). Analyses using green fluorescent protein (GFP)-fused proteins showed that Anp32a, FUBP1, and TRβ1 were localized in the nucleus, whereas transaldolase 1, CDC42-ep4, CK-B, and Coronin 1B were distributed in both the cytoplasm and nucleus. Using a digitonin-permeabilized in vitro transport assay, we demonstrated that, with the exception of CK-B, these proteins were transported into the nucleus by importin α5 together with importin β and Ran. Further, we found that leptomycin B (LMB) treatment increased nuclear CK-B-GFP signals, suggesting that CK-B enters the nucleus and is then exported in a CRM1-dependent manner. Thus, we identified a comprehensive set of candidate proteins that are transported into the nucleus in a manner dependent on importin α5, which enhances our understanding of nucleocytoplasmic signaling in neural cells.
许多转运因子，如输入蛋白和输出蛋白，已被识别出来，并且核质运输的分子机制也已得到表征。然而，每个转运因子所携带的特定分子以及表征各种蛋白质进出细胞核运动的时间特征，仍有待阐明。在这里，我们使用蛋白质组学方法来识别通过输入蛋白α5转运到成年小鼠脑细胞细胞核中的分子。我们总共鉴定出48种蛋白质，其中我们选择了7种进行更深入的表征：酸性（富含亮氨酸）核磷蛋白32家族成员A（Anp32a）、远上游元件结合蛋白1（FUBP1）、甲状腺激素受体β1（TRβ1）、转醛醇酶1、CDC42效应蛋白4（CDC42-ep4）、冠蛋白1B和脑特异性肌酸激酶（CK-B）。使用绿色荧光蛋白（GFP）融合蛋白进行的分析表明，Anp32a、FUBP1和TRβ1定位于细胞核中，而转醛醇酶1、CDC42-ep4、CK-B和冠蛋白1B则分布在细胞质和细胞核中。使用洋地黄皂苷通透的体外运输试验，我们证明，除了CK-B外，这些蛋白质通过输入蛋白α5与输入蛋白β和Ran一起转运到细胞核中。此外，我们发现，莱普霉素B（LMB）处理增加了核CK-B-GFP信号，表明CK-B进入细胞核，然后以CRM1依赖的方式输出。因此，我们鉴定出了一组全面的候选蛋白质，它们以依赖于输入蛋白α5的方式转运到细胞核中，这增强了我们对神经细胞核质信号传导的理解。
