van Hemert Martijn J, Niemantsverdriet Maarten, Schmidt Thomas, Backendorf Claude, Spaink Herman P
Section Molecular Cell Biology, Institute of Biology, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands.
J Cell Sci. 2004 Mar 15;117(Pt 8):1411-20. doi: 10.1242/jcs.00990. Epub 2004 Mar 2.
Nucleocytoplasmic transport of proteins plays an important role in the regulation of many cellular processes. Differences in nucleocytoplasmic shuttling can provide a basis for isoform-specific biological functions for members of multigene families, like the 14-3-3 protein family. Many organisms contain multiple 14-3-3 isoforms, which play a role in numerous processes, including signalling, cell cycle control and apoptosis. It is still unclear whether these isoforms have specialised biological functions and whether this specialisation is based on isoform-specific ligand binding, expression regulation or specific localisation. Therefore, we studied the subcellular distribution of 14-3-3 sigma and 14-3-3 zeta in vivo in various mammalian cell types using yellow fluorescent protein fusions and isoform-specific antibodies. 14-3-3 sigma was mainly localised in the cytoplasm and only low levels were present in the nucleus, whereas 14-3-3 zeta was found at relatively higher levels in the nucleus. Fluorescence recovery after photobleaching (FRAP) experiments indicated that the 14-3-3 proteins rapidly shuttle in and out of the nucleus through active transport and that the distinct subcellular distributions of 14-3-3 sigma and 14-3-3 zeta are caused by differences in nuclear export. 14-3-3 sigma had a 1.7x higher nuclear export rate constant than 14-3-3 zeta, while import rate constants were equal. The 14-3-3 proteins are exported from the nucleus at least in part by a Crm1-dependent, leptomycin B-sensitive mechanism. The differences in subcellular distribution of 14-3-3 that we found in this study are likely to reflect a molecular basis for isoform-specific biological specialisation.
蛋白质的核质运输在许多细胞过程的调控中起着重要作用。核质穿梭的差异可为多基因家族成员（如14-3-3蛋白家族）的亚型特异性生物学功能提供基础。许多生物体含有多种14-3-3亚型，它们在众多过程中发挥作用，包括信号传导、细胞周期控制和细胞凋亡。目前尚不清楚这些亚型是否具有专门的生物学功能，以及这种专门化是否基于亚型特异性配体结合、表达调控或特定定位。因此，我们使用黄色荧光蛋白融合物和亚型特异性抗体，研究了14-3-3σ和14-3-3ζ在体内各种哺乳动物细胞类型中的亚细胞分布。14-3-3σ主要定位于细胞质中，细胞核中仅存在低水平，而14-3-3ζ在细胞核中的含量相对较高。光漂白后荧光恢复（FRAP）实验表明，14-3-3蛋白通过主动运输快速穿梭进出细胞核，14-3-3σ和14-3-3ζ不同的亚细胞分布是由核输出差异引起的。14-3-3σ的核输出速率常数比14-3-3ζ高1.7倍，而输入速率常数相等。14-3-3蛋白至少部分通过依赖Crm1的、对放线菌酮B敏感的机制从细胞核输出。我们在这项研究中发现的14-3-3亚细胞分布差异可能反映了亚型特异性生物学专门化的分子基础。
