Paschal B M, Gerace L
Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037, USA.
J Cell Biol. 1995 May;129(4):925-37. doi: 10.1083/jcb.129.4.925.
Protein import into the nucleus is a multistep process that requires the activities of several cytosolic factors. In this study we have purified a cytosolic factor that interacts with the nuclear pore complex glycoprotein p62. Isolation involved biochemical complementation of cytosol depleted of this activity by preadsorption with recombinant p62 and the use of a novel flow cytometry-based assay for quantitation of nuclear import. The purified activity (NTF2) is an apparent dimer of approximately 14-kD subunits and is present at approximately 10(6) copies per cell. We obtained a cDNA encoding NTF2 and showed that the recombinant protein restores transport activity to p62-pretreated cytosol. Our data suggest that NTF2 acts at a relatively late stage of nuclear protein import, subsequent to the initial docking of nuclear import ligand at the nuclear envelope. NTF2 interacts with at least one additional cytosolic transport activity, indicating that it could be part of a multicomponent system of cytosolic factors that assemble at the pore complex during nuclear import.
蛋白质导入细胞核是一个多步骤过程，需要几种胞质因子的参与。在本研究中，我们纯化了一种与核孔复合体糖蛋白p62相互作用的胞质因子。分离过程涉及通过用重组p62预吸附使缺乏该活性的胞质进行生化互补，并使用基于流式细胞术的新方法来定量核输入。纯化的活性物质（NTF2）是由大约14-kD亚基组成的明显二聚体，每个细胞中约有10^6个拷贝。我们获得了编码NTF2的cDNA，并表明重组蛋白可将转运活性恢复到经p62预处理的胞质中。我们的数据表明，NTF2在核蛋白导入的相对后期起作用，即在核输入配体最初停靠在核膜之后。NTF2与至少一种其他胞质转运活性相互作用，表明它可能是在核输入过程中在孔复合体处组装的多组分胞质因子系统的一部分。
