Greber U F, Senior A, Gerace L
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, CA 92037.
EMBO J. 1990 May;9(5):1495-502. doi: 10.1002/j.1460-2075.1990.tb08267.x.
One of a small number of polypeptides of the nuclear pore complex that have been identified is a major glycoprotein called gp210. Since it is very resistant to chemical extractions from membranes, gp210 was suggested to be integrated into nuclear membranes. In this study we have determined the membrane topology of this protein by biochemical and immunological approaches. We found that limited proteolysis of isolated nuclear envelopes with papain released a 200 kd water-soluble fragment of gp210 containing concanavalin A-reactive carbohydrate. Immunogold electron microscopy with a monoclonal antibody showed that this domain is localized on the lumenal side of nuclear membranes at pore complexes. Anti-peptide antibodies against two sequences near the C-terminus of gp210 were used to map possible membrane spanning and cytoplasmically disposed regions of this protein. From analysis of the protease sensitivity of these epitopes in sealed membrane vesicles, we determined that gp210 contains a small cytoplasmic tail and only a single membrane-spanning region. Thus, gp210 is a transmembrane protein with most of its mass, including the carbohydrate, located in the perinuclear space. This topology suggests that gp210 is involved primarily in structural organization of the pore complex, for which it may provide a membrane attachment site.
已被鉴定出的核孔复合体少数几种多肽之一是一种名为gp210的主要糖蛋白。由于它对从膜上进行化学提取具有很强的抗性，因此有人提出gp210整合到了核膜中。在本研究中，我们通过生化和免疫学方法确定了该蛋白的膜拓扑结构。我们发现，用木瓜蛋白酶对分离的核膜进行有限的蛋白水解会释放出一个200kd的gp210水溶性片段，该片段含有伴刀豆球蛋白A反应性碳水化合物。用单克隆抗体进行的免疫金电子显微镜观察表明，该结构域位于核膜孔复合体的腔面。针对gp210 C末端附近两个序列的抗肽抗体被用于绘制该蛋白可能的跨膜和胞质区域。通过分析这些表位在密封膜泡中的蛋白酶敏感性，我们确定gp210含有一个小的胞质尾巴，并且只有一个跨膜区域。因此，gp210是一种跨膜蛋白，其大部分质量，包括碳水化合物，位于核周空间。这种拓扑结构表明gp210主要参与孔复合体的结构组织，它可能为此提供一个膜附着位点。
