Abbondanza C, Rossi V, Roscigno A, Gallo L, Belsito A, Piluso G, Medici N, Nigro V, Molinari A M, Moncharmont B, Puca G A
Istituto di Patologia generale ed Oncologia, Facoltà di Medicina e Chirurgia, Seconda Università degli studi di Napoli, I-80138 Naples, Italy.
J Cell Biol. 1998 Jun 15;141(6):1301-10. doi: 10.1083/jcb.141.6.1301.
A 104-kD protein was coimmunoprecipitated with the estrogen receptor from the flowtrough of a phosphocellulose chromatography of MCF-7 cell nuclear extract. mAbs to this protein identified several cDNA clones coding for the human 104-kD major vault protein. Vaults are large ribonucleoprotein particles of unknown function present in all eukaryotic cells. They have a complex morphology, including several small molecules of RNA, but a single protein species, the major vault protein, accounts for >70% of their mass. Their shape is reminiscent of the nucleopore central plug, but no proteins of known function have been described to interact with them. Western blot analysis of vaults purified on sucrose gradient showed the presence of estrogen receptor co-migrating with the vault peak. The AER317 antibody to estrogen receptor coimmunoprecipitated the major vault protein and the vault RNA also in the 20,000 g supernatant fraction. Reconstitution experiments of estrogen receptor fragments with the major vault protein mapped the site of the interaction between amino acids 241 and 280 of human estrogen receptor, where the nuclear localization signal sequences are located. Estradiol treatment of cells increased the amount of major vault protein present in the nuclear extract and coimmunoprecipitated with estrogen receptor, whereas the anti-estrogen ICI182,780 had no effect. The hormone-dependent interaction of vaults with estrogen receptor was reproducible in vitro and was prevented by sodium molybdate. Antibodies to progesterone and glucocorticoid receptors were able to coimmunoprecipitate the major vault protein. The association of nuclear receptors with vaults could be related to their intracellular traffic.
一种104-kD的蛋白质与雌激素受体在MCF-7细胞核提取物的磷酸纤维素层析洗脱液中进行了共免疫沉淀。针对该蛋白质的单克隆抗体鉴定出了几个编码人104-kD主要穹窿蛋白的cDNA克隆。穹窿是存在于所有真核细胞中的功能未知的大型核糖核蛋白颗粒。它们具有复杂的形态，包括几个小分子RNA，但单一的蛋白质种类，即主要穹窿蛋白，占其质量的70%以上。它们的形状让人联想到核孔中央栓，但尚未描述有已知功能的蛋白质与它们相互作用。对在蔗糖梯度上纯化的穹窿进行蛋白质印迹分析显示，雌激素受体与穹窿峰共迁移。针对雌激素受体的AER317抗体在20,000 g上清液组分中也共免疫沉淀了主要穹窿蛋白和穹窿RNA。雌激素受体片段与主要穹窿蛋白的重组实验确定了人雌激素受体氨基酸241至280之间的相互作用位点，核定位信号序列位于该区域。用雌二醇处理细胞增加了核提取物中存在的并与雌激素受体共免疫沉淀的主要穹窿蛋白的量，而抗雌激素ICI182,780则没有作用。穹窿与雌激素受体的激素依赖性相互作用在体外是可重复的，并且被钼酸钠阻断。针对孕酮和糖皮质激素受体的抗体能够共免疫沉淀主要穹窿蛋白。核受体与穹窿的关联可能与其细胞内运输有关。
