Walser A, Rinke Y, Deppert W
Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie, Universität Hamburg, Federal Republic of Germany.
J Virol. 1989 Sep;63(9):3926-33. doi: 10.1128/JVI.63.9.3926-3933.1989.
The bulk of simian virus 40 (SV40) large T antigen in SV40-infected and -transformed cells localizes within the cell nucleus, while a minor fraction specifically associates with the plasma membrane (PM) and is exposed on the cell surface. PM-associated large T seems to span the lipid bilayer but, on the other hand, does not display typical features of a transmembrane protein. To further characterize the postulated transmembrane orientation of large T, we asked whether all large T molecules associated with the plasma membrane indeed are exposed on the cell surface. We compared the amount of cell surface-exposed large T, determined on living cells by a sensitive 3H-protein A-binding assay and by external immunoprecipitation, with that of total PM-associated large T extracted from isolated PM. We demonstrate that in mKSA cells (SV40-transformed BALB/c mouse fibroblasts), total PM-associated large T accounted for a substantial portion (ca. 2%) of total cellular large T. However, only 0.1 to 0.2% of it could be detected on the cell surface. Thus, only a minor fraction of PM-associated large T (less than 10%) is exposed on the surface of these cells. Interior PM-associated large T is stably associated with the plasma membrane, while the small fraction of surface-exposed large T is rapidly released from the cell surface.
在感染猴病毒40（SV40）并被其转化的细胞中，大部分SV40大T抗原定位于细胞核内，而一小部分则特异性地与质膜（PM）结合并暴露于细胞表面。与质膜结合的大T似乎跨越脂质双层，但另一方面，它并不表现出跨膜蛋白的典型特征。为了进一步表征大T假定的跨膜方向，我们研究了所有与质膜结合的大T分子是否确实暴露于细胞表面。我们通过灵敏的3H-蛋白A结合试验和外部免疫沉淀法在活细胞上测定细胞表面暴露的大T的量，并将其与从分离的质膜中提取的与质膜结合的大T总量进行比较。我们证明，在mKSA细胞（SV40转化的BALB/c小鼠成纤维细胞）中，与质膜结合的大T总量占细胞总大T的很大一部分（约2%）。然而，在细胞表面仅能检测到其中的0.1%至0.2%。因此，在这些细胞表面，只有一小部分与质膜结合的大T（不到10%）暴露在外。质膜内部结合的大T与质膜稳定结合，而一小部分表面暴露的大T则迅速从细胞表面释放。
