SV40 T-ANTIGEN AND THE EXOCYTOTIC PATHWAY
SV40 T-ANTIGEN AND THE EXOCYTOTIC PATHWAY
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DOI：
10.1002/j.1460-2075.1985.tb03806.x
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发表时间：
1985-01-01
期刊：
影响因子：
11.4
通讯作者：
SAMBROOK, J
中科院分区：
文献类型：
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作者：
SHARMA, S;RODGERS, L;SAMBROOK, J
将编码流感病毒血凝素15个氨基酸的信号肽和SV 40大T抗原C端694个氨基酸的嵌合基因插入牛乳头瘤病毒（BPV）表达载体，并导入NIH-3 T3细胞。获得表达高水平（约1000）的细胞系。5倍。106分子/细胞）的嵌合蛋白（HA-T抗原）。HA-T抗原的生化特性和细胞内定位与野生型T抗原进行了比较。野生型T抗原主要位于细胞核中，尽管在细胞表面上检测到一小部分。HA-T抗原仅存在于内质网（ER）中。在生物合成过程中，HA-T抗原跨ER的膜协同易位，信号肽被切割，富含甘露糖的寡糖连接到多肽上（T抗原在Asn 154处含有1个潜在的N-连接糖基化位点）。HA-T抗原不会变成末端糖基化或酰化，并且很少或没有抗原到达细胞表面。这些结果表明，T抗原不能被运输沿着胞吐途径。为了解释野生型T抗原在SV 40转化细胞表面的存在，提出了一种替代途径，涉及T抗原从细胞核转运到细胞表面。
A chimeric gene consisting of DNA coding for the 15-amino acid signal peptide of influenza virus hemagglutinin and the C-terminal 694 amino acids of SV40 large T antigen was inserted into a bovine papilloma virus (BPV) expression vector and introduced into NIH-3T3 cells. Cell lines were obtained that express high levels (.apprx. 5 .times. 106 molecules/cell) of the chimeric protein (HA-T antigen). The biochemical properties and intracellular localization of HA-T antigens were compared with those of wild-type T antigen. Wild-type T antigen is located chiefly in the cell nucleus, although a small fraction is detected on the cell surface. HA-T antigen is found exclusively in the endoplasmic reticulum (ER). During biosynthesis, HA-T antigen is co-translationally translocated across the membrane of the ER, the signal peptide is cleaved and a mannose-rich oligosaccharide is attached to the polypeptide (T antigen contains 1 potential N-linked glycosylation site at Asn154). HA-T antigen does not become terminally glycosylated or acylated and little or none reaches the cell surface. These results suggest that T antigen is incapable of being transported along the exocytotic pathway. To explain the presence of wild-type T antigen on the surface of SV40-transformed cells, an alternative route is proposed involving transport of T antigen from the nucleus to the cell surface.