Transport of an external Lys-Asp-Glu-Leu (KDEL) protein from the plasma membrane to the endoplasmic reticulum: studies with cholera toxin in Vero cells.
Transport of an external Lys-Asp-Glu-Leu (KDEL) protein from the plasma membrane to the endoplasmic reticulum: studies with cholera toxin in Vero cells.
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DOI：
10.1083/jcb.133.4.777
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发表时间：
1996-05
期刊：
影响因子：
--
通讯作者：
Söling HD
中科院分区：
文献类型：
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作者：
Majoul IV;Bastiaens PI;Söling HD
霍乱毒素（cholera toxin，CTX）的A2链含有一个羧基端Lys-Asp- Glu-Leu（KDEL）序列。因此，我们分析了免疫荧光法和亚细胞分级法在Vero细胞中的CTX是否可以用来证明KDEL蛋白质从高尔基体到ER的逆行运输。免疫荧光研究显示，在用CTX脉冲处理后，CTX-A和B亚基（CTX-A和CTX-B）在15-20分钟后（30分钟后最大）达到高尔基体样结构。在30和90分钟之间，CTX-A（而不是CTX-B）出现在中间室和ER中，而CTX-B易位到溶酶体中。亚细胞分级分离研究证实了这些结果：CTX摄取15分钟后，CTX-A仅与内体和高尔基体室相关。30分钟后，少量CTX-A以胰蛋白酶抗性形式出现在ER中，60分钟后，出现大量CTX-A。CTX-A似乎主要以其氧化形式（CTX-A1-S-S-CTX-A2）从高尔基体转运至ER，在ER中，CTX-A缓慢还原形成游离的CTX-A1和CTX-A2，如在N-乙基马来酰亚胺存在下，在CTX摄取开始后30和90分钟将细胞均质化的实验所示。在CTX在高尔基体中积累后应用诺考达唑抑制CTX-A在ER中的出现并延迟3 '，5' cAMP的增加，表明微管参与了高尔基体-ER的逆行转运。
The A2 chain of cholera toxin (CTX) contains a COOH-terminal Lys-Asp- Glu-Leu (KDEL) sequence. We have, therefore, analyzed by immunofluorescence and by subcellular fractionation in Vero cells whether CTX can used to demonstrate a retrograde transport of KDEL proteins from the Golgi to the ER. Immunofluorescence studies reveal that after a pulse treatment with CTX, the CTX-A and B subunits (CTX-A and CTX-B) reach Golgi-like structures after 15-20 min (maximum after 30 min). Between 30 and 90 min, CTX-A (but not CTX-B) appear in the intermediate compartment and in the ER, whereas the CTX-B are translocated to the lysosomes. Subcellular fractionation studies confirm these results: after CTX uptake for 15 min, CTX-A is associated only with endosomal and Golgi compartments. After 30 min, a small amount of CTX-A appears in the ER in a trypsin-resistant form, and after 60 min, a significant amount appears. CTX-A seems to be transported mainly in its oxidized form (CTX-A1-S-S-CTX-A2) from the Golgi to the ER, where it becomes slowly reduced to form free CTX A1 and CTX-A2, as indicated by experiments in which cells were homogenized 30 and 90 min after the onset of CTX uptake in the presence of N- ethylmaleimide. Nocodazol applied after accumulation of CTX in Golgi inhibits the appearance of CTX-A in the ER and delays the increase of 3',5'cAMP, indicating the participation of microtubules in the retrograde Golgi-ER transport.