Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions.
Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions.
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DOI：
10.1083/jcb.127.6.1617
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发表时间：
1994-12
期刊：
影响因子：
--
通讯作者：
Tsukita S
中科院分区：
文献类型：
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作者：
Furuse M;Itoh M;Hirase T;Nagafuchi A;Yonemura S;Tsukita S;Tsukita S
Occludin是一种位于紧密连接（TJ）处的完整膜蛋白，具有四个跨膜结构域和一个由255个氨基酸组成的长COO末端胞质结构域（结构域E）。免疫荧光和激光扫描显微镜显示，鸡全长occludin引入到人类和牛上皮细胞被正确地传递到并纳入到预先存在的TJ。用各种缺失突变体进行的进一步转染研究表明，结构域E，特别是其COOH-末端约150个氨基酸（结构域E358/504），对于闭合蛋白在TJ的定位是必需的。第二，结构域E在大肠杆菌中作为与谷胱甘肽-S-转移酶的融合蛋白表达，并且该融合蛋白显示出与多种膜外周蛋白中的ZO-1（220 kD）和ZO-2（160 kD）的复合物特异性结合。在体外结合分析，使用谷胱甘肽-S-转移酶融合蛋白的各种缺失突变体的结构域E缩小了必要的ZO-1/ZO-2协会到结构域E358/504的序列。此外，该区域与在E.杆菌我们的结论是，occludin本身可以定位在TJ和直接与ZO-1。ZO-1与TJ定位所必需的序列的重合表明，通过ZO-1与潜在的细胞骨架的关联是闭合蛋白定位于TJ所必需的。
Occludin is an integral membrane protein localizing at tight junctions (TJ) with four transmembrane domains and a long COOH-terminal cytoplasmic domain (domain E) consisting of 255 amino acids. Immunofluorescence and laser scan microscopy revealed that chick full- length occludin introduced into human and bovine epithelial cells was correctly delivered to and incorporated into preexisting TJ. Further transfection studies with various deletion mutants showed that the domain E, especially its COOH-terminal approximately 150 amino acids (domain E358/504), was necessary for the localization of occludin at TJ. Secondly, domain E was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and this fusion protein was shown to be specifically bound to a complex of ZO-1 (220 kD) and ZO-2 (160 kD) among various membrane peripheral proteins. In vitro binding analyses using glutathione-S-transferase fusion proteins of various deletion mutants of domain E narrowed down the sequence necessary for the ZO-1/ZO-2 association into the domain E358/504. Furthermore, this region directly associated with the recombinant ZO-1 produced in E. coli. We concluded that occludin itself can localize at TJ and directly associate with ZO-1. The coincidence of the sequence necessary for the ZO-1 association with that for the TJ localization suggests that the association with underlying cytoskeletons through ZO-1 is required for occludin to be localized at TJ.