DIRECT ASSOCIATION OF OCCLUDIN WITH ZO-1 AND ITS POSSIBLE INVOLVEMENTIN THE LOCALIZATION OF OCCLUDIN AT TIGHT JUNCTIONS

Occludin is an integral membrane protein localizing at tight junctions (TJ) with four transmembrane domains and a long COOH-terminal cytopla smic domain (domain E) consisting of 255 amino acids. Immunofluorescen ce and laser scan microscopy revealed that chick full-length occludin introduced into human and bovine epithelial cells was correctly delive red to and incorporated into preexisting TJ. Further transfection stud ies with various deletion mutants showed that the domain E, especially its COOH-terminal similar to 150 amino acids (domain E358/504), was n ecessary for the localization of occludin at TJ. Secondly, domain E wa s expressed in Escherichia coli as a fusion protein with glutathione-S -transferase, and this fusion protein was shown to be specifically bou nd to a complex of ZO-1 (220 kD) and ZO-2 (160 kD) among various membr ane peripheral proteins. In vitro binding analyses using glutathione-S -transferase fusion proteins of various deletion mutants of domain E n arrowed down the sequence necessary for the ZO-1/ZO-2 association into the domain E358/504. Furthermore, this region directly associated wit h the recombinant ZO-1 produced in E. coli. We concluded that occludin itself can localize at TJ and directly associate with ZO-1. The coinc idence of the sequence necessary for the ZO-1 association with that fo r the TJ localization suggests that the association with underlying cy toskeletons through ZO-1 is required for occludin to be localized at T J.