Direct association of occludin with ZO-1 and its possible involvement in 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 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.
Furuse
M
M
Department of Information Physiology, National Institute for Physiological Sciences, Okazaki, Aichi, Japan.
Itoh
M
M
Hirase
T
T
Nagafuchi
A
A
Yonemura
S
S
Tsukita
S
S
Tsukita
S
S
D000818:Animals; D002451:Cell Compartmentation; D016764:Cell Polarity; D002478:Cells, Cultured; D002645:Chickens; D003593:Cytoplasm / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D007365:Intercellular Junctions / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D062793:Occludin; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein
D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C569042:TJP1 protein, human; C569046:TJP2 protein, human; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein