OCCLUDIN - A NOVEL INTEGRAL MEMBRANE-PROTEIN LOCALIZING AT TIGHT JUNCTIONS

Recently, we found that ZO-1, a tight junction-associated protein, was concentrated in the so called isolated adherens junction fraction fro m the liver (Itoh, M., A. Nagafuchi, S. Yonemura, T. Kitani-Yasuda, Sa . Tsukita, and Sh. Tsukita. 1993. J. Cell Biol. 121:491-502). Using th is fraction derived from chick liver as an antigen, we obtained three monoclonal antibodies specific for a approximately 65-kD protein in ra ts. This antigen was not extractable from plasma membranes without det ergent, suggesting that it is an integral membrane protein. Immunofluo rescence and immunoelectron microscopy with these mAbs showed that thi s approximately 65-kD membrane protein was exclusively localized at ti ght junctions of both epithelial and endothelial cells: at the electro n microscopic level, the labels were detected directly over the points of membrane contact in tight junctions. To further clarify the nature and structure of this membrane protein, we cloned and sequenced its c DNA. We found that the cDNA encoded a 504-amino acid polypeptide with 55.9 kD. A search of the data base identified no proteins with signifi cant homology to this membrane protein. A most striking feature of its primary structure was revealed by a hydrophilicity plot: four putativ e membrane-spanning segments were included in the NH2-terminal half. T his hydrophilicity plot was very similar to that of connexin, an integ ral membrane protein in gap junctions. These findings revealed that an integral membrane protein localizing at tight junctions is now identi fied, which we designated as ''occludin.''