The Tight Junction Protein ZO-1 Establishes a Link between the Transmembrane Protein Occludin and the Actin Cytoskeleton*
- From the Departments of ‡Internal Medicine and‖Cell Biology, Yale School of Medicine, New Haven, Connecticut 06510 and the ¶Department of Plant and Microbial Biology, University of California, Berkeley, California 94720
Abstract
The tight junction protein ZO-1 belongs to a family of multidomain proteins known as the membrane-associated guanylate kinase homologs (MAGUKs). ZO-1 has been demonstrated to interact with the transmembrane protein occludin, a second tight junction-specific MAGUK, ZO-2, and F-actin, although the nature and functional significance of these interactions is poorly understood. To further elucidate the role of ZO-1 within the epithelial tight junction, we have introduced epitope-tagged fragments of ZO-1 into cultured MDCK cells and identified domains critical for the interaction with ZO-2, occludin, and F-actin. A combination of in vitroand in vivo binding assays indicate that both ZO-2 and occludin interact with specific domains within the N-terminal (MAGUK-like) half of ZO-1, whereas the unique proline-rich C-terminal half of ZO-1 cosediments with F-actin. Consistent with these observations, we found that a construct encoding the N-terminal half of ZO-1 is specifically associated with tight junctions, whereas the unique C-terminal half of ZO-1 is distributed over the entire lateral surface of the plasma membrane and other actin-rich structures. In addition, we have identified a 244-amino acid domain within the N-terminal half of ZO-1, which is required for the stable incorporation of ZO-1 into the junctional complex of polarized MDCK cells. These observations suggest that one functional role of ZO-1 is to organize components of the tight junction and link them to the cortical actin cytoskeleton.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grants DK45134 and CA66263 (to J. M. A.) and Grant DK34989 to the Yale Liver Center.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ Supported by National Institutes of Health Grant NRSA DK09261 and the Irwin M. Arias Postdoctoral Research Fellowship from the American Liver Foundation. To whom all correspondence should be addressed: Dept. of Internal Medicine, Digestive Diseases, Yale School of Medicine, 333 Cedar St., P. O. Box 208019, New Haven, CT 06520-8019. Tel.: 203-785-4133; Fax: 203-785-7273; E-mail:alan.fanning{at}yale.edu.
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↵2 L. L. Mitic, E. S. Schneeberger, A. S. Fanning, and J. M. Anderson, submitted for publication.
- Abbreviations:
- aa
-
amino acid(s)
- GST
-
glutathione S-transferase
- MAGUK
-
membrane-associated guanylate kinase homolog
- SH3
-
Src homology 3
- MDCK
-
Madin-Darby canine kidney
- PBS
-
phosphate-buffered saline
- PAGE
-
polyacrylamide gel electrophoresis
- PDZ
-
PSD95/dlg/ZO-1.
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- Received July 28, 1998.
- Revision received September 1, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
