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© The Rockefeller University Press, 0021-9525/1998//199 $5.00
The Journal of Cell Biology, Volume 141, Number 1, , 1998 199-208

Regular Articles

ZO-3, a Novel Member of the MAGUK Protein Family Found at the Tight Junction, Interacts with ZO-1 and Occludin



Julie Haskins, Lijie Gu, Erika S. Wittchen, Jennifer Hibbard, and Bruce R. Stevenson

Department of Cell Biology and Anatomy, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

A 130-kD protein that coimmunoprecipitates with the tight junction protein ZO-1 was bulk purified from Madin-Darby canine kidney (MDCK) cells and subjected to partial endopeptidase digestion and amino acid sequencing. A resulting 19–amino acid sequence provided the basis for screening canine cDNA libraries. Five overlapping clones contained a single open reading frame of 2,694 bp coding for a protein of 898 amino acids with a predicted molecular mass of 98,414 daltons. Sequence analysis showed that this protein contains three PSD-95/SAP90, discs-large, ZO-1 (PDZ) domains, a src homology (SH3) domain, and a region similar to guanylate kinase, making it homologous to ZO-1, ZO-2, the discs large tumor suppressor gene product of Drosophila, and other members of the MAGUK family of proteins. Like ZO-1 and ZO-2, the novel protein contains a COOH-terminal acidic domain and a basic region between the first and second PDZ domains. Unlike ZO-1 and ZO-2, this protein displays a proline-rich region between PDZ2 and PDZ3 and apparently contains no alternatively spliced domain. MDCK cells stably transfected with an epitope-tagged construct expressed the exogenous polypeptide at an apparent molecular mass of ~130 kD. Moreover, this protein colocalized with ZO-1 at tight junctions by immunofluorescence and immunoelectron microscopy. In vitro affinity analyses demonstrated that recombinant 130-kD protein directly interacts with ZO-1 and the cytoplasmic domain of occludin, but not with ZO-2. We propose that this protein be named ZO-3.

Abbreviations used in this paper: aa, amino acid; GUK, guanylate kinase; PVDF, polyvinylidene difluoride; SH3, src homology; VSV-G, vesicular stomatitis virus G protein.

The Rockefeller University Protein Sequencing Facility is supported in part by National Institutes of Health shared instrumentation grants and by funds provided by the US Army and Navy for purchase of equipment. This work was supported by operating grants to B.R. Stevenson from the Medical Research Council of Canada and the Kidney Foundation of Canada. B.R. Stevenson is a Senior Scholar of the Alberta Heritage Foundation for Medical Research.


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