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J. Cell Biol.,
Volume 141, Number 1, April 6, 1998 199-208
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.
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