Evidence for a functional interaction between cingulin and ZO-1 in cultured cells.
Cingulin, a protein component of the submembrane plaque of tight junctions (TJ), contains globular and coiled-coil domains and interacts in vitro with several TJ and cytoskeletal proteins, including the PDZ protein ZO-1. Overexpression of Xenopus cingulin in transfected Xenopus A6 cells resulted in the disruption of endogenous ZO-1 localization, suggesting that cingulin functionally interacts with ZO-1. Glutathione S-transferase pull-down experiments showed that a conserved ZO-1 interaction motif (ZIM) at the NH(2) terminus of cingulin is required for cingulin-ZO-1 interaction in vitro. An NH(2)-terminal region of cingulin, containing the ZIM, was sufficient, when fused to coiled-coil sequences, to target transfected cingulin to junctions. However, deletion of the ZIM did not abolish junctional localization of transfected cingulin in A6 cells, suggesting that cingulin can be recruited to TJ through multiple protein interactions. Interestingly, the ZIM was required for cingulin recruitment into ZO-1-containing adherens junctions of Rat-1 fibroblasts, indicating that cingulin junctional recruitment does not require the molecular context of TJ. Cingulin coiled-coil sequences enhanced the junctional accumulation of expressed cingulin head region in A6 cells, but purified recombinant cingulin did not form filaments under physiological conditions in vitro, suggesting that the cingulin coiled-coil domain acts primarily by promoting dimerization.
D'Atri
Fabio
F
Department of Molecular Biology, University of Geneva, 1211 Geneva 4, Switzerland.
Nadalutti
Fabio
F
Citi
Sandra
S
D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D000070559:Gizzard, Non-avian; D005982:Glutathione Transferase / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009218:Myosins / Q000302:isolation & purification / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection; D019459:Urothelium / Q000166:cytology / Q000502:physiology*; D014981:Xenopus; D029867:Xenopus Proteins; D062826:Zonula Occludens-1 Protein
C489228:CGN protein, Xenopus; D008565:Membrane Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C569045:TJP1 protein, Xenopus; C569044:Tjp1 protein, rat; D029867:Xenopus Proteins; D062826:Zonula Occludens-1 Protein; D005982:Glutathione Transferase; D009218:Myosins