The N and C Termini of ZO-1 Are Surrounded by Distinct Proteins and Functional Protein Networks

NIH, United States.
Journal of Biological Chemistry (Impact Factor: 4.57). 04/2013; 288(19). DOI: 10.1074/jbc.M113.466193
Source: PubMed


The proteins and functional protein networks of the tight junction remain incompletely defined. Among the currently known
proteins are barrier-forming proteins like occludin and the claudin family; scaffolding proteins like ZO-1; and some cytoskeletal,
signaling, and cell polarity proteins. To define a more complete list of proteins and infer their functional implications,
we identified the proteins that are within molecular dimensions of ZO-1 by fusing biotin ligase to either its N or C terminus,
expressing these fusion proteins in Madin-Darby canine kidney epithelial cells, and purifying and identifying the resulting
biotinylated proteins by mass spectrometry. Of a predicted proteome of ∼9000, we identified more than 400 proteins tagged
by biotin ligase fused to ZO-1, with both identical and distinct proteins near the N- and C-terminal ends. Those proximal
to the N terminus were enriched in transmembrane tight junction proteins, and those proximal to the C terminus were enriched
in cytoskeletal proteins. We also identified many unexpected but easily rationalized proteins and verified partial colocalization
of three of these proteins with ZO-1 as examples. In addition, functional networks of interacting proteins were tagged, such
as the basolateral but not apical polarity network. These results provide a rich inventory of proteins and potential novel
insights into functions and protein networks that should catalyze further understanding of tight junction biology. Unexpectedly,
the technique demonstrates high spatial resolution, which could be generally applied to defining other subcellular protein

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    • "This may contribute to formation of the distinct lipid microdomains known to exist at the tight junction (Nusrat et al., 2000). Finally, the identification of several other BAR domain–containing proteins as proximal to tight junctions, including BIN3 (Van Itallie et al., 2013), pacsin2, and IRSp53 (Fredriksson et al., 2015), suggests that there may be common themes in barrier regulation to be uncovered. "
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    ABSTRACT: Assembly and sealing of the tight junction barrier is critically dependent on the perijunctional actin cytoskeleton yet little is known about physical and functional links between barrier-forming proteins and actin. Here we identify a novel functional complex of the junction scaffolding protein ZO-1 and the F-BAR domain protein TOCA-1. Using MDCK epithelial cells, we show that an alternative splice of TOCA-1 adds a PDZ-binding motif which binds ZO-1, targeting TOCA-1 to barrier contacts. This isoform of TOCA-1 recruits the actin nucleation promoting factor N-WASP to tight junctions. CRISPR-mediated knockout of TOCA-1 results in increased paracellular flux and delayed recovery in a calcium switch assay. Knockout of TOCA-1 does not alter FRAP kinetics of GFP ZO-1 or occludin but longer term (12h) time lapse microscopy reveals strikingly decreased tight junction membrane contact dynamics in knockout cells compared with controls. Re-expression of TOCA-1 with, but not without, the PDZ binding motif rescues both altered flux and membrane contact dynamics. Ultrastructural analysis shows actin accumulation at the adherens junction in TOCA-1 knockout cells but unaltered freeze-fracture fibril morphology. Identification of the ZO-1/TOCA-1 complex provides novel insights into the underappreciated dependence of the barrier on the dynamic nature of cell-to-cell contacts and perijunctional actin. © 2015 by The American Society for Cell Biology.
    Preview · Article · Jun 2015 · Molecular biology of the cell
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    ABSTRACT: BioID is a unique method to screen for physiologically relevant protein interactions that occur in living cells. This technique harnesses a promiscuous biotin ligase to biotinylate proteins based on proximity. The ligase is fused to a protein of interest and expressed in cells, where it biotinylates proximal endogenous proteins. Because it is a rare protein modification in nature, biotinylation of these endogenous proteins by BioID fusion proteins enables their selective isolation and identification with standard biotin-affinity capture. Proteins identified by BioID are candidate interactors for the protein of interest. BioID can be applied to insoluble proteins, can identify weak and/or transient interactions, and is amenable to temporal regulation. Initially applied to mammalian cells, BioID has potential application in a variety of cell types from diverse species. Curr. Protoc. Protein Sci. 74:19.23.1-19.23.14. © 2013 by John Wiley & Sons, Inc.
    Full-text · Article · Nov 2013 · Current protocols in protein science / editorial board, John E. Coligan ... [et al.]
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    ABSTRACT: The tight junction is a multi-protein complex and is the apical most junctional complex in certain epithelial and endothelial cells. A great deal of attention has been devoted to the understanding of these proteins in contributing to the barrier function - that is, regulating the paracellular flux or permeability between adjacent cells. However, tight junction proteins are now recognized as having functions beyond the barrier. The focus of this review is to discuss the barrier function of the tight junction and to summarize the literature with a focus on the role of tight junction proteins in proliferation, transformation, and metastasis.
    No preview · Article · Jun 2013 · Cancer letters
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