Biotin ligase tagging with ZO-1 was applied to identify a more complete tight junction proteome.
Identical but also different proteins and functional networks were identified near the N and C ends of ZO-1.
The ends of ZO-1 are embedded in different functional subcompartments of the tight junction.
Biotin tagging with ZO-1 expands the tight junction proteome and defines subcompartments of the junction. 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 compartmentalization.
"BioID analysis: a powerful new tool to investigate host–pathogen interactions BioID is a powerful new method that detects close physical proximity between proteins in living cells at specific times chosen by the investigator (Roux et al., 2012; Van Itallie et al., 2013). Our BioID results provided compelling confirmation that SINC associates with the INM, by demonstrating SINC proximity to lamin B1 and specific INM proteins, including emerin, MAN1, LAP1, and LBR, as discussed later. "
[Show abstract][Hide abstract] 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.
Cancer letters 06/2013; 337(1). DOI:10.1016/j.canlet.2013.05.038 · 5.62 Impact Factor
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