The N- and C- termini of ZO-1 are surrounded by distinct proteins and functional protein networks.
ABSTRACT 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 which are within molecular dimensions of ZO-1 by fusing biotin ligase to either its N- or C-terminus, expressing these fusion proteins in MDCK epithelial cells and purifying and identifying the resulting biotinylated proteins by mass spectrometry. Out 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. 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 subcellular protein compartmentalization.
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ABSTRACT: Tight junctions are complex membrane structures that regulate paracellular movement of material across epithelia and play a role in cell polarity, signaling and cytoskeletal organization. In order to expand knowledge of the tight junction proteome, we used biotin ligase (BioID) fused to occludin and claudin-4 to biotinylate their proximal proteins in cultured MDCK II epithelial cells. We then purified the biotinylated proteins on streptavidin resin and identified them by mass spectrometry. Proteins were ranked by relative abundance of recovery by mass spectrometry, placed in functional categories, and compared not only among the N- and C- termini of occludin and the N-terminus of claudin-4, but also with our published inventory of proteins proximal to the adherens junction protein E-cadherin and the tight junction protein ZO-1. When proteomic results were analyzed, the relative distribution among functional categories was similar between occludin and claudin-4 proximal proteins. Apart from already known tight junction- proteins, occludin and claudin-4 proximal proteins were enriched in signaling and trafficking proteins, especially endocytic trafficking proteins. However there were significant differences in the specific proteins comprising the functional categories near each of the tagging proteins, revealing spatial compartmentalization within the junction complex. Taken together, these results expand the inventory of known and unknown proteins at the tight junction to inform future studies of the organization and physiology of this complex structure.PLoS ONE 01/2015; 10(3):e0117074. DOI:10.1371/journal.pone.0117074 · 3.53 Impact Factor
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ABSTRACT: The BioID proximity-based biotin labeling technique was recently developed for the characterization of protein–protein interaction networks . To date, this method has been applied to a number of different polypeptides expressed in cultured cells. Here we report the adaptation of BioID to the identification of protein–protein interactions surrounding the c-MYC oncoprotein in human cells grown both under standard culture conditions and in mice as tumor xenografts. Notably, in vivo BioID yielded > 100 high confidence MYC interacting proteins, including > 30 known binding partners. Putative novel MYC interactors include components of the STAGA/KAT5 and SWI/SNF chromatin remodeling complexes, DNA repair and replication factors, general transcription and elongation factors, and transcriptional co-regulators such as the DNA helicase protein chromodomain 8 (CHD8). Providing additional confidence in these findings, ENCODE ChIP-seq datasets highlight significant coincident binding throughout the genome for the MYC interactors identified here, and we validate the previously unreported MYC–CHD8 interaction using both a yeast two hybrid analysis and the proximity-based ligation assay. In sum, we demonstrate that BioID can be utilized to identify bona fide interacting partners for a chromatin-associated protein in vivo. This technique will allow for a much improved understanding of protein–protein interactions in a previously inaccessible biological setting.Journal of Proteomics 10/2014; 196. DOI:10.1016/j.jprot.2014.09.029 · 3.93 Impact Factor
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ABSTRACT: Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion.PLoS ONE 01/2015; 10(3):e0122886. DOI:10.1371/journal.pone.0122886 · 3.53 Impact Factor