title: Proteomic analysis of proteins surrounding occludin and claudin-4 reveals their proximity to signaling and trafficking networks.
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.
authors: Laboratory of Tight Junction Structure and Function, NHLBI, National Institutes of Health, Bethesda, MD, United States of America.
authors: Karin
authors: K
authors: Fredriksson
authors: Laboratory of Tight Junction Structure and Function, NHLBI, National Institutes of Health, Bethesda, MD, United States of America.
authors: Christina M
authors: CM
authors: Van Itallie
authors: Proteomics Core Facility, NHLBI, National Institutes of Health, Bethesda, MD, United States of America.
authors: Angel
authors: A
authors: Aponte
authors: Proteomics Core Facility, NHLBI, National Institutes of Health, Bethesda, MD, United States of America.
authors: Marjan
authors: M
authors: Gucek
authors: Laboratory of Tight Junction Structure and Function, NHLBI, National Institutes of Health, Bethesda, MD, United States of America.
authors: Amber J
authors: AJ
authors: Tietgens
authors: Laboratory of Tight Junction Structure and Function, NHLBI, National Institutes of Health, Bethesda, MD, United States of America.
authors: James M
authors: JM
authors: Anderson
mesh_terms: D000818:Animals; D001692:Biological Transport; D062506:Claudin-4 / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D006801:Humans; D013058:Mass Spectrometry; D062793:Occludin / Q000235:genetics / Q000378:metabolism*; D025941:Protein Interaction Mapping; D040901:Proteomics; D015398:Signal Transduction; D019108:Tight Junctions / Q000378:metabolism*; D019076:Transgenes; D022161:Transport Vesicles
chemical_list: C568157:CLDN4 protein, human; D062506:Claudin-4; C568985:OCLN protein, human; D062793:Occludin