title: The N and C termini of ZO-1 are surrounded by distinct proteins and functional protein networks.
abstract: BACKGROUND
  Biotin ligase tagging with ZO-1 was applied to identify a more complete tight junction proteome.
  
  
  RESULTS
  Identical but also different proteins and functional networks were identified near the N and C ends of ZO-1.
  
  
  CONCLUSIONS
  The ends of ZO-1 are embedded in different functional subcompartments of the tight junction.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratory of Tight Junction Structure and Function, NHLBI, National Institutes of Health, Bethesda, MD 20892, USA. Christina.vanitallie@nih.gov
authors.forename: Christina M
authors.initials: CM
authors.lastname: Van Itallie
authors.forename: Angel
authors.initials: A
authors.lastname: Aponte
authors.forename: Amber Jean
authors.initials: AJ
authors.lastname: Tietgens
authors.forename: Marjan
authors.initials: M
authors.lastname: Gucek
authors.forename: Karin
authors.initials: K
authors.lastname: Fredriksson
authors.forename: James Melvin
authors.initials: JM
authors.lastname: Anderson
mesh_terms: D022005:Adherens Junctions / Q000378:metabolism; D000818:Animals; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity; D004285:Dogs; D006801:Humans; D058977:Molecular Sequence Annotation; D025941:Protein Interaction Mapping; D060066:Protein Interaction Maps; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D062725:Tight Junction Proteins / Q000302:isolation & purification / Q000378:metabolism*; D019108:Tight Junctions / Q000378:metabolism; D062826:Zonula Occludens-1 Protein / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; C569042:TJP1 protein, human; D062725:Tight Junction Proteins; D062826:Zonula Occludens-1 Protein; D001710:Biotin; D019731:Carbon-Nitrogen Ligases
keywords: Adherens Junction; Mass Spectrometry (MS); Membrane; Proteomics; Tight Junctions; ZO-1
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