Article

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

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 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.

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