Abstract
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.
Highlights
Biotin ligase tagging with ZO-1 was applied to identify a more complete tight junction proteome
Of a predicted proteome of ϳ9000, 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
With the caveat that this method is dependent on both the presence of and access to lysines on target proteins, our results suggest that the use of biotin ligase tagging from ZO-1 provides a more complete set of functionally relevant proteins and insights into potentially pertinent networks than previous methods
Summary
Biotin ligase tagging with ZO-1 was applied to identify a more complete tight junction proteome. With the goal of identifying a more complete set of tight junction-associated proteins and to begin to define junctionassociated protein networks, we took advantage of a recently published technique [12] to identify proximal proteins in living cells In this method, a biotin ligase engineered to have lowered substrate specificity is fused to a protein of interest and expressed in cells. With the caveat that this method is dependent on both the presence of and access to lysines on target proteins, our results suggest that the use of biotin ligase tagging from ZO-1 provides a more complete set of functionally relevant proteins and insights into potentially pertinent networks than previous methods Proteins identified by this method include many recognized tight junction and adherens junction proteins. Along with the expected tight junction proteins, we identify a number of proteins that might not have been predicted based on current understanding of the tight junction; study of these proteins may provide new insights into the role of ZO-1 and functions of the tight junction
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
