Description:
<jats:p>Zonula occludens (ZO‐1) is a cytoplasmic scaffold protein that structurally and functionally tethers cytoskeletal components and membrane proteins at the tight junction. Interestingly, ZO‐1 has a poorly characterized ZU5 domain at the carboxy‐terminus that ZO‐2 and ZO‐3 proteins lack. The goal of this study is to examine ZO‐1‐ZU‐5 function in a structurally defined manner. To determine ZO‐1‐ZU5 domain function, EGFP‐ZO‐1, EGFP‐ZO‐1ΔZU5 and EGFP‐ZO‐1 constructs mutated within the ZU5 domain hydrophobic pocket were engineered and stably transfected into MDCK II cells. FRAP analyses show that EGFP‐ZO‐1ΔZU5 rapidly recovers and the mobile fraction (M<jats:sub>f</jats:sub>) is elevated compared to EGFP‐ZO‐1. The M<jats:sub>f</jats:sub> increases by 30% in EGFP‐ZO‐1ΔZU5 as a 50% decrease in t<jats:sub>1/2</jats:sub> is observed. Disruption of the ZU5‐hydrophobic pocket caused similar changes suggesting the ZU5 domain participates in stabilizing ZO‐1 at the tight junction. Macromolecular flux studies demonstrated that ZO‐1ΔZU5 expression enhances leak pathway permeability compared to EGFP‐ZO‐1. These data indicate that the ZU5 domain stabilizes ZO‐1 at the tight junction and contributes to development of the barrier to paracellular flux of small solutes.</jats:p>