Occludin localization at the tight junction requires the second extracellular loop

Occludin is a transmembrane protein of the tight junction with two extracel lular loops. Our previous demonstration that the extracellular loops are ad hesive suggested the possibility that they contribute to localizing occludi n at the tight junction. To address this question, truncated forms of occlu din were generated in which one or both of the extracellular loops were del eted. These constructs were expressed in both occludin-null Rat-1 fibroblas ts and in MDCK epithelial cells. The patterns of sensitivity to proteinase K suggested all constructs were present on the plasma membrane and retained the normal topology. In fibroblasts, all truncated forms of occludin coloc alized with ZO-1 at regions of cell-cell contact, demonstrating that even i n the absence of tight junctions cytoplasmic interactions with ZOs is suffi cient to cluster occludin. In MDCK cell monolayers, both full-length and oc cludin lacking the first extracellular loop colocalized with ZO-1 at the ti ght junction. In contrast, constructs lacking the second, or both, extracel lular loops were absent from tight junctions and were found only on the bas olateral cell surface. By freeze-fracture electron microscopic analysis, ov erexpression of full length occludin induced side-to-side aggregation of fi brils within the junction, while excess occludin on the lateral membrane di d not form fibrils. These results suggest that the second extracellular dom ain is required for stable assembly of occludin in the tight junction and t hat occludin influences the structural organization of the paracellular bar rier.