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Abstract:
Differential centrifugation of Triton X-100 or CHAPS lysates from control and cholesterol
(CH)-depleted MDCK II cells, segregated integral tight junction (TJ) proteins associated with
detergent-resistant membranes (DRMs) into two groups. Group A proteins (occludin,
claudin-2 and -3) were detected in large, intermediate and small aggregates in both
detergents, whereas group B proteins (claudin-1, -4 and -7) were observed in small
aggregates in TX-100 and in intermediate and small aggregates in CHAPS. Depletion of CH
altered the distribution of group A and B proteins among the three size categories in a
detergent-specific manner. In lysates produced with octyl glucoside, a detergent that
selectively extracts proteins from DRMs, group A proteins were undetectable in large
aggregates and CH depletion did not alter the distribution of either group A or B proteins in
intermediate or small aggregates. Neither occludin (group A) nor claudin-1 (group B) was in
intimate enough contact with CH to be cross-linked to [3H]-photo-cholesterol. However,
antibodies to either TJ protein co-immunoprecipitated caveolin-1, a CH-binding protein.
Unlike claudins, occludin's presence in TJs and DRMs did not require palmitoylation.
Equilibrium density centrifugation on discontinuous OptiPrep gradients revealed detergentrelated
differences in the densities of TJ-bearing DRMs. There was little or no change in
those densities after CH depletion. Removing CH from the plasma membrane increased
tyrosine and threonine phosphorylation of occludin, and transepithelial electrical
resistance (TER) within 30 min. After 2 h of CH efflux, phospho-occludin levels and TER
fell below control values. We conclude that the association of integral TJ proteins with
DRMS, pelleted at low speeds, is partially CH-dependent. However, the buoyant density of
TJ-associated DRMs is a function of the detergent used and is insensitive to decreases in CH.
