BREFELDIN-A (BFA) INHIBITS BASOLATERAL MEMBRANE (BLM) DELIVERY AND DIMERIZATION OF TRANSCOBALAMIN-II RECEPTOR IN HUMAN INTESTINAL EPITHELIAL CACO-2 CELLS - BFA EFFECTS ON BLM CHOLESTEROL CONTENT

Brefeldin A (BFA) treatment of Caco-2 cells (5 mu g/ml for 12 h) reduc ed by 90% the cholesterol, but not the phospholipid (PL), levels of th e basolateral membrane (BLM), thus altering its PL/cholesterol molar r atio from 2.6 to 22.0, and decreasing its steady state fluorescent ani sotropy (r(s)) from 0.27 to 0.15. BFA treatment for 12 h also resulted in complete loss of transcobalamin II receptor (TC II-R) activity/pro tein levels in the BLM and the disappearance of trans-Golgi network (T GN) morphology as revealed by confocal immunofluorescence microscopy u sing antibody to TGN 38. However, BFA treatment had no effect on eithe r total cellular cholesterol, TC II-R activity, or PL levels. When cel ls treated with BFA for 12 h were exposed to BFA-free medium for 0-24 h, all of the effects were reversed, including reappearance of normal TGN morphology. TC H-R delivered to the BLM. during this period was pr ogressively sialylated and changed its physical state from a monomer ( 8 h) to a dimer (12 h), coinciding with increased delivery (11-53 pmol ) of cholesterol to the BLM and an increase in the BLM r(s) from 0.15 to 0.21. These results indicate that cholesterol, but not FL, delivery to the BLM of Caco-2 cells is BFA-sensitive, and cholesterol, by infl uencing the higher order of the BLM, is essential for TC II-R dimeriza tion.