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 microg/ml for 12 h) reduced by 90% the cholesterol, but not the phospholipid (PL), levels of the basolateral membrane (BLM), thus altering its PL/cholesterol molar ratio from 2.6 to 22.0, and decreasing its steady state fluorescent anisotropy (rs) from 0.27 to 0.15. BFA treatment for 12 h also resulted in complete loss of transcobalamin II receptor (TC II-R) activity/protein levels in the BLM and the disappearance of trans-Golgi network (TGN) morphology as revealed by confocal immunofluorescence microscopy using antibody to TGN 38. However, BFA treatment had no effect on either total cellular cholesterol, TC II-R activity, or PL levels. When cells 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 II-R delivered to the BLM during this period was progressively 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 rs from 0.15 to 0.21. These results indicate that cholesterol, but not PL, delivery to the BLM of Caco-2 cells is BFA-sensitive, and cholesterol, by influencing the higher order of the BLM, is essential for TC II-R dimerization.
Bose
S
S
Division of Gastroenterology and Hepatology, Medical College of Wisconsin and Veterans Affairs Medical Center, Milwaukee, Wisconsin 53226, USA.
Chapin
S J
SJ
Seetharam
S
S
Feix
J
J
Mostov
K E
KE
Seetharam
B
B
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001485:Basement Membrane / Q000187:drug effects / Q000378:metabolism; D020126:Brefeldin A; D018938:Caco-2 Cells; D002784:Cholesterol / Q000378:metabolism*; D003517:Cyclopentanes / Q000494:pharmacology*; D019281:Dimerization; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism; D018942:Macrolides; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D003517:Cyclopentanes; D008051:Lipid Bilayers; D018942:Macrolides; D009844:Oligosaccharides; D011956:Receptors, Cell Surface; C028176:transcobalamin receptor; D020126:Brefeldin A; D002784:Cholesterol; D019158:N-Acetylneuraminic Acid