Gh. Hansen et al., Cholesterol depletion of enterocytes - Effect on the Golgi complex and apical membrane trafficking, J BIOL CHEM, 275(7), 2000, pp. 5136-5142
Intestinal brush border enzymes, including aminopeptidase N and sucrase-iso
maltase, are associated with "rafts" (membrane microdomains rich in cholest
erol and sphingoglycolipids), To assess the functional role of rafts in the
present work, we studied the effect of cholesterol depletion on apical mem
brane trafficking in enterocytes, Cultured mucosal explants of pig small in
testine were treated for 2 h with the cholesterol sequestering agent methyl
-beta-cyclodextrin and lovastatin, an inhibitor of hydroxymethylglutaryl-co
enzyme A reductase, The treatment reduced the cholesterol content >50%. Mor
phologically, the Gels complex/trans-Golgi network was partially transforme
d into numerous 100-200 nm vesicles. By immunogold electron microscopy, ami
nopeptidase N was localized in these Golgi-derived vesicles as well as at t
he basolateral cell surface, indicating a partial missorting. Biochemically
, the rates of the Golgi-associated complex glycosylation and association w
ith rafts of newly synthesized aminopeptidase N were reduced, and less of t
he enzyme had reached the brush border membrane after 2 h of labeling. In c
ontrast, the basolateral Na+/K+-ATPase was neither missorted nor raft-assoc
iated. Our results implicate the Golgi complex/trans-Golgi network in raft
formation and suggest a close relationship between this event and apical me
mbrane trafficking.