Dissecting the role of the Golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface

In this study, we compared the transport of newly synthesized cholesterol w ith that of influenza virus hemagglutinin (HA) from the endoplasmic reticul um to the plasma membrane. The arrival of cholesterol on the cell surface w as monitored by cyclodextrin removal, and HA transport was monitored by sur face trypsinization and endoglycosidase H digestion. We found that disassem bly of the Golgi complex by brefeldin A treatment resulted in partial inhib ition of cholesterol transport while completely blocking HA transport. Furt her, microtubule depolymerization by nocodazole inhibited cholesterol and H A transport to a similar extent. When the partitioning of cholesterol into lipid rafts was analyzed, we found that newly synthesized cholesterol began to associate with low-density detergent-resistant membranes rapidly after synthesis, before it was detectable on the cell surface, and its raft assoc iation increased further upon chasing. When cholesterol transport was block ed by using 15 degrees C incubation, the association of newly synthesized c holesterol with low-density detergent-insoluble membranes was decreased and cholesterol accumulated in a fraction with intermediate density. Our resul ts provide evidence for the partial contribution of the Golgi complex to th e transport of newly synthesized cholesterol to the cell surface and sugges t that detergent-resistant membranes are involved in the process.