Intracellular Transport of Low Density Lipoprotein Derived Free Cholesterol Begins at Clathrin-Coated Pits and Terminates at Cell Surface Caveolae

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Abstract
Free cholesterol (FC) is selectively internalized from low-density lipoprotein (LDL) by confluent fibroblast monolayers (Fielding & Fielding (1995) Biochemistry 34, 14237−14244). The kinetics of transport of LDL-derived 3H-FC within the cell were studied by density-gradient ultracentrifugal fractionation and in terms of the effects of inhibitors of endocytosis and intracellular transport. By these criteria, the initial uptake of LDL-FC was mediated by the cell-surface clathrin-coated pits. FC label then appeared in clathrin-coated dense vesicles. Uncoating of clathrin from these vesicles led to the appearance of label in a light density fraction and, subsequently, in an intermediate density fraction coincident with protein markers of the trans-Golgi network in these cells. 3H-FC was finally transported to the plasma membrane via a temperature-sensitive, probably microtubule-dependent pathway. These data are consistent with a role for the trans-Golgi network as an intermediate compartment in intracellular FC transport. They provide further evidence of a role for cell-surface caveolae in FC efflux.