Rapid intracellular transport of LDL-derived cholesterol to the plasma membrane in cultured fibroblasts

The kinetics of low density lipoprotein (LDL) cholesterol transport to the plasma membrane of Chinese hamster ovary (CHO) cells was studied. LDL was reconstituted with 3H]cholesteryl linoleate and added to CHO cells in a pulse-chase experiment. The internalization and lysosomal cleavage of reconstituted LDL (rLDL) 3H]cholesteryl linoleate to free 3H]cholesterol occurred with a half-time of 37 min after a 30-min lag. The rate of transport of released 3H]cholesterol to the plasma membrane was measured by brief (20-30 sec) cholesterol oxidase treatment of intact, adherent cells: the half-time of transport was 42 min. The similarity in the rate of free cholesterol release from rLDL and transport of this cholesterol to the plasma membrane suggests very rapid transport of rLDL cholesterol from the lysosome to the plasma membrane. Cells were shown to be intact throughout the cholesterol oxidase treatment by the absence of cell-derived lactate dehydrogenase (LDH) activity or K+ in the assay buffer.