INTERNALIZED PLASMA-MEMBRANE CHOLESTEROL PASSES THROUGH AN ENDOSOME COMPARTMENT THAT IS DISTINCT FROM THE ACID VESICLE-LYSOSOME COMPARTMENT

Cholesterol from the plasma membrane of MA-10 Leydig tumor cells is in ternalized into the cell and either esterified or used as substrate fo r steroid hormone synthesis. In the present studies we show that chlor oquine and sphinganine cause LDL cholesterol and cholesteryl esters to accumulate in the cells. A lysosome fraction contained the excess cho lesterol and cholesteryl esters. Both inhibitors blocked the conversio n of plasma membrane cholesterol into intracellular cholesteryl esters and caused dose-dependent inhibition of dibutyryl-cAMP-stimulated pro gesterone synthesis. Radiolabeled cholesterol applied to the plasma me mbrane of MA-10 cells accumulated in the lysosome fraction of chloroqu ine and sphinganine-treated cells. Evidence that these inhibitors did not require the Golgi was provided by experiments using brefeldin A. E xperiments utilizing a fluorescent cholesterol analogue and a lysosoma l marker indicated that cholesterol entered the cells in structures th at were different than the acidic vesicle-lysosome compartment. Consis tent with this observation was the observation that the peak fluoresce nce fractions of cells subjected to density gradient centrifugation wa s of lower density than the lysosome fraction. (C) 1997 Academic Press .