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 internalized into the cell and either esterified or used as substrate for steroid hormone synthesis. In the present studies we show that chloroquine and sphinganine cause LDL cholesterol and cholesteryl esters to accumulate in the cells. A lysosome fraction contained the excess cholesterol and cholesteryl esters. Both inhibitors blocked the conversion of plasma membrane cholesterol into intracellular cholesteryl esters and caused dose-dependent inhibition of dibutyryl-cAMP-stimulated progesterone synthesis. Radiolabeled cholesterol applied to the plasma membrane of MA-10 cells accumulated in the lysosome fraction of chloroquine and sphinganine-treated cells. Evidence that these inhibitors did not require the Golgi was provided by experiments using brefeldin A. Experiments utilizing a fluorescent cholesterol analogue and a lysosomal marker indicated that cholesterol entered the cells in structures that were different than the acidic vesicle-lysosome compartment. Consistent with this observation was the observation that the peak fluorescence fractions of cells subjected to density gradient centrifugation was of lower density than the lysosome fraction.
Porpaczy
Z
Z
Department of Internal Medicine, University of Oklahoma Health Sciences Center, Oklahoma City 73190, USA.
Tomasek
J J
JJ
Freeman
D A
DA
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D020126:Brefeldin A; D003994:Bucladesine / Q000494:pharmacology; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism*; D002738:Chloroquine / Q000494:pharmacology; D002784:Cholesterol / Q000031:analogs & derivatives / Q000378:metabolism*; D002788:Cholesterol Esters / Q000378:metabolism; D008078:Cholesterol, LDL / Q000378:metabolism; D003517:Cyclopentanes / Q000494:pharmacology; D011992:Endosomes / Q000378:metabolism*; D005456:Fluorescent Dyes; D006056:Golgi Apparatus; D007984:Leydig Cell Tumor / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D051379:Mice; D011374:Progesterone / Q000096:biosynthesis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology; D014407:Tumor Cells, Cultured
C077527:7-nitrobenz-2-oxa-1,3-diazol-4-ylcholesterol; D002788:Cholesterol Esters; D008078:Cholesterol, LDL; D003517:Cyclopentanes; D005456:Fluorescent Dyes; D011500:Protein Synthesis Inhibitors; D020126:Brefeldin A; D011374:Progesterone; D003994:Bucladesine; D002738:Chloroquine; D002784:Cholesterol; D009327:4-Chloro-7-nitrobenzofurazan; D013110:Sphingosine; C005682:safingol