CELL-FREE TRANSFER OF CHOLESTEROL FROM LYSOSOMES TO PHOSPHOLIPID-VESICLES

The objective of this work was to develop a cell-free system for study ing the transfer of cholesterol from lysosomes to membrane acceptor pa rticles. The methods involved: 1) loading of CHO cells at 15 degrees C with [H-3]cholesteryl oleate-reconstituted LDL, such that it accumula ted undegraded in endosomes; 2) homogenization of cells, followed by p reparation of an endosome-lysosome donor fraction; 3) incubation of th e donor fraction at 37 degrees C in a defined cytosol-like medium cont aining acceptor particles of egg phosphatidylcholine small unilamellar vesicles (PC-SUV); and 4) measurement of cholesteryl oleate (GO) hydr olysis and transfer of the resulting free cholesterol (FC) to vesicles . During cell-free incubation LDL-loaded endosomes fused with lysosome s leading to the lysosomal hydrolysis of LDL cholesteryl ester. Maxima l hydrolysis of approximately 50% was achieved in 4-8 h. This hydrolys is was inhibited by lysosomotropic agents, proton ionophores, or remov al of ATP and GTP from the medium, indicating that it took place in se aled lysosomes. In the absence of PC-SUV, the release of LDL-derived F C from lysosomes was less than or equal to 10%/8 h. This was increased to a maximum of 25-30%/8 h at 3 mg/ml of PC-SUV. In contrast, the rel ease of undegraded CO was 5-15%/8 h and not stimulated by PC-SUV, sugg esting that the transfer of FC to PC-SUV was selective and not due to the uncontrolled release of lysosomal contents. In comparisons between CHO-K1 cells and sterol transport-defective CHO(2-2) cells, lysosomes from the latter cell were 35% less efficient as donors of cholesterol for transfer to egg phosphatidylcholine small unilamellar vesicles, i ndicating that these methods reproduce an important aspect of sterol t rafficking in cells. In addition, this result suggests thai the mutati on in CHO(2-2) has a direct effect on the lysosomes of these cells.