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.