Lysosomal membrane cholesterol dynamics

Although the majority of exogenous cholesterol and cholesterol ester enters the cell by LDL-receptor-mediated endocytosis and the lysosomal pathway, t he assumption that cholesterol transfers out of the lysosome by rapid (minu tes), spontaneous diffusion has heretofore not been tested. As shown herein , lysosomal membranes were unique among known organellar membranes in terms of cholesterol content, cholesterol dynamics, and response to cholesterol- mobilizing proteins. First, the lysosomal membrane cholesterol:phospholipid molar ratio, 0.38, was intermediate between those of the plasma membrane a nd other organellar membranes. Second, a fluorescence sterol exchange assay showed that the initial rate of spontaneous sterol transfer out of lysosom es and purified lysosomal membranes was extremely slow, tilt > 4 days. This was > 100-fold longer than that reported in intact cells (2 min) and 40-60 -fold longer than from any other known intracellular membrane. Third, when probed with several cholesterol-binding proteins, the initial rate of stero l transfer was maximally increased nearly 80-fold and the organization of c holesterol in the lysosomal membrane was rapidly altered. Nearly half of th e essentially nonexchangeable sterol in the lysosomal membrane was converte d to rapidly (t(1/2) = 6 min; fraction = 0.06) and slowly (t(1/2) = 154 min ; fraction = 0.36) exchangeable sterol domains/pools. In summary, the data revealed that spontaneous cholesterol transfer out of the lysosome and lyso somal membrane was extremely slow, inconsistent with rapid spontaneous diff usion across the lysosomal membrane. In contrast, the very slow spontaneous transfer of sterol out of the lysosome and lysosomal membrane was consiste nt with cholesterol leaving the lysosome earlier in the endocytic process a nd/or with cholesterol transfer out of the lysosome being mediated by addit ional process(es) extrinsic to the lysosome and lysosomal membrane.