The Fate of Cholesterol Exiting Lysosomes

Abstract

Cholesterol released from ingested low density lipoproteins in lysosomes moves both to the plasma membrane and to the endoplasmic reticulum (ER) where it is re-esterified. Whether cholesterol can move directly from lysosomes to ER or first must traverse the plasma membrane has not been established. To examine this question, the endocytic pathway of rat hepatoma cells was loaded at 18°C with low density lipoproteins (LDL) labeled with [ 3 H]cholesteryl linoleate, and the label then was chased at 37°C. The hydrolysis of the accumulated ester proceeded linearly for several hours. Almost all of the released [ 3 H]cholesterol moved to the plasma membrane rapidly and without a discernable lag. In contrast, the re-esterification in the ER of the released [ 3 H]cholesterol showed a characteristic lag of 0.5-1 h. These data are inconsistent with direct cholesterol transfer from lysosomes to ER; rather, they suggest movement through the plasma membrane.Furthermore, we found that progesterone, imipramine and 3-␤-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A) strongly inhibited the re-esterification of lysosomal cholesterol in the ER. However, contrary to previous reports, they did not block transfer of [ 3 H]cholesterol from lysosomes to the cell surface. Therefore, the site of action of these agents was not at the lysosomes. We suggest instead that their known ability to block cholesterol movement from the plasma membrane to the ER accounts for the inhibition of lysosomal cholesterol esterification.These findings are consistent with the hypothesis that cholesterol released from lysosomes passes through the plasma membrane on its way to the ER rather than proceeding there directly. As a result, ingested cholesterol is subject to the same homeostatic regulation as the bulk of cell cholesterol, which is located in the plasma membrane.Cell cholesterol levels are tightly regulated by homeostatic mechanisms. For example, when cells ingest cholesterol in the form of low density lipoprotein (LDL), 1 sterol biosynthesis is reduced and excess cholesterol is converted to cholesteryl esters for storage (1, 2). While these processes are well understood, the associated pathways of intracellular cholesterol movement are obscure. It appears that cholesterol moves bidirectionally between the plasma membrane and the ER (3). Furthermore, the cholesterol released from ingested LDL in lysosomes moves rapidly to the plasma membrane (4). Since the cholesterol derived from the degradation of LDL is reesterified by ACAT (1, 2), lysosomal sterol must also be transported to the ER.Two recent studies addressed the question of the pathway taken by cholesterol from lysosomes to ER. One suggested direct movement of cholesterol from lysosomes to ER by a mechanism that is inhibited by amphiphiles (5). The other concluded that approximately 70% of lysosomal cholesterol passes through the plasma membrane prior to esterification, the remainder moving directly between the lysosomes and the ER (6).In the present study, we have analyzed the fate of inge...

The Sterol-sensing Domain of the Niemann-Pick C1 (NPC1) Protein Regulates Trafficking of Low Density Lipoprotein Cholesterol

“…Cells were loaded with [ 3 H]CL-LDL at 16 -18°C. At this temperature, there is no significant hydrolysis of LDL cholesteryl esters and trafficking of free cholesterol from late endosomes is prevented (32). Brief incubations with CD enabled sampling of cholesterol predominantly associated with the PM pool, rather than with intracellular membranes (29,33).…”

Acyl-coenzyme A:cholesterol acyltransferase promotes oxidized LDL/oxysterol-induced apoptosis in macrophages

“…Amphipathic amines, such as U18666A, block cholesterol esterification by inhibiting cholesterol trafficking without having a direct effect on ACAT activity (49)(50)(51)(52) (Fig. 4).…”

Cholesterol Movement in Niemann-Pick Type C Cells and in Cells Treated with Amphiphiles

“…ER and cell cholesterol were then determined as described (30). Panel A, cell cholesterol in normal fibroblasts treated with 2.5 M U18666A (q) or 80 M imipramine (E).…”

“…Determinations of cholesterol and protein mass (28,31), the activity of acid lysosomal ␤-galactosidase (30), and in vivo cholesterol esterification (29) were as described. ER cholesterol was taken as the total cholesterol esterified when whole cell homogenates were reacted to completion with 30 M […”