Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 278, Issue 29, Pages 27180-27189Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M300542200
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Funding
- NCI NIH HHS [CA23108] Funding Source: Medline
- NHLBI NIH HHS [HL 36709] Funding Source: Medline
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We previously studied the early trafficking of low density lipoprotein (LDL)-derived cholesterol in mutant Chinese hamster ovary cells defective in Niemann-Pick type C1 (NPC1) using cyclodextrin ( CD) to monitor the arrival of cholesterol from the cell interior to the plasma membrane ( PM) (Cruz, J. C., Sugii, S., Yu, C., and Chang, T.-Y. ( 2000) J. Biol. Chem. 275, 4013 - 4021). We found that newly hydrolyzed cholesterol derived from LDL first appears in certain CD-accessible pool(s), which we assumed to be the PM, before accumulating in the late endosome/lysosome, where NPC1 resides. To determine the identity of the early CD-accessible pool( s), in this study, we performed additional experiments, including the use of revised CD incubation protocols. We found that prolonged incubation with CD (> 30 min) caused cholesterol in internal membrane compartment( s) to redistribute to the PM, where it became accessible to CD. In contrast, a short incubation with CD ( 5 - 10 min) did not cause such an effect. We also show that one of the early compartments contains acid lipase (AL), the enzyme required for liberating cholesterol from cholesteryl ester in LDL. Biochemical and microscopic evidence indicates that most of the AL is present in endocytic compartment( s) distinct from the late endosome/lysosome. Our results suggest that cholesterol is liberated from LDL cholesteryl ester in the hydrolytic compartment containing AL and then moves to the NPC1-containing late endosome/lysosome before reaching the PM or the endoplasmic reticulum.
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