1998;140:39C47. carrier. These outcomes claim that in Rab11-overexpressing cells, deposition of cholesterol in recycling endosomes results in its impaired esterification, presumably due to defective recycling of cholesterol to the plasma membrane. The findings point to the importance of the recycling endosomes in regulating cholesterol and Ascomycin (FK520) sphingolipid trafficking and cellular cholesterol homeostasis. INTRODUCTION Cholesterol is an essential constituent of membranes in mammalian cells and a precursor for steroid hormone and bile acid synthesis. Cellular cholesterol levels are tightly regulated at the level of synthesis, esterification, and exchange with plasma lipoproteins (Brown and Goldstein, 1999 ; Simons and Ikonen, 2000 ). The route of low-density lipoprotein (LDL)-cholesterol uptake is hitherto the best characterized cellular cholesterol-trafficking pathway. The role of the LDL receptor in LDL internalization, the breakdown of the lipoprotein particle in acidic organelles, and the homeostatic mechanisms regulating the LDL-receptor levels have been unraveled (Brown and Goldstein, 1986 ). However, the contribution of other endocytic routes on cholesterol transport and balance and their interplay with the LDL-receptor route are so far poorly understood at the molecular level. The endocytic organelles have been mainly defined based on the flow of different cargo molecules to early, recycling, and late compartments. Internalized molecules are initially transported to early endosomes (also termed sorting endosomes) from where they can be delivered to late endosomes and lysosomes for degradation or become recycled to the plasma membrane either directly or via a recycling endosomal membrane system (Gruenberg and Maxfield, 1995 ; Mellman, 1996 ). Recycling endosomes are considered to be cholesterol enriched (Gagescu (Palo Alto, CA). Green fluorescent protein (GFP)-wtRab5, GFP-wtRab6, GFP-wtRab7, and GFP-wtRab11 were as described previously (White (1983) for 24 h before labeling. To analyze esterification in the presence of LDL, cells grown in culture medium were washed with phosphate-buffered saline (PBS) and labeled with [3H]oleic acid (5 Ci/ml) in serum-free, 2% defatted BSA medium supplemented with 50 g/ml LDL for 4 h. After labeling, the cells were washed with ice-cold PBS on ice and scraped into PBS, harvested by centrifugation, and resuspended in 2% NaCl. Aliquots were removed for determining the protein concentration. A chromatography recovery standard was added (2.5C5 nCi of [14C]cholesteryl oleate) and the lipids extracted with 2 ml of methanol and 1 ml of chloroform as described previously (Bligh and Dyer, 1959 Ascomycin (FK520) ). After subsequent centrifugation, 1/10 of the supernatant was Ascomycin (FK520) removed for liquid scintillation counting to determine the [14C]cholesteryl oleate radioactivity. The extracted lipids were Ascomycin (FK520) separated by thin layer chromatography on silica gel plates by using hexane/diethyl ether/acetic acid (80:20:1) as the solvent. The cholesteryl ester band was determined based on the comigration of a cholesteryl ester standard, scraped, and 3H and 14C radioactivity measured by liquid scintillation counting. The results were corrected for the volume and procedural losses based on the recovery of 14C radioactivity and plotted against the total amount of protein in the sample. The protein concentration was determined according to Lowry (1951) . To analyze esterification in delipidated cells, cells grown in 5% LPDS medium for 24 h were EZH2 washed with PBS and labeled with [3H]oleic acid (5 Ci/ml) in serum-free, 2% defatted BSA medium for 4 h. Lipids were Ascomycin (FK520) analyzed as described above. To analyze esterification in cells loaded with cholesterol/m-CD-complex the cells were initially delipidated as described above and labeled with [3H]oleic acid (5 Ci/ml) in serum-free, 2% defatted BSA medium for 4 h. During the labeling, cholesterol/m-CD-complex prepared as described previously (Leppimaki em et al. /em , 2000 ) was added at 50 g/ml concentration of cholesterol at staggered time points.