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Transport of plasma membrane-derived cholesterol and the function of Niemann-Pick C1 Protein¹

VOLKER WIEGAND^*, TA-YUAN CHANG, JEROME F. STRAUSS, III, FALK FAHRENHOLZ^* and GERALD GIMPL^*,2

^* Institute of Biochemistry, Johannes Gutenberg-University of Mainz, Mainz, Germany;
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire, USA; and
Center for Research on Reproduction and Women’s Health, University of Pennsylvania, Philadelphia, Pennsylvania, USA

²Correspondence: Institute of Biochemistry, Johannes Gutenberg-University of Mainz, Becherweg 30, D-55099 Mainz, Germany. E-mail: gimpl{at}mail.uni-mainz.de

SPECIFIC AIMS

The gene responsible for 95% of Niemann-Pick C (NPC) disease encodes a multiple, membrane-spanning protein, designated NPC1, which regulates the sorting and trafficking of endocytosed cholesterol. The prominent cellular feature of NPC disease is extensive sequestration of endocytosed cholesterol in lysosomes or late endosomes. Using a novel, fluorescent cholesterol derivative, we have visualized for the first time the fate of the plasma membrane-derived cholesterol in dependence on the function of the NPC1 protein.

PRINCIPAL FINDINGS

1. The fluorescent probe 6-dansyl-cholestanol (DChol) faithfully mimics cholesterol
Cholesterol was labeled with a "dansyl" group at position 6 (Fig. 1 A). DChol could be readily incorporated into MßCD. This water-soluble inclusion complex allowed a very efficient labeling of cells and was used in all experiments of the present study. To prove whether DChol faithfully mimics cellular cholesterol, we compared the behavior of DChol versus [³H]cholesterol with respect to its (i) efflux kinetics, (ii) raft association, and (iii) esterification rate in CHO cells. The MßCD-mediated efflux followed biexponential kinetics, indicating two cholesterol pools for [³H]cholesterol and DChol (Fig. 1C ). DChol behaves similar to [³H]cholesterol with respect to its distribution in Triton X-100-insoluble lipid domains (Fig. 1D ). After 24 h of incubation with DChol-MßCD or [³H]cholesterol-MßCD, 45% of each of the steroids had been esterified in CHO cells (Fig. 1E ). When the cells were pretreated with the ACAT inhibitor 58–035, the esterification of DChol was inhibited (Fig. 1E , inset).

View larger version (34K): [in this window] [in a new window]   Figure 1. Structure and characterization of DChol. A) Structure; B) excitation and emission spectrum; C) efflux kinetics of DChol versus [3H]cholesterol. Chinese hamster ovary (CHO) cells were dual-labeled with 6 µM [3H]cholesterol-methyl-ß-cyclodextrin (MßCD) and DChol-MßCD in medium containing acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitor 58–035 (400 ng/ml) for 18 h at 37°C. The efflux was started by adding prewarmed (37°C) acceptor medium. Sterol efflux was expressed as the fraction of initial sterol ([3H]cholesterol or DChol) remaining in the cells. D) "Raft" association of DChol and [3H]cholesterol. CHO cells (1.8x107 cells) were dual-labeled with DChol-MßCD and [3H]cholesterol-MßCD for 2 h at 37°C. Then, the cells were lysed in 2% Triton X-100 at 4°C and were fractionated using a 0–40% OptiprepTM step gradient. (Upper) An aliquot of each of the fractions was analyzed by immunoblotting with an anticaveolin antibody. The protein content and the amounts of both sterols (lower) were determined in each of the 800 µl fractions. E) Esterification rate of DChol versus [3H]cholesterol in CHO cells. Cells were incubated with [3H]cholesterol-MßCD or DChol-MßCD. After 1-h labeling at 37°C, the cells were washed, were further incubated at 37°C for the indicated times, and then were harvested. Lipids were extracted and separated by silica gel thin-layer chromatography (TLC). (Inset) TLC analysis of lipid extracts from CHO cells that were incubated for 8 or 24 h in the absence or presence of 58–035. The data are means ± SD for triplicate determinations.

2. The itinerary of DChol: from the plasma membrane to the endoplasmic reticulum (ER)/Golgi vesicles and lipid droplets
Trafficking of DChol from the plasma membrane into the ER occurred within minutes. In 20–40% of CHO cells, DChol appeared in the trans-Golgi region. After chasing CHO cells for 1–2 h, DChol was typically distributed within vesicles and droplets. DChol was scarcely transported to lysosomes. Most of the DChol containing fluorescent structures represented lipid droplets as a result of their costaining with Nile Red, a marker of neutral lipid deposits. The influx of DChol was not dependent on ongoing adenosine 5'-triphosphate synthesis.

3. In cells lacking ACAT activity, DChol is found in the ER
To test the influence of ACAT activity on the distribution of DChol, we pretreated CHO cells with the ACAT inhibitor 58–035. This treatment inhibited the esterification of DChol (Fig. 1E , inset) and suppressed the appearance of fluorescence in lipid droplets. Instead, DChol was localized to the ER network. DChol remained in the ER even after an incubation time of 24 h or longer. To rule out the possibility that this distribution pattern was a result of a nonspecific effect of the drug, we also examined the distribution of DChol in the CHO mutant cell line (AC29) deficient in ACAT activity. Again, DChol was found in the ER, and fluorescence in lipid droplets was not observed.

4. In progesterone-pretreated cells, DChol was observed in enlarged lipid droplets
Administration of progesterone to cells is known to induce a NPC-like phenotype. When CHO cells were pulse-labeled with DChol and chased for 2 h, DChol resided in many small (0.5 µm) droplets. In contrast, in CHO cells pretreated with progesterone, DChol resided in markedly larger droplets after the same 2-h chase period. The diameter of these droplets was 1 µm and further increased to 2–3 µm when the chase time was prolonged to 3 h. In untreated cells, the size of the DChol-containing droplets remained constant when the chase time was increased.

5. Translocation of plasma membrane-derived DChol into enlarged lipid droplets is a typical feature of the NPC1 phenotype
The transport of DChol was studied in CT43 cells, CHO mutants deficient in a functional NPC1 protein. NPC1–enhanced green fluorescent protein (EGFP) was typically localized in tubulovesicular structures (Fig. 2 A), which represent late endosomal compartments, in CT43 cells expressing the NPC1–EGFP. In CT43 cells, DChol was translocated to droplets of similar size as those found in progesterone-treated cells. During chase times from 3 to 6 h, the DChol droplets enlarged from 0.5 µm to 2–3 µm (Fig. 2B ). When the NPC phenotype of CT43 cells was corrected by transfection with NPC1, DChol was again found in small-sized (0.5 µm diameter) droplets (Fig. 2C ). Finally, we analyzed the translocation of plasma membrane-derived DChol into normal fibroblasts versus NPC fibroblasts. The fibroblasts were pulse-labeled with DChol and were chased for 4 h (Fig. 2D, E ) or 14 h (Fig. 2F ). In normal fibroblasts, DChol was observed to be transported to small lipid droplets after an incubation time of 4 h (Fig. 2D ) or 14 h (not shown). In contrast, in human NPC fibroblasts, DChol was translocated to enlarged (1 µm-sized) lipid droplets (Fig. 2E ). After an incubation period of 14 h, DChol accumulated in significantly enlarged lipid droplets (Fig. 2F ), similar to that observed in progesterone-treated CHO cells or in the NPC-like CT43 cells (Fig. 2B ).

View larger version (126K): [in this window] [in a new window]   Figure 2. Distribution of DChol in lipid droplets is dependent on the function of NPC1. A) Confocal image of CT43 cells transfected with NPC1GFP (CT43-NPC1GFP) shows the distribution of NPC1GFP in tubules (arrowheads) and vesicles. B) CT43 cells and C) CT43 cells transfected with NPC1 (CT43-NPC1) were pulse-labeled with DChol-MßCD for 20 min at 19°C and were chased for (B) 2 h, (C) 3 h, or (B) 5 h (typical droplet in left inset) at 37°C. Distribution of DChol in (D) human fibroblasts or (E, F) NPC fibroblasts. D) Normal fibroblasts or (E, F) NPC fibroblasts were labeled with DChol for (D, E) 4 h or (F) 14 h. Original bars in (A–C, E, F) 5 µm; (D) 3 µm; (insets) 1 µm.

CONCLUSIONS

We provided the following lines of evidence that the new cholesterol probe DChol mimics cholesterol: (i) The influx of DChol into CHO cells occurred rapidly by an energy-independent mechanism via the ER. (ii) The cyclodextrin-mediated efflux of DChol from CHO cells occurred from two kinetic pools as observed for [³H]cholesterol. (iii) DChol behaved like [³H]cholesterol with respect to its presence in Triton X-100-insoluble lipid domains. (iv) DChol showed the same kinetics of esterification by ACAT as [³H]cholesterol. (v) Following inhibition of ACAT, the unesterified DChol accumulated in the ER in accordance with earlier predictions for cholesterol.

Cholesterol can move from the plasma membrane to intracellular compartments via two competing pathways: the ER or the endocytic pathway (Fig. 3 ). The cessation of tubulovesicular trafficking in NPC1 organelles was suggested to be causative for the NPC1 defects. The trafficking of DChol to enlarged droplets was typically observed in cells that have been pretreated with progesterone or suffer from a loss of NPC1 activity (e.g., in CT43 cells or in human NPC fibroblasts). The enlargement of DChol droplets probably occurred by fusion of small-sized droplets. When the NPC phenotype in CT43 cells was corrected by transfection with NPC1–EGFP, DChol was again targeted to "normal", 0.5-µm diameter droplets. Conclusively, the activity of NPC1 maintains the small size of the DChol droplets, possibly by preventing their fusion. NPC1 organelles may help to remove the cholesterol from different sources, such as the plasma membrane, the ER, endosomes/lysosomes, or lipid droplets. In this respect, NPC1 tubules could represent the transport device for long-distance movements through the cell.

View larger version (23K): [in this window] [in a new window]   Figure 3. Cholesterol trafficking pathways and the NPC1-sorting organelle. A) Influx of plasma membrane-derived cholesterol occurs by vesicular pathways (yellow; receptor-mediated and constitutive endocytosis) and by less-defined transport routes via the ER and Golgi compartments. The NPC1-sorting organelle is primarily localized in late endosomes and plays a central role in distribution of cholesterol and glycolipids. B) Model of intracellular cholesterol (red) transport mediated by the tubulovesicular NPC1 compartment (blue, ER; black line, plasma membrane). The NPC1 organelle could transport cholesterol by shuttling whole organelle-associated cholesterol structures or in the form of raft domains (red lines). Lys, Lysosomes; LE, late endosome; EE, early-sorting endosome; ERC, endocytic recycling compartment; R, low-density lipoprotein (LDL) receptor (LDLR); CE, cholesteryl ester.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0818fje; to cite this article, use FASEB J. (February 19, 2003) 10.1096/fj.02-0818fje

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