The Niemann-Pick C1 Protein Resides in a Vesicular Compartment Linked to Retrograde Transport of Multiple Lysosomal Cargo*

Niemann-Pick C disease (NP-C) is a neurovisceral lysosomal storage disorder. A variety of studies have highlighted defective sterol trafficking from lysosomes in NP-C cells. However, the heterogeneous nature of additional accumulating metabolites suggests that the cellular lesion may involve a more generalized block in retrograde lysosomal trafficking. Immunocytochemical studies in fibroblasts reveal that theNPC1 gene product resides in a novel set of lysosome-associated membrane protein-2 (LAMP2)(+)/mannose 6-phosphate receptor(−) vesicles that can be distinguished from cholesterol-enriched LAMP2(+) lysosomes. Drugs that block sterol transport out of lysosomes also redistribute NPC1 to cholesterol-laden lysosomes. Sterol relocation from lysosomes in cultured human fibroblasts can be blocked at 21 °C, consistent with vesicle-mediated transfer. These findings suggest that NPC1(+) vesicles may transiently interact with lysosomes to facilitate sterol relocation. Independent of defective sterol trafficking, NP-C fibroblasts are also deficient in vesicle-mediated clearance of endocytosed [14C]sucrose. Compartmental modeling of the observed [14C]sucrose clearance data targets the trafficking defect caused by mutations in NPC1 to an endocytic compartment proximal to lysosomes. Low density lipoprotein uptake by normal cells retards retrograde transport of [14C]sucrose through this same kinetic compartment, further suggesting that it may contain the sterol-sensing NPC1 protein. We conclude that a distinctive organelle containing NPC1 mediates retrograde lysosomal transport of endocytosed cargo that is not restricted to sterol.

fatal, neurodegenerative lysosomal storage disorder (1). Human NPC1 (2) and its ortholog in the mouse (3) have recently been identified by positional and complementation cloning. NPC1 is predicted to be a polytopic membrane-spanning protein with a domain of striking homology to the putative sterolsensing regions of other proteins that are essential for managing cellular cholesterol homeostasis (2,4). NPC1 also has a terminal di-leucine motif that targets proteins to late endocytic compartments (2).
The most recognized cellular lesion in NP-C cells is impaired lysosomal relocation of endocytosed low density lipoprotein (LDL)-derived cholesterol to other intracellular sites, such as the plasma membrane and endoplasmic reticulum (5)(6)(7). Cholesterol accumulates in lysosomes as well as the Golgi apparatus (8,9). Induction of cholesterol-mediated homeostatic responses is proposed to be delayed because of deficient enrichment of regulatory sterol pools in the endoplasmic reticulum (10,11).
NP-C is also characterized by lysosomal accumulation of other metabolites. Phospholipids, glycosphingolipids, sphingoid bases, cystine, and glycoproteins, as well as cholesterol, are stored in a tissue-specific pattern (1,12). To date, it has been assumed that the transport defect in NP-C cells is specific to cholesterol and that other metabolites accumulate secondarily to cholesterol. However, in the brain, where functional impairment is progressive and ultimately fatal, massive accumulation of glycolipids occurs, with little, if any, excessive cholesterol storage (1,12). Extensive glycolipid storage in fetal human NP-C liver further suggests that this storage is an early event in the development of the disorder (12). These glycolipid storage anomalies do not readily integrate into a unified pathogenic mechanism involving a primary defect in sterol trafficking. The possibility that the NP-C lesion might alternatively reflect a more generalized defect in trafficking out of lysosomes has not been previously explored.
Here we provide evidence that sterol transport from lysosomes is vesicle-mediated, as suggested by a previous report (13). We show that NPC1 resides in a novel set of vesicles that appear to interact with cholesterol-filled lysosomes. We further show that mutations in NPC1 do not exclusively affect sterol transport, but rather, target a vesicle-mediated retrograde transport pathway, potentially affecting a wide range of endocytosed material. Cholesterol, in addition to being a cargo, appears to modulate NPC1 vesicle-mediated transport from lysosomes.
Tissue Culture-Normal and NP-C fibroblasts were derived from volunteers and confirmed patients of the Developmental and Metabolic Neurology Branch under the guidelines approved by the NINDS Intramural Review Board. Five different normal cell lines (ENZ123, ENZ125, ENZ143, GM5565, and GM1652) and three NP-C cell lines (GM3123, DMN92.31, and DMN87.57) were used. A null mutant NP-C cell line (DMN98.16), negative for NPC1 mRNA and protein expression, 2 was used solely in immunocytochemical studies.
Fibroblasts were cultured in Eagle's minimal essential medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 2 mM glutamine, and 100 units of penicillin/streptomycin/ml in humidified 95% air and 5% CO 2 at 37°C. For biochemical analyses, fibroblasts seeded at a density of 80,000 cells/well in plastic 6-well dishes (Costar, Cambridge, MA) were incubated for 5-7 days in McCoy's medium with 5% LPDS supplemented as above. For immunocytochemical analyses, fibroblasts were seeded at a density of 20,000 cells/well in McCoy's/5% LPDS medium in 9.5-cm glass microscope wells (Nunc, Inc., Naperville, IL) coated with human fibronectin.
Biochemical Measurement of Fluid-phase Endocytosis-Fluid-phase endocytosis was measured using [ 14 C]sucrose as described (14 -16). Briefly, cells were washed once with McCoy's/5% LPDS medium and then incubated for 3 h at 37°C in prewarmed medium containing 5 Ci of [ 14 C]sucrose (specific activity, 11,000 dpm/pmol). The labeling medium was removed, and the cells were washed six times with ice-cold serum-free McCoy's medium. For reflux experiments, cells were recultured in medium at 37°C. At specified times, the medium was transferred to tubes and centrifuged at 4°C for 5 min at 1500 rpm (to pellet any cells present). Cell monolayers were washed with cold serum-free McCoy's medium and then solubilized in 1% SDS for 30 min. Total cell protein was determined by the method of Lowry et al. (17). Kinetic Data Analysis-[ 14 C]Sucrose data were analyzed using Win-SAAM, the Windows version of the SAAM (Simulation, Analysis and Modeling) program (18 -20) and a four-compartment model. Compartments 1-3 represent intracellular pools of [ 14 C]sucrose that turn over at distinct rates; compartment 4 represents the culture medium. This is a modified version of the model proposed by Blomhoff et al. (21), which contained compartments that turned over in 10, 18, and 330 min and were considered to represent early, late and terminal (lysosomal) endocytic compartments, respectively. Because only a few sampling times were available before 30 min, we retained the first compartment in the model but fixed its turnover to be 10 min. Loss of tracer from compartments 1 and 2 is directed to the medium, with no assumptions made about the pathway of delivery involved. Based on our immunocytochemical findings (see under "Results"), we directed loss from compartment 3 (lysosomes) back into compartment 2 (late endosomes) rather than into the medium.
Pilot studies indicated that [ 14 C]sucrose loading for 3 h provided optimal conditions for egress studies. The model was set up to simulate the loading period so as to determine the distribution of [ 14 C]sucrose among the intracellular compartments by the end of loading. To simulate cellular clearance, the value for compartment 4 (culture medium) was set to zero, and the amount of tracer remaining in compartments 1, 2, and 3 (expressed as a percentage of dose in cells at the end of loading) was calculated for 24 h. Calculated values were compared with the observed data. Transfer rates for pathways, other than loss from compartment 1, were adjusted iteratively by the software until the calculated curves fit each set of experimental data.
Calculations-Data from each study (normal cell line and NP-C cell line) were fit simultaneously by the model. Differences were introduced in specific pathways until the model fit both the normal and NP-C data (or normal Ϯ LDL data). The model was also used to predict the pool sizes at steady state. The latter were calculated by simulating a constant input into compartment 1 for each study. The values for the pool sizes were expressed relative to a mass of 100% in compartment 1 of normal cells.
Statistics-Because the normal and NP-C cell lines were studied in pairs, differences were tested using the paired t test. Differences in transfer rates between normal and NP-C cells were expressed as a percentage of normal in each study (rather than the absolute value because this varied with each cell line).
NPC1 Antipeptide Antibodies-Polyclonal antibodies were raised against keyhole limpet hemocyanin-conjugated synthetic peptides representing residues 75-93 (NPC1-L), 147-165 (NPC1-N), and 1256 -1274 (NPC1-C) of the human NPC1 protein (Fig. 1). The peptides were synthesized on an automated Symphony/Multiplex peptide synthesizer using HBYU/NMP protocol (FMOC chemistry) and purified by reversephase high performance liquid chromatography. The sequences were verified by amino acid analysis, analytical high pressure liquid chromatography, and electrospray mass spectrometry. New Zealand White rabbits were injected subcutaneously with 1 mg of keyhole limpet hemocyanin-conjugated peptide (in 0.5 ml of phosphate-buffered saline) mixed thoroughly into an emulsion with 0.5 ml of Freund's complete adjuvant. For subsequent boosts, keyhole limpet hemocyanin-conjugated peptide (0.5 mg in Freund's incomplete adjuvant) was injected subcutaneously 3 and 6 weeks later. Sera obtained from these animals contained polyclonal antibodies against NPC1 protein. Western blot analysis of human fibroblast cell lines reveals that all three NPC1 antipeptide antisera identify a 165-kDa protein. 3 Immunocytochemical Analyses-Cells in glass chamber slides were washed in phosphate-buffered saline and fixed in 3% paraformaldehyde for 30 min. Cells were immunolabeled using an indirect procedure in which all incubations (quench, primary and secondary antibodies, and washes) were performed in blocker solution containing filipin (0.05%) and goat IgG (2.5 mg/ml). Primary antibodies used were NPC1 (1:1000), LAMP2 (1:40), and MPR (1:1000). Secondary fluorescein isothiocyanate-and LRSC-labeled antibodies were used at 1:50 dilution. Fluorescence was viewed with a Zeiss laser scanning confocal microscope using a krypton-argon Omnichrome laser with excitation wavelengths of 488 and 568 nm for fluorescein isothiocyanate and LRSC, respectively. Filipin fluorescence was viewed using an argon ion laser (Coherent) with an excitation wavelength of 351 nm.

NPC1 Protein Resides in a Distinct Vesicular
Compartment-We established the intracellular localization of NPC1 in cultured human fibroblasts using antibodies against three distinct 19-amino acid peptide sequences of the protein (Fig. 1). Although all three antisera immunostained similar structures, NPC1-C antiserum was routinely used because it provided the brightest staining. Wild type and some mutated forms of NPC1 localized in granular structures (Fig. 2, A and B). NPC1 immunostaining is specific because it is blocked by co-incubation with NPC1-C peptide and is absent in "null mutant" NP-C fibroblasts that do not express NPC1 (Fig. 2C).
Drugs That Block Transport of Cholesterol out of Lysosomes Induce Lysosomal Sequestration of NPC1 Protein-Because the documented mutations of NPC1 indicate that it plays a critical role in transporting endocytosed cholesterol from lysosomes, one might expect that this protein can interact with cholesterol-enriched lysosomes. We tested whether drugs such as U18666A (25) and progesterone (26), which block cholesterol transport out of lysosomes, might also alter the cellular distribution of NPC1. Cytochemical analysis reveals that in addition to the sequestration of cholesterol in lysosomes (25,26), U18666A (Fig. 5) and progesterone (not shown) also trap the majority of NPC1 protein within these same organelles. When progesterone is washed out of cells at 37°C, to re-establish cholesterol transport (26), distinct NPC1(ϩ)/LAMP2(ϩ) and NPC1 (Ϫ)/LAMP2(ϩ) vesicles cleared of cholesterol are formed (data not shown). These observations suggest that a normally transient interaction of NPC1 with cholesterol-laden lysosomes is required to relocate cholesterol to other cellular sites.
Cholesterol Transport From Lysosomes Is Temperature-dependent-The localization of NPC1 to LAMP2(ϩ) vesicles and its drug-induced relocation to cholesterol-filled lysosomes suggest that sterol transport from lysosomes is vesicle-mediated. To further evaluate this potential mode of trafficking, proges-terone was again used to reversibly accumulate LDL-derived cholesterol in lysosomes (7,26). During the subsequent progesterone washout phase, the temperature was reduced to determine whether lysosomal cholesterol relocation could be blocked at temperatures that perturb vesicular trafficking (27). Cytochemical analysis reveals that the pool of cholesterol sequestered in lysosomes (Fig. 6A) was readily mobilized during progesterone washout at 37°C (Fig. 6B) but not at 21°C (Fig. 6C).
In similar experiments, the relocation of lysosomal [ 3 H]cholesterol to the plasma membrane was also blocked below 21°C (Fig. 7). Taken together, these results suggest that NPC1 participates in a vesicle-mediated transport of cholesterol from lysosomes.
Clearance of Endocytosed [ 14 C]Sucrose Is Impaired in NP-C Fibroblasts-Because a vesicle-mediated mechanism of sterol transport from lysosomes allows potential cotransport of addi-  1. NPC1 antipeptide antibodies. The NPC1 protein (2) contains as many as 16 transmembrane domains (black stripes). The "sterol-sensing" region of NPC1 is contained within transmembrane domains 4 -8 (boxed). The carboxyl terminus contains a di-leucine motif (LLNF). The location of the peptide sequences within the predicted domains of the NPC1 protein used to generate polyclonal antibodies are shown. NPC1-L, peptide within the leucine zipper region (striped box; residues 73-94) of the unique NPC1 amino-terminal region (residues 55-165); NPC1-N, a different peptide within the unique NPC1 amino-terminal region; NPC1-C, peptide within the carboxyl-terminal tail.
tional lysosomal cargo, we examined whether NP-C cells are also defective in clearing other endocytosed material. [ 14 C]Sucrose, a fluid-phase marker for vesicular transport (14 -16), was used to monitor endocytic trafficking. We tested whether mutated NPC1 alters vesicular transport in the absence of lysosomal cholesterol storage. Lysosomes of normal and NP-C fibroblasts were cleared of sterol by extended incubation in lipoprotein-deficient medium prior to loading with [ 14 C]sucrose (8) and then were maintained in such medium during subsequent [ 14 C]sucrose loading and clearance. As illustrated in Fig.  8, clearance of endocytosed [ 14 C]sucrose was delayed in all NP-C cell lines examined. The difference in clearance was apparent at 30 min and was maintained thereafter.
Compartmental Modeling Predicts That NP-C Fibroblasts Are Defective in Clearance of [ 14 C]Sucrose from a Late Endocytic Compartment-In order to identify the cellular compartments through which sucrose transport might be altered by a nonfunctional NPC1 protein, we constructed a compartmental model for the endocytic uptake and release of tracer [ 14 C]sucrose in normal and NP-C fibroblasts (Fig. 9). In normal fibroblasts, the relative accumulation of endocytosed cargo (Table I) and the calculated turnover times of compartments 1, 2, and, 3 (10 min, 1 h, and Ն24 h, respectively) correspond to previously reported values for early endocytic, late endocytic, and lysosomal compartments (15,16,21,28).
In NP-C fibroblasts, the calculated rates of [ 14 C]sucrose efflux from intracellular compartments 1 and 3 were identical to those for normal cells, but the rate from compartment 2 was Normal human fibroblasts were incubated in McCoy's/5% LPDS medium at 37°C for 4 days and then incubated in fresh medium containing LDL (50 g/ml) for 24 h to enrich cellular membranes with sterol. Fibroblasts were immunostained for MPR (green) and NPC1 (red). Note that the intracellular distribution of NPC1 is quite different from that of MPR, with little apparent colocalization. retarded (47% of normal, as shown in Fig. 9 for egress data displayed in Fig. 8). Release from compartment 2 into the medium was significantly lower in NP-C cells compared with normal cells (0.71 Ϯ 0.23/h versus 0.44 Ϯ 0.17/h (mean Ϯ S.D.), respectively; p Ͻ 0.01 for eight paired experiments). Thus, the rate of transport from compartment 2 into the medium is nearly one-half the rate seen in normal cells (62.6 Ϯ 14.4%; n ϭ 8). Uptake into compartment 1 from the medium was 3.63E-5 Ϯ 0.8E-5/h in normal versus 4.35E-5 Ϯ 0.9E-5/h in NP-C fibroblasts (n ϭ 8). Movement into compartment 3 from compartment 2 (0.54 Ϯ 0.16/h) and release from compartment 3 (0.05 Ϯ 0.01/h) were the same in both normal and NP-C cells. The modeling reveals that the impaired movement from late endosomes back into the medium accounts for the observed delayed clearance of [ 14 C]sucrose from NP-C cells (as depicted in Fig. 8).
The compartmental modeling also predicts the NP-C phenotype, namely, excessive lysosomal storage of endocytosed cargo (see Table I). Most (88%) of the excess endocytosed cargo retained in the three NP-C fibroblast lines studied is predicted to accumulate in compartment 3 (lysosomes). The [ 14 C]sucrose mass predicted to accumulate in lysosomes of NP-C cells is about 2-fold greater than normal cells, consistent with the previously reported 2-fold increase in the fluid-phase volume (29,30) and endocytosed sterol content (6,7,10) of NP-C lysosomes.
Cellular Cholesterol Enrichment Retards Clearance of Endocytosed [ 14 C]Sucrose from Normal Fibroblasts-We explored the possibility that endocytosed cholesterol is not only a cargo of the NPC1mediated retrograde lysosomal transport pathway but may also modulate this pathway through the sterol-sensing domain of the NPC1 protein. Lipoprotein-depleted normal fibroblasts were incubated in the absence or presence of LDL during both the endocytic uptake and subsequent clearance of [ 14 C]sucrose. As seen in Fig.  10, cellular sterol enrichment significantly retarded [ 14 C]sucrose clearance in normal fibroblasts.
In order to identify the cellular compartments potentially targeted by this cellular sterol enrichment, we applied the same compartmental model (Fig. 9). Sterol enrichment significantly reduced [ 14 C]sucrose release from compartment 2 back into the medium (0.71 Ϯ 0.23/h versus 0.57 Ϯ 0.21/h, p Ͻ 0.05; for (-)LDL versus (ϩ)LDL, respectively) in three different normal fibroblast lines. Consistent with a recent study (31), cellular sterol enrichment did not alter the rate of trafficking of bulk flow markers between early endocytic compartments and the cell surface. DISCUSSION The well documented lesions in lysosomal sterol processing that occur in Niemann-Pick C disease have established a role for NPC1 in sterol transport out of lysosomes. The identification of putative sterol-sensing domains in the recently cloned NPC1 gene further links this protein to cellular sterol processing (2). Our present studies reveal that in cultured human fibroblasts, NPC1 identifies a novel vesicle that appears to interact with cholesterol-filled lysosomes. We provide evidence that NPC1 mediates a vesicular form of transport out of lysosomes and that the cargo transported along this pathway is not restricted to sterol.
We have shown that NPC1 protein (Fig. 2) resides in a novel set of vesicles that are LAMP2(ϩ) (Fig. 3) and MPR(Ϫ) (Fig. 4) and that do not accumulate endocytosed lysosomal markers (Fig. 3). The ganglioside G M2 exclusively localizes to NPC1(ϩ) vesicles and not to lysosomes containing endocytosed cholesterol, confirming their unique identity. 4 We found that drugs that block sterol transport out of lysosomes (U18666A and progesterone) also trap NPC1 in cholesterol-laden lysosomes (Fig. 5). Sterol relocation from lysosomes can be blocked by low temperature (Figs. 6 and 7) and drugs (13) that affect vesicular trafficking. Taken together, these findings suggest that NPC1 vesicles transiently interact with lysosomes to transfer sterol to other cellular sites. Definitive characterization of the novel NPC1(ϩ) vesicular compartment as well as other organelles that may participate in retrograde lysosomal transport is cur-4 E. J. Blanchette-Mackie, manuscript in preparation.
FIG. 5. U18666A induces NPC1 to colocalize with endocytosed cholesterol in lysosomes. Normal human fibroblasts were incubated in McCoy's/5% LPDS medium at 37°C for 4 days, washed in phosphate-buffered saline, and then incubated in fresh medium containing LDL (50 g/ml) and U18666A (2 g/ ml) for 24 h to allow endocytosed LDLderived cholesterol to accumulate in lysosomes. Cells were immunostained for NPC1 (red) and cytochemically stained with filipin to reveal cholesterol (green). The diagonal line marks the position at which the cell was scanned in the x-z axis. A portion of the x-z scan (bracketed by the arrows) is shown in B-D. A merge (B) of the NPC1 image (red, as seen in C) and the cholesterol image (green, as seen in D) shows extensive yellow color, indicating that the two are largely colocalized. rently under investigation.
Several independent lines of research have provided evidence for retrograde lysosomal transport. Lysosomal membrane proteins appear to continuously cycle between lysosomes and the plasma membrane via endosomal compartments (32)(33)(34)(35)(36)(37)(38)(39). Endocytosed fluid-phase cargo also transfers from lysosomes back to late endocytic compartments (40,41). Physical interactions between MPR(ϩ)-late endosomes and lysosomes that allow content mixing have been reported (41)(42)(43)(44)(45)(46). The NPC1(ϩ) cholesterol-laden lysosome induced by drugs in the current studies (Fig. 5) could represent a hybrid organelle analogous to that which forms from the fusion of a late endosome with a lysosome (44,45). Drugs that retard cholesterol transport from lysosomes may allow fusion of NPC1 vesicles with lysosomes but block their subsequent fission. The observed reappearance of NPC1 vesicles as well as presumed lysosomes cleared of sterol after removal of progesterone from cell cultures (data not shown) is consistent with a reversible mechanism of drug interdiction involving blocked fission of a hybrid organelle.
NP-C fibroblasts were found to be defective in vesicle-mediated retrograde transport of [ 14 C]sucrose in the absence of lysosomal cholesterol storage (Fig. 8). Compartmental modeling was used to identify the kinetic compartment associated with this intracellular trafficking defect. The modeling targets the defect in retrograde transport to a late endocytic compartment (Fig. 9) and predicts that this defect is primarily responsible for the lysosomal storage (Table I)  Cell extracts were prepared and fractionated in 10% Percoll gradients as described (7). High density ␤-hexosaminidase-enriched fractions were designated as lysosomes, and low density alkaline phosphataseenriched fractions were designated as plasma membrane. A notable block in transfer of endocytosed cholesterol from lysosomes to the plasma membrane was seen below 21°C, the temperature at which vesicular transport is blocked. Note that delayed progesterone washout at low temperatures cannot account for the observed reduction in relocation of lysosomal [ 3 H]cholesterol. The half-life of cellular [ 3 H]progesterone washout is 40 min at 37°C and less than twice that at 16°C. cytic compartment likely represents the immunocytochemically defined NPC1 vesicles.
The role that endocytosed sterol plays in NP-C disease may be quite different than previously envisioned. The defect in vesicle-mediated egress of both [ 14 C]sucrose and cholesterol suggests that the NPC1-mediated transport pathway is not restricted to sterol mobilization. Defective cellular clearance of a fluid-phase marker in the absence of lysosomal cholesterol storage in NP-C suggests that generalized, bulk retrograde transport of vesicular components is impaired. Several studies have provided evidence that endocytosed lipids (47) and proteins (48) recycle to the cell surface from late endocytic compartments. Increasingly, attention has focused on the sorting and trafficking of endocytosed plasma membrane glycolipids (49). The defect in retrograde vesicular transport that we presently report could account for the excessive G M2 accumulation seen in endosomal compartments of some NP-C cell lines (50). Recently we demonstrated that endocytosed G M2 is a specific marker for the NPC1 vesicle, 4 consistent with a role for this organelle in the intracellular trafficking of both glycolipids and cholesterol. Disruption of glycolipid transport might contribute to the cellular pathology associated with NP-C. Glycolipids, such as G M2 , accumulate extensively in the absence of excessive cholesterol storage in NP-C neurons (51,52) and in the whole brain (1,12). Interestingly, glycolipids and cholesterol are the major lipid components of membrane microdomains found in rafts; detergent-insoluble, glycolipid-enriched complexes; and caveolae (49). Preliminary cellular extraction studies show that NPC1 has the solubility properties of a raftassociated protein. 5  Sucrose associated with the cell monolayer and medium was measured as described. Each data point represents the mean value Ϯ S.D. of triplicate culture wells. The graph represents the results of a single representative paired experiment. Data points are shown as squares for normal fibroblasts (GM5565) and as diamonds for NP-C fibroblasts (GM3123). The calculated fits to the data, using the model shown in Fig. 9, are represented by solid and dashed curves for normal and NP-C fibroblasts, respectively. The model is available at htp://biomodel.georgetown.edu/model.  We also provide preliminary evidence that endocytosed sterol itself may modulate functions of NPC1. Cellular sterol enrichment retards the clearance of endocytosed [ 14 C]sucrose from normal fibroblasts (Fig. 10) and, like NPC1 mutations, specifically targets a late endocytic compartment (Fig. 9). Thus, in addition to being cargo, endocytosed sterol appears to alter the rate of vesicular trafficking along this pathway, possibly interacting with the sterol-sensing domain of NPC1.
In conclusion, the present findings suggest that a novel NPC1 organelle mediates retrograde lysosomal trafficking of a potentially wide spectrum of lysosomal cargo. The tissue-specific pattern of metabolites that accumulate in NP-C disease likely reflects the diverse cargo that is transported along this pathway. In the liver, where endocytosed lipoprotein processing can be significant, sterol may represent a major portion of the endocytosed cargo utilizing this pathway. Alternatively, in neurons and other types of cells in which little sterol may be exogenously derived, NPC1 may predominantly regulate retroendocytic glycolipid trafficking. ]sucrose in the absence or presence of LDL (50 g/ml) for 3 h at 37°C to load endocytic compartments with the tracer. Cells were washed and then incubated in McCoy's/5% LPDS medium in the absence or presence of LDL (50 g/ml) at 37°C for the indicated times. [ 14 C]Sucrose associated with the cell monolayer and medium was measured as described. Each data point represents the mean Ϯ S.D. of triplicate culture wells. The graph represents the results of a single representative paired experiment using normal fibroblasts (GM5565). Data points are shown as squares and diamonds for cells in the absence and presence of LDL, respectively. The calculated fits to the data, using the model shown in Fig. 9, are represented by solid and dashed curves for cells in the absence and presence of LDL, respectively.