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Originally published In Press as doi:10.1074/jbc.M314160200 on January 27, 2004

J. Biol. Chem., Vol. 279, Issue 15, 15571-15578, April 9, 2004
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The ABCA1 Transporter Modulates Late Endocytic Trafficking

INSIGHTS FROM THE CORRECTION OF THE GENETIC DEFECT IN TANGIER DISEASE*

Edward B. Neufeld{ddagger}§, John A. Stonik{ddagger}, Stephen J. Demosky, Jr.{ddagger}, Catherine L. Knapper{ddagger}, Christian A. Combs¶, Adele Cooney||, Marcella Comly||, Nancy Dwyer||, Joan Blanchette-Mackie||, Alan T. Remaley{ddagger}, Silvia Santamarina-Fojo{ddagger}, and H. Bryan Brewer, Jr.{ddagger}

From the {ddagger}Molecular Disease Branch, NHLBI, NHLBI Light Microscopy Core Facility, and ||Laboratory for Cellular Biology and Biochemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, December 24, 2003 , and in revised form, January 26, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have previously established that the ABCA1 transporter, which plays a critical role in the lipidation of extracellular apolipoprotein acceptors, traffics between late endocytic vesicles and the cell surface (Neufeld, E. B., Remaley, A. T., Demosky, S. J., Jr., Stonik, J. A., Cooney, A. M., Comly, M., Dwyer, N. K., Zhang, M., Blanchette-Mackie, J., Santamarina-Fojo, S., and Brewer, H. B., Jr. (2001) J. Biol. Chem. 276, 27584-27590). The present study provides evidence that ABCA1 in late endocytic vesicles plays a role in cellular lipid efflux. Late endocytic trafficking was defective in Tangier disease fibroblasts that lack functional ABCA1. Consistent with a late endocytic protein trafficking defect, the hydrophobic amine U18666A retained NPC1 in abnormally tubulated, cholesterol-poor, Tangier disease late endosomes, rather than cholesterol-laden lysosomes, as in wild type fibroblasts. Consistent with a lipid trafficking defect, Tangier disease late endocytic vesicles accumulated both cholesterol and sphingomyelin and were immobilized in a perinuclear localization. The excess cholesterol in Tangier disease late endocytic vesicles retained massive amounts of NPC1, which traffics lysosomal cholesterol to other cellular sites. Exogenous apoA-I abrogated the cholesterol-induced retention of NPC1 in wild type but not in Tangier disease late endosomes. Adenovirally mediated ABCA1-GFP expression in Tangier disease fibroblasts corrected the late endocytic trafficking defects and restored apoA-I-mediated cholesterol efflux. ABCA1-GFP expression in wild type fibroblasts also reduced late endosome-associated NPC1, induced a marked uptake of fluorescent apoA-I into ABCA1-GFP-containing endosomes (that shuttled between late endosomes and the cell surface), and enhanced apoA-I-mediated cholesterol efflux. The combined results of this study suggest that ABCA1 converts pools of late endocytic lipids that retain NPC1 to pools that can associate with endocytosed apoA-I, and be released from the cell as nascent high density lipoprotein.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cholesterol plays a critical role in the sorting and trafficking of other membrane lipids and proteins and serves to stabilize the structure of membrane domains required for signal transduction (1). The maintenance of optimal sterol levels for normal cellular function requires complex homeostatic cellular mechanisms that regulate cholesterol synthesis, intracellular trafficking, uptake, and efflux. The ABCA11 transporter plays a pivotal role in the energy-dependent efflux of cellular cholesterol and choline-containing phospholipids to apoA-I, the initial step in the formation of the nascent HDL particle (2-6). The role of ABCA1 in the maintenance of normal cellular sterol levels and HDL formation is strikingly illustrated when the transporter is mutated in Tangier disease (7-9). This rare human genetic disorder is characterized by excess cholesterol ester accumulation in macrophages, low serum HDL levels, and increased risk of coronary heart disease (10).

Studies of rare genetic diseases of cholesterol metabolism have revealed several proteins residing in late endosomes, and lysosomes play key roles in cellular cholesterol trafficking and metabolism. Niemann-Pick C proteins 1 and 2 (11, 12), which are defective in NP-C disease (13), redistribute LDL-derived lysosomal cholesterol to other cellular sites including the plasma membrane and the endoplasmic reticulum. MLN64, structurally related to steroidogenic acute regulatory protein defective in congenital adrenal hyperplasia, traffics lysosomal cholesterol to mitochondria (14). ATP-binding cassette protein G1, although not yet linked to a genetic defect, also appears to reside in late endocytic vesicles and play a role in cholesterol trafficking (15). Oxysterol-binding protein-related protein ORP1L, also involved in sterol metabolism, resides in late endosomes and plays a role in macrophage late endosome membrane dynamics (16).

To date, the cellular site(s) of function of the human ABCA1 transporter remain(s) to be determined. Our recent studies have established that ABCA1 resides on the plasma membrane as well as in endocytic vesicles that can shuttle between late endocytic compartments and the cell surface (17, 18). Based on these findings, we proposed that endosomal ABCA1 might play a role in the apoA-I-mediated efflux of cellular lipids (17). Several lines of research have provided evidence to support an endocytic pathway for the ABCA1-mediated cellular lipidation of apoA-I. Takahashi and Smith (19) first provided evidence that cellular cholesterol efflux involves endocytosis and resecretion of apoA-I. More recently, Smith et al. (20) have shown that apoA-I colocalizes with ABCA1-containing endosomes. Additional support for our conceptualization has been provided by recent studies that apoA-I-mediated lipid efflux is defective in the lysosomal storage diseases NP-C (21) and Niemann-Pick type B disease (22).

In the present study, we examined the functionality of ABCA1 residing in endocytic vesicles in human Tangier disease fibroblasts that lack a functional ABCA1 transporter. These studies revealed defective lipid and protein trafficking in late endocytic vesicles in Tangier disease fibroblasts that can be corrected by adenovirally mediated expression of GFP-tagged ABCA1. Our present findings suggest that ABCA1 in late endocytic vesicles (late endosomes and lysosomes) can mobilize late endocytic lipids for apoA-I-mediated cellular efflux.

The ABCA1 transporter residing in late endocytic vesicles appears to convert pools of late endocytic lipids, which would otherwise associate with NPC1, to pools that can associate with apoA-I to form the nascent HDL particle.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—{alpha}-Minimal essential medium (AMEM) was purchased from BIOSOURCE (Rockville, MD). Fetal bovine serum (FBS) was obtained from HyClone Laboratories, Inc. (Logan, UT). Lipoprotein-deficient bovine serum (LPDS) was prepared by Intracel Corp. (Rockville, MD). Glass microscope culture wells (Lab-Tek) were purchased from Thomas Scientific. Filipin was purchased from Polysciences (Warrington, PA). U18666A (3-{beta}-(2-(diethylamino))ethoxy)androst-5-en-17-one), generously supplied by Dr. W. Andrus, The Upjohn Co., was stored as a 10 mg/ml stock solution in ethanol at -20 °C. Mouse anti-human LAMP2 antibodies, developed by Dr. J. T. August, were obtained from the Developmental Studies Hybridoma Bank maintained by the University of Iowa (Iowa City, IA). Alexa-labeled secondary anti-human IgG antibodies, Alexa-568, and BODIPY-sphingomyelin were obtained from Molecular Probes (Eugene, OR).

Tissue Culture—Wild type and Tangier disease fibroblasts were derived from volunteers and confirmed patients of the Molecular Disease Branch under the guidelines approved by the NHLBI Intramural Review Board, National Institutes of Health. The diagnosis of Tangier disease was based on clinical presentation (<5 mg/dl HDL cholesterol) and confirmed by biochemical assay (cholesterol efflux from skin fibroblasts <5% of wild type controls) (23). Four different wild type cell lines and five Tangier disease cell lines, including the proband (24), were used. All five Tangier disease fibroblasts presented similar cellular phenotypes. Fibroblasts were cultured in AMEM supplemented with 10% FBS, 2 mM glutamine, and 100 units of penicillin/streptomycin/ml in humidified 95% air and 5% CO2 at 37 °C. For biochemical analyses, fibroblasts seeded at a density of 5 x 104 cells/well in plastic 24-well dishes (Costar, Cambridge, MA) were incubated for 5-7 days in AMEM medium as above. For immunocytochemical analyses, fibroblasts were seeded at a density of 20,000 cells/well in AMEM, 5% LPDS medium in 9.5-cm glass microscope wells (Nunc, Inc., Naperville, IL). Some wild type and Tangier disease fibroblasts were transiently infected with adenovirus (Adv-ABCA1-GFP) containing the expression plasmid pTRE2 (Clontech, Palo Alto, CA), encoding a chimeric ABCA1-GFP protein (pTRE2-ABCA1-GFP) (18).

Isolation and Fluorescent Labeling of Apolipoprotein—Lipoproteins were isolated by sequential ultracentrifugation as described previously (25). ApoA-I purified from human plasma (26) was over 99% pure, as determined by SDS-PAGE and amino-terminal sequence analysis. The succinimidyl ester of Alexa 568 (Molecular Probes, Eugene, OR) was conjugated to apoA-I, according to the manufacturer's instructions. Alexa 568-labeled human apoA-I was purified by gel filtration and then spin-concentrated (Vivascience, Hannover, Germany) to 1 mg/ml in phosphate-buffered saline.

Lipid Efflux Assay—Cholesterol efflux was performed as described previously (17). Nearly confluent cells were labeled with [3H]cholesterol for 32 h, washed, infected with AdvABCA1-GFP for 1 h, and then incubated for 16 h in AMEM containing 1 mg/ml BSA (AMEM/BSA) in the presence or absence of 10 µg/ml apoA-I or 10 µg/ml Alexa 568-apoA-I. Percentage efflux was calculated by subtracting the radioactive counts of blank media (AMEM/BSA) from the radioactive counts in the presence of apoA-I, and then dividing the result by the sum of the radioactive counts in the medium plus the cell fraction.

Transport of Endocytosed Sphingomyelin—The transport of sphingomyelin along the endocytic pathway was assessed as described by Pagano and Chen (27). Briefly, wild type and Tangier disease fibroblasts maintained in lipoprotein-depleted serum, as described above, were incubated with 5 µM BODIPY-sphingomyelin in Ham's F-12 medium without serum for 30 min at 37 °C, washed, chased in Ham's F-12 medium without serum for 60 min at 37 °C, washed, and then incubated in 5% defatted BSA for 30 min at 37 °C. After washing, cells were fixed in 3% paraformaldehyde for 10 min, washed, and then imaged.

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 were performed either in blocker solution containing filipin (0.05%) and goat IgG (2.5 mg/ml) or 10% FBS in phosphate-buffered saline containing saponin (0.2%). Primary antibodies used were raised against NPC1 (11) and human LAMP2. Secondary Alexa 568-labeled antibodies were used at 1:100 dilution. Fluorescence was viewed with a x40 (NA 1.3) oil immersion objective on a Zeiss 410 or 510 laser scanning confocal microscope, using a UV laser (Enterprise model Coherent, Inc.), an argon laser, and a HeNe laser, with excitation wavelengths of 364, 488, and 543 or 568 nm, for filipin, enhanced green fluorescent protein, and BODIPY, and Alexa 568 fluorescence, respectively.

Time-lapse Confocal Fluorescence Microscopy—For live cell imaging, cells prepared in glass chamber slides were maintained at 37 °C in an enclosed chamber using a feedback heater-blower (World Precision Instruments, Sarasota, FL). Time-lapse images were obtained using a 60x (NA 1.4) oil objective on an Olympus IX-70 microscope equipped with a cooled charge-coupled device camera (Orca ER) and a spinning disc confocal head. The optical slice thickness was ~1 µM. Monochromatic 488 and 568 nm excitation light for GFP and Alexa 568 was provided by argon laser excitation controlled with an acousto-optical tunable filter, and detected using 505-540- and 580-620-nm BP emission filters, respectively. A total of 60 GFP, or DiI, or Alexa 568 images were acquired at a rate of 1/s. For dual color imaging, GFP and Alexa 568 images were obtained sequentially at an acquisition rate determined by their respective intensities. Capture, animation, and export to QuickTime movie were performed using the Metamorph software (Universal Imaging, Downington, PA). Zoom, pseudocoloring, drawing, and text in QuickTime movies were added using Adobe AfterEffects software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cholesterol Is Retained in Late Endocytic Vesicles in Tangier Disease Fibroblasts—We first examined the distribution of cellular cholesterol, revealed by cholesterol-specific cytochemical staining with filipin (11), in wild type and Tangier disease human skin fibroblasts (Fig. 1A). Under conditions of cholesterol loading, Tangier disease fibroblasts accumulated cholesterol in perinuclear vesicles (Fig. 1A, c), established previously to represent late endocytic compartments (11). To determine whether cholesterol in late endocytic vesicles can traffic to the surface, intracellular pools of cholesterol that traffic to the plasma membrane were depleted in living wild type and Tangier disease fibroblasts, using cyclodextrin as an extracellular acceptor (28). As shown in Fig. 1A, the excess cholesterol retained in perinuclear vesicles in Tangier disease fibroblasts could not be depleted by cyclodextrin (Fig. 1A, d). This finding suggests that trafficking of cholesterol from late endocytic vesicles to the cell surface is perturbed in Tangier disease cells.



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  		FIG. 1.
Late endocytic vesicles in Tangier disease fibroblasts accumulate lipids that impair their movement toward the cell surface. A, cholesterol accumulates in perinuclear late endocytic vesicles in Tangier disease fibroblasts. Wild type (a and b) and Tangier disease (c and d) fibroblasts maintained in LPDS medium for 7 days were then incubated with LPDS medium containing 50 µg/ml LDL for 24 h (a and c) and then transferred to LPDS medium containing 2% CD for 24 h (b and d) prior to fixation and staining with the cholesterol-specific cytochemical stain filipin, as described under "Experimental Procedures." Note the massive accumulation (c) and retention (d) of cholesterol in perinuclear vesicles in Tangier disease fibroblasts. B, the motility of Tangier disease late endocytic vesicles is impaired. Late endosomes and lysosomes in living wild type (a) and Tangier disease (b) fibroblasts were labeled by incubation with 10 µg/ml DiI-LDL for 24 h followed by a 4-h washout. Note the perinuclear accumulation of DiI-LDL-labeled late endocytic vesicles in Tangier disease (b) fibroblasts. C, cholesterol retained in late endocytic vesicles in Tangier disease fibroblasts resides in detergent-resistant membrane. Wild type (a and b) and Tangier disease (c and d) fibroblasts maintained in LPDS medium for 7 days were then fixed as described under "Experimental Procedures." Some cells (b and d) were then extracted in 1% Triton X-100 at 4 °C prior to cytochemical staining for cholesterol with filipin as described under "Experimental Procedures." Note the retention of detergent-resistant cholesterol in perinuclear vesicles in Tangier disease (d) fibroblasts. D, sphingomyelin is retained in late endocytic vesicles in Tangier disease fibroblasts. Living wild type (a) and Tangier disease (b) fibroblasts maintained in LDPS medium for 7 days were incubated with 5 µM/ml BODIPY-SM as described under "Experimental Procedures." Note the accumulation of fluorescent sphingomyelin in perinuclear punctate structures as well as in the hypertrophied Golgi in Tangier disease (b) fibroblasts.

 
The Motility of Late Endocytic Vesicles in Tangier Disease Fibroblasts Is Impaired—We have shown above that Tangier disease fibroblasts accumulate cholesterol in late endocytic compartments and have impaired trafficking of cholesterol to the cell surface. Excess cholesterol in late endocytic vesicles has been reported to impair their movement from the perinuclear region toward the cell periphery (29). To determine whether late endosomes in Tangier disease fibroblasts exhibit impaired motility, living wild type and Tangier disease fibroblasts were incubated with DiI-LDL, in order to label late endocytic vesicles, and then monitored by time-lapse confocal fluorescence microscopy. DiI-LDL-labeled late endocytic vesicles in living Tangier disease fibroblasts remained clustered in the perinuclear region (Fig. 1B) and exhibited impaired movement toward the cell surface (Movie 1, see Supplemental Material). These findings suggest that the cellular cholesterol sequestered in Tangier disease fibroblast late endocytic vesicles impairs their movement toward the cell surface.

Cholesterol Retained in Tangier Disease Late Endocytic Vesicles Is Detergent-resistant—We next assessed the ability of detergent to extract cholesterol from late endocytic compartments in lipoprotein-depleted wild type and Tangier disease fibroblasts (Fig. 1C). Cholesterol retained in cells after cold Triton X-100 extraction has been shown to be associated with membrane microdomains enriched with sphingomyelin and other sphingolipids (30). As shown in Fig. 1C, the majority of the cholesterol that accumulates in late endocytic vesicles in Tangier disease fibroblasts is detergent-resistant and thus appears to be associated with sphingolipids.

Sphingomyelin Trafficking Is Perturbed in Tangier Disease Late Endocytic Vesicles—We next examined whether sphingomyelin was also retained in late endocytic compartments in Tangier disease fibroblasts (Fig. 1D). BODIPY-sphingomyelin was incorporated into the plasma membrane of living, lipoprotein-depleted, wild type and Tangier disease fibroblasts, and then allowed to traffic to intracellular compartments. Fluorescent sphingolipids incorporated into the plasma membrane of wild type fibroblasts trafficked to the Golgi, as reported previously (27) (Fig. 1D). Tangier disease fibroblasts presented a distinctive phenotype. Plasma membrane-derived BODIPY-sphingomyelin was retained in endocytic vesicles as well as in a markedly hypertrophied Golgi (Fig. 1D). Schmitz and co-workers (31) have reported previously that the Golgi is hypertrophied in Tangier disease fibroblasts.

Cholesterol Retained in Tangier Disease Late Endocytic Vesicles Recruits NPC1—The sterol-sensing NPC1 protein resides in a distinctive subset of late endosomes (11, 32) that plays a critical role in the relocation of endocytosed LDL-derived lysosomal cholesterol to the plasma membrane and the endoplasmic reticulum (13). To probe further the functionality of ABCA1 in late endocytic compartments, we examined the effect of lipoprotein depletion on the distribution of cholesterol and NPC1 protein in Tangier disease fibroblasts. Maintenance of wild type fibroblasts in lipoprotein-free medium depletes cholesterol and NPC1 from late endocytic vesicles (32), as shown in Fig. 2. LDL uptake in lipoprotein-depleted wild type fibroblasts enriches the membranes of late endocytic vesicles with cholesterol, which then retains NPC1 (32), as shown in Fig. 2. The excess cholesterol that accumulates in Tangier disease late endocytic vesicles (Fig. 1) might be expected to retain excess NPC1. As shown in Fig. 2, cholesterol retained in late endocytic vesicles of lipoprotein-depleted Tangier disease fibroblasts did indeed retain NPC1. Most interesting, the structure of late endocytic vesicles in ABCA1-mutant Tangier disease fibroblasts is abnormal (Fig. 3). Late endocytic vesicles in Tangier disease fibroblasts often appear to be twisted, interconnected, and often dilated tubular structures.



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  		FIG. 2.
Cholesterol retained in Tangier disease late endocytic vesicles recruits NPC1. Wild type (A-F) and Tangier disease (G-I) fibroblasts maintained in LPDS medium for 7 days were incubated for an additional 24 h in LPDS medium in the absence (A-C and G-I) or presence (D-F) of 50 µg/ml LDL and then fixed and stained with filipin to reveal the cellular distribution of cholesterol (C, F, and I) and immunostained for NPC1 (red, A, D, and G) or LAMP2 for late endosomes and lysosomes (green, B, E, and H), as described under "Experimental Procedures." Note the retention of NPC1 (G) in cholesterol-enriched (I) late endocytic vesicles (H) in sterol-depleted Tangier disease fibroblasts.

 



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  		FIG. 3.
ApoA-I reduces NPC1 in wild type but not in Tangier disease late endocytic compartments. Wild type (A-F) and Tangier disease (G-L) fibroblasts were maintained in LPDS medium for 4-5 days and then incubated for an additional 24 h in LPDS medium containing 50 µg/ml LDL for 24 h. Cells were then incubated in medium containing 0.1% BSA in the absence (A-C and G-I) or presence (D-F and J-L) of 10 µg/ml apoA-I for 24 h, and then fixed and stained with filipin to reveal the cellular distribution of cholesterol (C, F, I, and L) and immunostained for NPC1 (red, A, D, G, and J) and LAMP2 (green, B, E, H, and K) as described under "Experimental Procedures." Note the reduction in late endosomal NPC1 in the presence of apoA-I in wild type (D versus A) but not in Tangier disease (J versus G) fibroblasts, as well as the massive accumulation of NPC1 (G and J) and cholesterol (I and L) in Tangier disease late endocytic vesicles (H and K). The tubulated late endocytic vesicles seen in Tangier disease fibroblasts (J-L, arrowheads) are shown enlarged in (J'-L'). Note that the large, late endocytic vesicle, whose lumen is indicated by the asterisk, is marked by LAMP2 (K') and NPC1 (J') and is cholesterol-enriched on its surface membrane (L'). The twisted late endocytic tubule (K') indicated by the small arrowhead also contains NPC1 (J') and cholesterol (L').

 
ApoA-I-mediated Mobilization of Late Endocytic Cholesterol Is Defective in Tangier Disease Fibroblasts—To explore further the potential role of the ABCA1 transporter in the efflux of cholesterol from late endocytic vesicles, we tested the effect of apoA-I on the distribution of cellular cholesterol and NPC1. Incubation with apoA-I markedly reduced the amount of NPC1 residing in late endocytic vesicles in wild type fibroblasts (Fig. 3). In Tangier disease fibroblasts, on the other hand, cholesterol and NPC1 remained sequestered in late endocytic vesicles in the presence of apoA-I (Fig. 3). These findings are consistent with the concept that ABCA1 in late endosomes converts pools of cholesterol that retain NPC1 to pools that lipidate apoA-I.

ABCA1-GFP Expression in Wild Type and Tangier Disease Fibroblasts Stimulates Efflux of Cellular Cholesterol and Mobilizes NPC1 from Late Endocytic Compartments—We have reported previously that stable expression of ABCA1-GFP in a HeLa cell line that lacks endogenous expression of ABCA1 restored apoA-I-mediated efflux of cellular cholesterol and choline-containing phospholipids (17, 33). To gain further insight into the role of ABCA1 in late endocytic trafficking, wild type and Tangier disease fibroblasts were infected with a recombinant adenovirus that expresses ABCA1-GFP (18). As shown in Fig. 4A, ABCA1-GFP resides on the cell surface and in cholesterol-enriched late endocytic vesicles in both wild type and Tangier disease fibroblasts. As shown in Fig. 5, expression of ABCA1-GFP in wild type fibroblasts enhanced apoA-I-mediated cholesterol efflux. Concomitant with increased cellular cholesterol efflux, ABCA1-GFP expression reduced NPC1 retention in late endocytic vesicles in wild type fibroblasts (Fig. 4C).



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  		FIG. 4.
ABCA1-GFP expression in wild type and Tangier disease fibroblasts. A, ABCA1-GFP resides in cholesterol-enriched late endocytic vesicles. Wild type (a-c) and Tangier disease (d-f) fibroblasts were infected with AdvABCA1-GFP (green, a and d) and then fixed after 48 h and immunostained for LAMP2 (red, b and e), a marker for late endocytic vesicles, and stained with filipin (blue, c and f) to reveal the cellular distribution of cholesterol, as described under "Experimental Procedures." Note that ABCA1 (green) colocalizes with both LAMP2 (red) and cholesterol (blue) in both wild type and Tangier disease fibroblasts. B, ABCA1-GFP trafficking in fibroblasts. As seen in Movie 2 (see Supplemental Material), as well as this single frame (a) from Movie 2, the intracellular distribution and trafficking of ABCA1-GFP expressed in wild type (left) and Tangier disease (right) fibroblasts are similar. In Tangier disease fibroblasts, ABCA1-GFP is expressed on the plasma membrane and in endocytic vesicles (b, c, and d; single frames from Movie 3). Moreover, ABCA1-GFP expression restores late endocytic vesicle motility in Tangier disease fibroblasts, as seen in Movie 3 (see Supplemental Material). C, ABCA1-GFP reduces NPC1 in both wild type and Tangier disease late endocytic vesicles. Uninfected wild type (a and b) and Tangier disease fibroblasts (c and d), as well as wild type (e and f) and Tangier disease (g and h) fibroblasts infected with AdvABCA1-GFP (green, e and g) were fixed and immunostained for NPC1 (red, b, d, f, and h), as described under "Experimental Procedures." Note the reduction in cellular NPC1 immunofluorescence in both wild type and Tangier disease fibroblasts concomitant with ABCA1-GFP expression.

 



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  		FIG. 5.
ABCA1-GFP expression in Tangier disease fibroblasts restores apoA-I-mediated cellular cholesterol efflux. [3H]Cholesterol-labeled wild type and Tangier disease fibroblasts infected with Tet-Off recombinant adenovirus as well as wild type and Tangier disease fibroblasts infected with both Tet-Off adenovirus and AdvABCA1-GFP were incubated in medium containing 0.1% BSA in the absence or presence of apoA-I. The % of cell-associated cholesterol removed by apoA-I was measured as described under "Experimental Procedures" is shown above. Values are presented as mean ± S.D. of a representative experiment. Note expression of ABCA1-GFP enhances sterol efflux in wild type cells and corrects the defective efflux in Tangier disease fibroblasts.

 
Expression of ABCA1-GFP in Tangier disease fibroblasts restored apoA-I-mediated cholesterol efflux to normal levels (Fig. 5). Thus, we have established for the first time that the genetic defect in Tangier disease can be corrected by expression of ABCA1-GFP. As anticipated, concomitant with the normalized cellular cholesterol efflux, ABCA1-GFP expression reduced NPC1 retention in Tangier disease fibroblast late endocytic vesicles (Fig. 4C). Time-lapse confocal microscopy revealed that the distribution and trafficking of ABCA1-GFP in Tangier disease fibroblasts are indistinguishable from that observed in wild type human fibroblasts (Fig. 4B, Movie 2, see Supplemental Material). Expression of ABCA1-GFP restores normal motility to Tangier disease late endocytic vesicles (Fig. 4B, Movies 2 and 3, see Supplemental Material). The combined results of these studies support a role for the ABCA1 transporter in the conversion of cholesterol in late endocytic compartments from pools that recruit NPC1 to late endocytic vesicles to pools that can lipidate apoA-I and be removed from the cell.

ABCA1-GFP Expression Stimulates Uptake of ApoA-I into ABCA1-GFP-containing Endosomes—We next determined if exogenously supplied apoA-I is internalized into ABCA1-GFP-containing endosomes in fibroblasts. To this end, we synthesized Alexa 568-tagged apoA-I, which supports ABCA1-mediated cellular cholesterol efflux equally as well as native apoA-I (wild type fibroblasts, 7.31 ± 0.67% versus 7.20 ± 0.21%; Tangier disease fibroblasts, 0.49 ± 0.44% versus 0.18 ± 0.27%, mean ± S.D., % of total cellular [3H]cholesterol effluxed to apoA-I and Alexa 568-apoA-I, respectively).

Cellular uptake of the functional Alexa 568-apoA-I was markedly enhanced in ABCA1-GFP-expressing compared with non-ABCA1-GFP-expressing wild type fibroblasts (Fig. 6A) and in ABCA1-GFP-expressing Tangier disease fibroblasts as well (data not shown). Alexa 568-apoA-I colocalized with ABCA1-GFP on the cell surface as well as in perinuclear late endocytic vesicles (Fig. 6, A and B). These findings show that exogenous apoA-I can indeed traffic to ABCA1-containing endosomes.



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  		FIG. 6.
ABCA1-GFP expression stimulates apoA-I uptake into ABCA1-containing endosomes. A, wild type human fibroblasts infected with Tet-Off adenovirus alone (a-c) or with Tet-Off adenovirus and AdvABCA1-GFP (d-f) for 36 h were then incubated with 10 µg/ml Alexa 568-tagged apoA-I for an additional 12 h and then fixed, as described under "Experimental Procedures." ABCA1-GFP is expressed at the plasma membrane and in endocytic vesicles (green, d). Note the marked uptake of uptake of Alexa 568-tagged apoA-I (red, e) in fibroblasts expressing ABCA1-GFP and the extensive colocalization of Alexa 568-tagged apoA-I in ABCA1-GFP-containing endosomes in the merged image (yellow, f). B, apoA-I traffics to and from the plasma membrane in ABCA1-GFP-containing endosomes. Wild type human fibroblasts infected Tet-Off adenovirus and AdvABCA1-GFP (d-f) for 36 h were then incubated with 10 µg/ml Alexa 568-tagged apoA-I for an additional 12 h, washed, and observed by time-lapse confocal microscopy at 37 °C, as described under "Experimental Procedures." ABCA1-GFP was expressed on the cell surface as well as in early and late endocytic vesicles (a). ApoA-I (b, red) traffics in ABCA1-GFP-containing endosomes (a, green) (Movie 4; see Supplemental Material). Note the extensive colocalization of apoA-I and ABCA1-GFP, seen as yellow in c, and the merged image of a and b (Movies 4-6; see Supplemental Material). The boxed regions in the merged image (c) are shown enlarged in (d-f). The intracellular ABCA1-GFP-containing vesicle, pseudocolored yellow (d), shuttles apoA-I to the site at the cell surface (e) denoted by the arrowhead (d and e, Movie 5, see Supplemental Material). The intracellular ABCA1-GFP-containing vesicle, pseudocolored yellow (f and g, at the arrow), shuttles apoA-I from the cell periphery (f) to the perinuclear region enriched with late endocytic vesicles (g) (as shown in Movie 6, see Supplemental Material).

 
Time-lapse confocal microscopy confirmed the extensive colocalization of apoA-I and ABCA1-GFP in endocytic compartments (Fig. 6B, Movies 4-6, see Supplemental Material) and revealed that ABCA1-containing vesicles can shuttle apoA-I to (Movie 5, see Supplemental Material) and from (Movie 6, see Supplemental Material) the plasma membrane. These time-lapse confocal studies also revealed vesicular trafficking pathways for ABCA1 and apoA-I that link perinuclear late endocytic vesicles to endosomes that dock at the cell surface (Movies 4-6, see Supplemental Material).

NPC1 Sorting Is Defective in Tangier Disease Fibroblasts—The potential role of the ABCA1 transporter in protein sorting in late endocytic compartments was examined by testing the effect of the hydrophobic amine U18666A on the subcellular distribution of NPC1. U18666A blocks cholesterol and NPC1 transport out of the hybrid organelles formed after the fusion of NPC1-late endosomes with lysosomes (11). As anticipated, U18666A induced accumulation of cholesterol in the lysosomes of both wild type and Tangier disease fibroblasts (Fig. 7). Surprisingly, however, unlike wild type cells, U18666A did not trap NPC1 in the cholesterol-laden lysosomes of Tangier disease fibroblasts. Instead, NPC1 protein remained sequestered in abnormally tubulated, cholesterol-poor, late endosomes in U18666A-treated Tangier disease fibroblasts (Fig. 7). This finding suggests that the sorting of NPC1 protein in late endocytic compartments is perturbed when the ABCA1 transporter is mutated. The observed defect in the sorting of NPC1 in Tangier disease fibroblast late endocytic vesicles is consistent with the localization and functionality of the ABCA1 transporter at this site.



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  		FIG. 7.
In Tangier disease fibroblasts, U18666A traps NPC1 in cholesterol-poor late endocytic tubules and not in cholesterol-laden lysosomes. A, wild type (a-c) and Tangier disease (d-f) fibroblasts were incubated in LPDS medium for 7 days, then incubated with LPDS medium containing 50 µg/ml LDL and 10 µg/ml U18666A for 24 h, and then fixed and stained with filipin to reveal the cellular distribution of cholesterol (blue, c and f) and immunostained for NPC1 (red, a and d) and LAMP2 (green, b and e), as described under "Experimental Procedures." Note, as established previously (28), U18666A treatment of wild type fibroblasts sequesters NPC1 (red, a) in cholesterol-laden (blue, c) LAMP (+) lysosomes (green, b). In Tangier disease fibroblasts, in contrast, NPC1 is sequestered in cholesterol-poor late endocytic tubules, whereas cholesterol is trapped in vesicular lysosomes devoid of NPC1. B, details of features in Tangier disease fibroblast shown in A. The tubule indicated by the arrow in d, e, and f is shown enlarged as d', e', and f ' in B. Note the tubule contains NPC1 (d', red) as well as the late endocytic marker LAMP2 (e', green) but is devoid of luminal cholesterol (f ', blue). The lysosome indicated by the arrowhead in d, e, and f is shown enlarged as d'', e'', and f '' in B. Note the punctate vesicle at the tip of the arrowhead is a lysosome marked by LAMP2 (e'', green) and is cholesterol-laden (f '', blue) but is devoid of NPC1 (d''). The merge of images d, e, and f is shown enlarged as g.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our present studies reveal that ABCA1-mediated modulation of late endocytic lipid and protein trafficking plays a role in cellular cholesterol trafficking and metabolism. We have shown that in Tangier disease fibroblasts, both classes of lipids known to efflux from cells in an ABCA1-dependent manner, cholesterol and sphingomyelin, were found to be retained in detergent-resistant membranes in late endocytic vesicles. The retention of plasma membrane-derived fluorescent sphingolipid analogs in late endocytic vesicles in lysosomal storage disease fibroblasts has been shown to require the presence of excess cholesterol (34). Lipoprotein-depleted Tangier disease fibroblasts present a distinctive phenotype, because both endocytosed cholesterol and endocytosed fluorescent sphingomyelin are retained in late endocytic vesicles. Lipid-enriched Tangier disease late endocytic vesicles were observed to be abnormally clustered in the perinuclear region. Time-lapse confocal microscopy revealed impaired motility of Tangier disease late endocytic vesicles, consistent with previous reports of impaired late endocytic motility induced by cholesterol accumulation (29). ABCA1-GFP expression in Tangier disease fibroblasts restored endosomal vesicle motility as well as apoA-I-mediated cholesterol efflux. Taken together, these findings strongly suggest a specific role for ABCA1 in the mobilization of both cholesterol and sphingomyelin from late endosomes, consistent with a previous report (21). Additional evidence for a functional role of the ABCA1 transporter in late endocytic compartments is provided by the present finding of abnormal morphology and protein trafficking in Tangier disease late endocytic vesicles.

We established previously the localization of NPC1 to late endocytic vesicles that are distinctive from cholesterol-enriched (wild type) or cholesterol-laden (NP-C disease) lysosomes (11, 32). NPC1 in late endosomes functions to redistribute LDL-derived cholesterol from lysosomes to other cellular sites including the plasma membrane and endoplasmic reticulum (13). In wild type fibroblasts, cholesterol enrichment of late endocytic vesicles by LDL uptake recruits NPC1 to its site of cellular function in late endosomes (32). In Tangier disease fibroblasts, cholesterol-enriched late endocytic vesicles retained NPC1. ABCA1-GFP expressed in both wild type and Tangier disease fibroblasts was found to traffic to cholesterol-enriched late endocytic vesicles and to reduce NPC1 immunostaining concomitant with increased apoA-I-mediated cellular cholesterol efflux. Expression of ABCA1-GFP also markedly stimulated endocytosis of a functional fluorescent apoA-I into ABCA1-GFP-containing vesicles. Time-lapse confocal microscopy revealed the existence of vesicular trafficking pathways that can shuttle apoA-I in ABCA1-containing late endosomes to and from the cell surface.

Our present findings are consistent with the model illustrated in Fig. 8. ABCA1 and apoA-I on the cell surface are internalized into early endosomes and can either recycle back to the plasma membrane or traffic to late endocytic compartments. ABCA1 in late endocytic vesicles converts late endocytic pools of cholesterol that retain NPC1 to pools that together with phospholipids can associate with apoA-I. The "lipidated" apoA-I in late endocytic vesicles traffics to the cell surface and is released as the nascent HDL particle. Thus, a pool of lipid-poor apoA-I is predicted to traffic from the cell surface to late endocytic vesicles and, in an ABCA1-dependent manner, return to the cell surface in a lipid-rich state. We cannot exclude the possibility that ABCA1-mobilized endocytic lipids can traffic to other cellular sites where they can also associate with apoA-I.



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  		FIG. 8.
Model for pathway of ABCA1-mediated intracellular lipidation of apoA-I. ApoA-I (red) binds to the cell surface and is internalized along with ABCA1 (green) into early endosomes. The portion of ABCA1 and apoA-I that does not recycle to the cell surface is delivered to late endocytic compartments. ABCA1 in late endocytic vesicles converts pools of late endocytic cholesterol (yellow) that recruit NPC1 to pools that associate with apoA-I. Lipidated apoA-I in the lumen of the vesicle traffics back to the cell surface where it is released as the nascent HDL particle. ABCA1 at the cell surface and in early endocytic compartments may also mediate the lipidation of apoA-I.

 
ABCA1 in late endocytic vesicles appears to provide the opportunity for the removal of excess endocytosed LDL-derived cholesterol from the cell, prior to its potential distribution to other cellular sites by NPC1. Late endocytic ABCA1 likely plays a critical role in the macrophage, where unregulated uptake of oxidized LDL into late endocytic compartments via scavenger receptors underlies foam cell formation.

ApoA-I has been shown recently (35, 36) to retard calpain-mediated ABCA1 degradation at the cell surface. Thus, apoA-I may be required to allow delivery of intact, functional ABCA1 from the plasma membrane to late endocytic compartments. Consistent with this concept, we found that apoA-I decreased the retention of NPC1 to late endocytic vesicles in cholesterol-enriched wild type fibroblasts.

Although the primary substrates and mechanism of action of ABCA1 have not been precisely defined, the current consensus concept of ABCA1 functionality involves modification of membrane lipid microdomains (37, 38). Our current findings suggest that ABCA1-mediated modifications in late endocytic membranes underlie lipidation of a cellular pool of apoA-I. The plasma membrane as well as early endocytic vesicles represent additional potential sites of ABCA1-mediated lipidation of apoA-I. Support for this concept is provided by the recent demonstration that the ABC transporters, P-glycoprotein, MRP1, and breast cancer resistance protein, reside and function in lysosomes as well as at the plasma membrane (39). We speculate that the ABCA1 transporter mediates membrane modifications that may be linked to site-specific downstream effector pathways at different subcellular sites.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains Movies 1-6. Back

§ To whom correspondence should be addressed: NHLBI, Molecular Disease Branch, 10/7N115, National Institutes of Health, 10 Center Dr., Bethesda, MD 20892. Tel.: 301-496-3195; Fax: 301-402-0190; email: neufelde{at}mail.nih.gov.

1 The abbreviations used are: ABCA1, ATP-binding cassette protein A1; BSA, bovine serum albumin; DiI-LDL, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-low density lipoprotein; FBS, fetal bovine serum; GFP, green fluorescent protein; HDL, high density lipoprotein; LAMP2, lysosomal-associated membrane protein 2; MRP1, multidrug resistance protein 1; NP-C, Niemann-Pick type C disease; NPC1, Niemann-Pick C1 protein; AMEM, {alpha}-Minimal essential medium; LPDS, lipoprotein-deficient bovine serum; BODIPY, 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene. Back



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 ABSTRACT
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 DISCUSSION
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