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Originally published In Press as doi:10.1074/jbc.M507733200 on October 4, 2005

J. Biol. Chem., Vol. 280, Issue 48, 39942-39949, December 2, 2005
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Intracellular Lipidation of Newly Synthesized Apolipoprotein A-I in Primary Murine Hepatocytes*

Jovana Maric, Robert S. Kiss, Vivian Franklin, and Yves L. Marcel1

From the Lipoprotein and Atherosclerosis Research Group, University of Ottawa Heart Institute, Departments of Biochemistry, Microbiology, and Immunology, and Pathology and Laboratory Medicine, University of Ottawa, Ottawa, Ontario K1Y 4W7, Canada

Received for publication, July 15, 2005 , and in revised form, September 28, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatocytes, which are the main site of apolipoprotein (apo)A-I and ATP-binding cassette transporter A1 (ABCA1) expression, are also the main source of circulating high density lipoprotein. Here we have characterized the intracellular lipidation of newly synthesized apoA-I, in primary hepatocytes cultured with [3H]choline to label choline-phospholipids, low density lipoprotein-[3H]cholesterol to label the cell surface, or [3H]mevalonate to label de novo synthesized cholesterol. Phospholipidation of apoA-I is significant and most evident in endoplasmic reticulum (ER) and medial Golgi, both in the lumen and on the membrane fractions of the ER and medial Golgi. In the presence of cycloheximide, endogenous apoA-I is substantially phospholipidated intracellularly but acquires some additional lipid after export out of the cell. In cells labeled with low density lipoprotein-[3H]cholesterol, intracellular cholesterol lipidation of apoA-I is entirely absent, but the secreted apoA-I rapidly accumulates cholesterol after secretion from the cell in the media. On the other hand, de novo synthesized cholesterol can lipidate apoA-I intracellularly. We also showed the interaction between apoA-I and ABCA1 in ER and Golgi fractions. In hepatocytes lacking ABCA1, lipidation by low density lipoprotein-cholesterol was significantly reduced at the plasma membrane, phospholipidation and lipidation by de novo synthesized sterols were both reduced in Golgi compartments, whereas ER lipidation remained mostly unchanged. Therefore, the early lipidation in ER is ABCA1 independent, but in contrast, the lipidation of apoA-I in Golgi and at the plasma membrane requires ABCA1. Thus, we demonstrated that apoA-I phospholipidation starts early in the ER and is partially dependent on ABCA1, with the bulk of lipidation by phospholipids and cholesterol occurring in the Golgi and at the plasma membrane, respectively. Finally, we showed that the previously reported association of newly synthesized apoA-I and apoB (Zheng, H., Kiss, R. S., Franklin, V., Wang, M. D., Haidar, B., and Marcel, Y. L. (2005) J. Biol. Chem. 280, 21612–21621) occurs after secretion at the cell surface.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Liver and intestine, which are the major sites of expression for apoA-I (1, 2) and ABCA1 (3–8), are the main contributors to high density lipoprotein (HDL)2 synthesis and secretion. The recent targeted inactivation of the hepatic ABCA1 demonstrated unequivocally that the liver is indeed the major site of synthesis, accounting for 83% of the circulating HDL (9). However, it still remains unclear whether apoA-I synthesized in the hepatocyte is lipidated intracellularly and/or at the cell surface. Banerjee and Redman (10, 11) showed that in the avian hepatocyte the initial lipidation of the protein occurs in Golgi fractions but the newly formed lipoprotein particles are not immediately mature. Chisholm and colleagues (12) reported that in HepG2 cells half of the lipidation of apoA-I occurs intracellularly and continues after secretion at the cell surface. Although in chicken livers, the newly secreted apoA-I accumulates lipids necessary to obtain the structure of a mature HDL, this is not the case in primary hepatocytes and HepG2 cells where lipidpoor apoA-I has often been found in the medium (13, 14).

In previous work (15), we addressed this issue indirectly, comparing the phospholipidation of endogenously synthesized apoA-I and exogenously added apoA-I. We showed that endogenous apoA-I consistently acquired more phospholipids and formed more HDL size particles than exogenous apoA-I (15). The phospholipidation of newly synthesized apoA-I is for the most part dependent on ABCA1, but the transfer of cholesterol to apoA-I is controlled by more complex pathways less dependent on ABCA1 (16). Unlike apoB, which is degraded if not sufficiently lipidated, apoA-I can be secreted as poorly lipidated complexes, perhaps even as a lipid-free protein. As indicated above, endogenously synthesized apoA-I forms particles of the same size in ABCA1-deficient hepatocytes and control cells, only it forms much fewer of the large particles in the absence of ABCA1 (15). Therefore apoA-I secretion is not controlled by lipid availability and the type of particle formed is dictated by the apoA-I structure and its ability to form dimers and tetramers that accommodate specific amounts of lipids. Here, we investigated the different steps of apoA-I lipidation as it traffics from the endoplasmic reticulum (ER) to the Golgi apparatus, until its secretion. We have identified early steps of phospholipidation in the ER that are independent of ABCA1 activity, and we characterized the intracellular compartments that contribute to the transfer of cholesterol to apoA-I during secretion. We also define the site of apoA-I association with apoB-containing lipoproteins.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—[1,2-3H]Cholesterol, [5-3H]mevalonolactone-Rs, [methyl-3H]choline chloride were obtained from PerkinElmer Life Sciences. Williams Medium E, HepatoZYME-SFM, and antibiotic-antimycotic were purchased from Invitrogen. Rabbit polyclonal anti-human apoA-I antibody was purchased from Calbiochem. Monoclonal antibodies directed against human apoA-I (a combination of 4H1 (against the extreme N terminus) and 5F6 (against the central region)) were obtained as previously described (17, 18) and biotinylated with Sulfo-NHS-Biotin from Pierce. Polyclonal anti-mouse apoB antibody was purchased from Biodesign International. Streptavidin-horseradish peroxidase conjugate and protein G-Sepharose were obtained from Amersham Biosciences. Primary anti-mouse apoB antibody was a gift from Dr. Ross Milne (University of Ottawa Heart Institute). Rabbit anti-mouse TGN38 antibody was obtained from BD Biosciences. Rabbit anti-mouse EEAI antibody was obtained from Affinity Bioreagents. Rabbit anti-mouse mannosidase II (ManII) antibody was a gift from Dr. Zemin Yao (University of Ottawa). Rabbit anti-mouse calnexin antibody was obtained from Stressgen Bioreagents. Sheep anti-mouse IgG, horseradish peroxidase-linked whole antibodies and donkey anti-rabbit IgG, horseradish peroxidase-linked whole antibodies were obtained from Amersham Biosciences. Nycodenz gradient maker, fibronectin, cycloheximide, stigmasterol, Sil-A derivatization chemical, and collagenase were obtained from Sigma. Complete protease inhibitor mixture was obtained from Roche. Acrylamide-Bis solution and SDS were obtained from Bio-Rad. Eco-Lite scintillation liquid and TEMED were obtained from ICN Biomedicals. SuperSignal chemiluminescent substrate was obtained from Pierce.

Cell Culture—Primary mouse hepatocytes were isolated from C57BL/6 ABCA1+/+ and C57BL/6 ABCA1–/– mice (a kind gift from Dr. Edward M. Rubin, DOE Joint Genome Institute, Berkeley, CA) by collagenase liver perfusion. The cells were plated on fibronectin-coated 10-cm plates and grown in Williams media (Invitrogen). [3H]Choline (10 µCi/ml) and [3H]mevalonate (15 µCi/ml) were directly added to loading media (hepatozyme media with 1% L-glutamine and 1% antimycotic antibiotic reagent). [3H]Cholesterol (10 µCi/ml) was dried under nitrogen into a thin film, then solubilized in a small volume of ethanol. LDL was added to the [3H]cholesterol and equilibrated with the LDL for one-half hour before adding to loading media. Five hours after the initial seeding, cells were incubated with loading media labeled with [3H]choline, [3H]mevalonate, or [3H]cholesterol. The next day (24 h later) the labeled media was removed and the cells were infected for 1 h with a recombinant adenovirus encoding apoA-I (Ad-AI) or control luciferase (Ad-Luc) at a multiplicity of infection of 75:1 plaque-forming units per cell in Williams Medium E without serum (19). After the 1-h infection, the adenovirus containing media was removed and the radioactive media returned for another 24 h.

Cellular Fractionation and Preparation of Nycodenz Gradient—The subcellular fractionation protocol was essentially as described in Tran et al. (20). At the end of the labeling period, the cells were washed twice with Williams media. At time 0, cycloheximide in hepatozyme media was added to cells (3.55 µl/ml of 100 mM stock). At the appropriate time point (0, 20, 40, and 60 min), the cells were washed twice with cold PBS and then scraped with a plastic scraper. The collected cells were then spun down at 2,000 x g for 5 min at 15 °C. The pellet was resuspended in membrane solubilization buffer (10 mM Tris-HCl, pH 7.4, 250 mM sucrose in ddH2O, Complete protease inhibitor mixture, 5 µM EDTA, pH 8) and homogenized using a ball bearing homogenization apparatus. The cell homogenate was then transferred to 15-ml glass Corex tubes and centrifuged at 9000 x g for 10 min at 5 °C, and the supernatant was kept. Nycodenz stock solution (27.6% Nycodenz in 10 mM Tris-HCl, pH 7.4, 3 mM KCl, 1 mM EDTA) and saline buffer were used to prepare four Nycodenz solutions of increasing percent concentrations (10, 14.66, 19.33, and 24%) and 2.5 ml of each were loaded from the bottom of the tube (Beckman Polyallomer Centrifuge Tubes) in decreasing percentage order. The tubes were then sealed with a piece of parafilm, and a linear gradient was formed by placing the tubes horizontally for 45 min at room temperature. The tubes were then centrifuged at 37,000 x g for 4 h at 15 °C (Beckman L8–70 M Ultracentrifuge). The supernatant was layered on top of the created Nycodenz gradient and centrifuged at 37,000 x g, for 1.5 h at 15 °C (SW41 rotor). Following the spin, each tube was fractionated into 15 aliquots. These aliquots were stored at 4 °C for Western blotting and determination of radioactive content.

Separation of Lumenal and Membrane-bound Fractions—To separate microsomal lumen from membranes, the 15 aliquots were grouped into 3 microsomal fractions (aliquots 1–3 distal Golgi fraction, 4–8 medial Golgi fraction, and 9–15 ER fraction). 1.5 ml were removed from each fraction and 1.5 ml of 0.2 M Na2CO3, pH 12.4, were added to each condition. The samples were allowed to mix on a rotator for 30 min at room temperature. The samples were transferred to open-end tubes (Beckman Polyallomer Thick Wall 3.2 ml tubes) and centrifuged at 70,000 x g for 30 min at 15 °C (TLA 100.4/TLA110). The supernatant, representing the lumenal content of each intracellular compartment, was collected and the pH adjusted to 8 with 75 µl of 2.5 N HCl. The pellet representing the membrane fraction was resuspended in 250 µl of membrane solubilization buffer.

Immunoprecipitation—Immunoprecipitation was preformed on subcellular and media samples collected during the fractionation procedure. 90 µl of anti-apoA-I antibody (Calbiochem) was added to 3 ml of sample. The samples were incubated at 4 °C overnight with continuous mixing. The next day, 100 µl of protein G-Sepharose (Amersham Biosciences) was added to the samples and the samples were incubated overnight at 4 °C. The next day, samples were centrifuged at 3,000 x g at 10 °C for 10 min (Sorvall RT 6000D). The supernatant was discarded and the pellet washed 3 times. When immunoprecipitating apoB with rabbit anti-apoB antibody (Biodesign) the same procedure was followed except that protein A-Sepharose (Amersham Biosciences) was used. When apoA-I was immunoprecipitated from a membrane sample, the sample was treated with 1% Triton X-100 for 30 min at 4 °C. The solubilized sample was then centrifuged at 75,000 x g for 30 min at 15 °C (TLA 100.4/TLA110) and the supernatant loaded on a gel.

Western Blotting—Protein samples from aliquots and fractions were separated on a 3–15% SDS-PAGE and then transferred to a nitrocellulose membrane (Bio-Rad). The membrane was blocked (5% skim milk in 0.5% Tween PBS) for 30 min and washed three times for 15 min in 0.5% Tween PBS. Primary mouse anti-human apoA-I antibodies 5F6 and 4H1 (University of Ottawa Heart Institute) were added at 1:1000 dilution. Primary anti-mouse apoB antibody (a gift from Dr. Ross Milne) was added at 1:1000 dilution. Rabbit anti-mouse TGN38 antibody (BD Biosciences) was added at 1:1000 dilution. Rabbit anti-mouse EEAI antibody (Affinity Bioreagents) was added at 1:1000 dilution. Rabbit anti-mouse ManII antibody (a gift from Dr. Zemin Yao) was added at 1:2000 dilution. Rabbit anti-mouse calnexin antibody (Stressgen Bioreagents) was added at 1:3000 dilution. After an overnight incubation at 4 °C, the membrane was washed three times at 15 min in 0.5% Tween-PBS. Secondary antibodies were added at 1:5000 dilution and the membrane was incubated for 1 h at room temperature. For apoA-I and apoB, sheep anti-mouse IgG horseradish peroxidase-linked whole antibodies were used (Amersham Biosciences). For all the remaining proteins, donkey anti-rabbit IgG horseradish peroxidase-linked whole antibodies were used (Amersham Biosciences). The membrane was washed 3 times for 15 min in 1x PBS with 0.5% Tween. The membrane was then washed with SuperSignal chemiluminescent substrate (Pierce) for 5 min and exposed to film.

Lipid Extraction and TLC Analysis—Lipids associated with immunoprecipitated samples (1 ml) were extracted by the method of Bligh and Dyer (21). To determine the amount of [3H]choline incorporated into sphingomyelin and phosphatidylcholine, phospholipids were separated by thin layer chromatography (TLC) using a polar solvent system containing CHCl3:methanol:acetic acid:formic acid:H2O (105:45:18:6:3, v/v). The lipids on the TLC plate were stained with iodine, and the appropriate bands scraped. The associated radioactivity was determined by scintillation counting. To determine the amount of [3H]mevalonate incorporated into cholesterol versus cholesteryl esters, the non-polar solvent system hexane:diethyl ether:acetic acid (105:45:1.5) was used.



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  		FIGURE 1.
Subcellular fractionation on Nycodenz gradient. Western blot of subcellular marker proteins. 15 fractions from the density gradient (see "Experimental Procedures") were separated by SDS-PAGE, and analyzed by Western blot. TGN38 is a distal Golgi marker protein. EEAI is a marker of early endosomes. ManII is a marker of medial Golgi. Cnx is an ER protein marker.

 
Gas Chromatography Analysis—For GC analysis, samples from the immunoprecipitation of apoA-I and media samples were resuspended in PBS and the lipids extracted (21). The lipid extract was divided in two. One sample for unesterified cholesterol determination was dried under nitrogen and stored at –20 °C. The other sample for total cholesterol determination was hydrolyzed with alcoholic KOH at 60 °C for 1. Cholesterol was extracted with hexane and evaporated under nitrogen and frozen at –20 °C. Stigmasterol was added as an internal standard and the dried samples were treated with Sil-A derivatization chemical (Sigma) prior to GC-MS analysis.

Isolation of LDL—LDL was isolated from a peripheral blood sample by sequential density ultracentrifugation. The concentration of LDL was calculated using the Markwell Lowry protein assay.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Primary mouse hepatocytes were isolated from C57BL/6 ABCA1+/+ mice by collagenase liver perfusion, and cultured as described under "Experimental Procedures." The cells were lysed and subcellular fractions isolated by Nycodenz gradient centrifugation. Antibodies against specific intracellular marker proteins were used to identify the various fractions (Fig. 1). The protein markers EEAI and TGN38 identify early endosomes and distal Golgi (dGolgi) microsomes, respectively. ManII and calnexin are markers of medial Golgi (mGolgi) and ER microsomes, respectively. The first three fractions were pooled to represent dGolgi with some early endosome vesicles. Fractions 4–6 and 9–15 were pooled to represent mGolgi and ER vesicles, respectively.

Steady State ApoA-I Lipidation with [3H]Choline-phospholipids or LDL-derived [3H]Cholesterol and ApoA-I-mediated Export of Cholesterol—To determine whether phospholipids and cholesterol become associated with apoA-I in ER and Golgi compartments, primary mouse hepatocytes were labeled with [3H]choline or LDL-[3H]cholesterol, infected with either Ad-Luc or adenovirus expressing apoA-I, and labeling was pursued by continued culture in labeling medium. The cells were then washed, homogenized, and subcellular fractions isolated. ApoA-I was immunoprecipitated and the associated radioactive lipid measured. We observed very significant acquisition of choline-labeled lipids by apoA-I in the ER compartment (Fig. 2). The presence of phospholipidated apoA-I was also evident in the mGolgi, although radioactivity was consistently lower in Golgi compared with ER. Under the same conditions, when the cells were labeled with [3H]cholesterol derived from LDL, lipidation with cholesterol was almost completely absent. We have shown previously that cholesterol delivered with LDL preferentially labels the cell surface pool, including the recycling endosome compartment (16). Therefore the absence of cholesterol labeling of ER and Golgi by exogenous cholesterol explains these negative results.



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  		FIGURE 2.
ApoA-I lipidation in the lumens of the subcellular fractions of ABCA1 WT hepatocytes. Primary mouse hepatocytes were isolated from C57BL/6 ABCA1 WT cells and labeled with [3H]choline (10 µCi/ml) and LDL-[3H]cholesterol (10 µCi/ml) incorporated in LDL (5 µg/ml) as described under "Experimental Procedures." The lumenal apoA-I was immunoprecipitated using anti-apoA-I primary antibody and protein G-Sepharose beads and radioactivity were counted. Results are presented as the mean ± S.D. of three experiments.

 
ApoA-I Protein Localization following Cycloheximide Treatment— Pulse-chase experiments were performed, where the cells were cultured as described above and at time 0 treated with cycloheximide to inhibit any new production of apoA-I (Fig. 3). The cells were collected at 0, 20, and 40 min and subcellular fractions isolated as described. ApoA-I was located both on the membrane fractions and in the lumen of ER, mGolgi, and dGolgi for the first hour following cycloheximide addition and accumulated in the media (Fig. 3A). Densitometric quantification of Western blot bands show that initially, apoA-I is located in all three intracellular compartments, mainly in the dGolgi and mGolgi, whereas the ER seems to contain the smallest amount, a result compatible with a previous report of the rapid transit of apoA-I (10). With time the ER compartment emptied and apoA-I transits into mGolgi and dGolgi. After 40 min, apoA-I leaves the mGolgi to accumulate in the media.

The subcellular fractions were treated with sodium carbonate to release the proteins residing inside the ER and Golgi vesicles and centrifuged to separate lumen and membrane compartments. Western blotting (Fig. 3B) shows at 0 and 20 min the presence of a large amount of membrane-bound apoA-I in the mGolgi fraction, which contrast with the low level of membrane-bound apoA-I in dGolgi. At 40 min in the dGolgi, we observed only the presence of lumenal apoA-I, which suggests that the progressive lipidation of apoA-I caused the release of apoA-I as a soluble complex.

Intracellular Lipidation of ApoA-I in ABCA1 WT Cells after Cycloheximide Treatment—Hepatocytes from C57BL/6 ABCA1+/+ mice were labeled with [3H]choline, [3H]cholesterol, or [3H]mevalonate and infected with Ad-human-AI as described above. Phospholipid and cholesterol lipidation of apoA-I was assessed after addition of cycloheximide for 0, 20, and 40 min.

In [3H]choline-labeled cells, immunoprecipitation of apoA-I in the different subcellular fractions shows that the phospholipidation of apoA-I starts in the ER, and continues through the Golgi leading to the secretion of phospholipidated apoA-I (Fig. 4A). Clearly, apoA-I binds phospholipids during or soon after its translation, and ER apoA-I, despite its low concentration, retains a high level of labeled phospholipids throughout the chase. ApoA-I remained significantly lipidated during transit through the mGolgi, but its clearance from the dGolgi (Fig. 3B) may explain the low level of apoA-I-associated phospholipids in that fraction (Fig. 4A). After 20 min, it is evident that some of the intracellularly phospholipidated apoA-I left the cell and could be found in the media, where it continues to accumulate.



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  		FIGURE 3.
ApoA-I levels in subcellular fractions and media following cycloheximide treatment of ABCA1 WT hepatocytes. Primary mouse hepatocytes from C57BL/6 WT were isolated and treated with cycloheximide (3.55 µl/ml of 100 mM stock) at time 0. Whole cell (A) and media (B) proteins were separated by SDS-PAGE and analyzed by Western blots for apoA-I. The results were quantified using Quantity One software. Results are presented as the mean ± S.D. of three experiments and representative Western blots are presented. The proteins of membrane (C) and lumen (D) fractions were separated by SDS-PAGE and blotted for apoA-I.

 



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  		FIGURE 4.
Time course of apoA-I lipidation in the lumens of intracellular fractions and in effluxed media following cycloheximide treatment of ABCA1 WT cells. Hepatocytes from C57BL/6 ABCA1 WT mice were isolated and grown in Williams media for 5 h. Cells were then labeled with: A, [3H]choline (10 µCi/ml); B, LDL-[3H]cholesterol (10 µCi/ml) incorporated in LDL (5 µg/ml); and C, [3H]mevalonate (15 µCi/ml) as described under "Experimental Procedures" and cycloheximide were administered to cells (3.55 µl/ml of 100 mM stock) at time 0. The lumenal apoA-I and media apoA-I from the three intracellular fractions were immunoprecipitated using anti-apoA-I primary antibody and protein G-Sepharose. The samples were washed three times in PBS and radioactivity was counted. Results are presented as the mean ± S.D. of three independent experiments.

 



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  		FIGURE 5.
Association of phosphatidylcholine (PC) and sphingomyelin (SM) with apoA-I immunoprecipitated from the lumens of intracellular fractions following cycloheximide treatment of WT cells. Hepatocytes from C57BL/6 ABCA1 WT mice were grown in Williams media for 5 h. Cells were labeled with [3H]choline (10 µCi/ml) as described under "Experimental Procedures" and treated with cycloheximide (3.55 µl/ml of 100 mM stock) at time 0. Lumenal apoA-I was immunoprecipitated and the associated lipids were extracted, separated by TLC, and radioactivity counted. Results are presented as the mean ± S.D. of one representative experiment, from three experiments performed.

 
The distribution of [3H]choline label in the phospholipids associated with apoA-I was analyzed by TLC. The lumenal fraction was isolated from the ER and Golgi compartments, and the lipids associated with immunoprecipitated apoA-I were extracted and separated by TLC. Phosphatidylcholine associated with apoA-I early in the ER lumen and peaked at 20 min (Fig. 5). Some sphingomyelin radioactivity started to associate with apoA-I early in the ER, but the majority was seen in the mGolgi (Fig. 5), where sphingomyelin is synthesized. Very little phosphatidylcholine or sphingomyelin radioactivity was measured in dGolgi.

The specificity of cholesterol binding by apoA-I was strikingly different when the cells were labeled with LDL-[3H]cholesterol (Fig. 4B), a protocol that preferentially labels the cell surface compartment (16). At all three time points, intracellular lipidation was almost non-existent. However, secreted apoA-I in the media was able to accumulate a large amount of cholesterol at the plasma membrane. This lipidation was significant at time 0 and continued to increase over the next 40 min, reflecting the transfer of cholesterol to secreted apoA-I at the level of plasma membrane and recycling endosomes. The absence of cholesterol-labeled apoA-I in the dGolgi fraction suggest that secreted apoA-I does not recycle to this fraction. To ascertain whether or not apoA-I can acquire cholesterol in the early stages of synthesis and transport, the cells were cultured with [3H]mevalonate to label de novo synthesized cholesterol, which originates in the ER and transits along the pathway of apoA-I transport. The results showed that de novo synthesized cholesterol does lipidate apoA-I early in ER and remain associated to apoA-I in Golgi compartments as well as after the secretion of the lipoprotein particle out of the cell (Fig. 4C). The amount of de novo synthesized cholesterol bound to apoA-I in ER remains constant throughout the secretion pathway. This lipidation is significant and complements the acquisition of cholesterol by apoA-I that occurs at the cell surface. TLC analysis of the lipids immunoprecipitated with apoA-I showed that [3H]mevalonate was almost exclusively found in the cholesterol fractions with very little if any with cholesteryl esters (data not shown).

It is clear that hepatic apoA-I secretion is accompanied by the export of cholesterol and phospholipids. To provide a quantitative evaluation, we measured by GC-MS the net mass of cholesterol associated with the immunoprecipitated apoA-I after a 3-h time point. In the total intracellular lumenal compartments, we found a total of 11.7 ng of cholesterol/µg of total secreted apoA-I. In all the membrane compartments, we found 2.5 ng of cholesterol/µg of secreted apoA-I. In the media, we measured 6.7 ng of cholesterol/µg of secreted apoA-I.

Intracellular Lipidation of ApoA-I in ABCA1 Knock-out Cells—The importance of hepatic ABCA1 in the lipidation of newly synthesized apoA-I has been documented by us and others (9, 15, 16), and cellular localization and traffic of ABCA1 are also well characterized (22, 23). However, we only have indirect evidence of intracellular lipidation of apoA-I by ABCA1 during secretion and no information on the sites of lipidation. To address these questions, primary mouse hepatocytes were isolated from C57BL/6 ABCA1–/–, which were cultured in Williams media for 5 h. During labeling with [3H]choline, [3H]cholesterol, or [3H]mevalonate, the cells were infected with adenovirus expressing apoA-I. As described previously, phospholipid and cholesterol lipidation of apoA-I in the absence of ABCA1 was assessed in the different subcellular fractions, which were isolated following the addition of cycloheximide for 0, 20, and 40 min.

ABCA1 deficiency resulted in a very strong decrease in apoA-I phospholipidation in the Golgi fractions and medium (Fig. 6A). Strikingly the high initial phospholipidation of apoA-I observed in ER of ABCA1 WT cells was not inhibited in the ABCA1 knock-out cells (Fig. 6A). In fact, apoA-I phospholipidation in ER is evident at least for the first 40 min following cycloheximide treatment, indicating that ER phospholipid acquisition by apoA-I is ABCA1 independent. In contrast, phospholipidation of apoA-I in the Golgi fractions was almost completely inhibited at all time points. As well, little phospholipidated apoA-I accumulated in the media outside the cell. We must conclude that the major site of ABCA1-dependent phospholipidation for newly synthesized apoA-I is in the Golgi, in keeping with previous evidence of the presence of ABCA1 in this organelle (23).

Lipidation of apoA-I by LDL-derived [3H]cholesterol was almost completely absent in the intracellular compartments of ABCA1 WT cells. As expected, intracellular cholesterol lipidation of apoA-I was not further reduced by the absence of ABCA1, but LDL-cholesterol lipidation of apoA-I secreted in the media was abolished (Fig. 6B). Lipidation of apoA-I by de novo synthesized cholesterol in ABCA1 WT cells was significant (Fig. 4C). This lipidation decreased significantly by about 60–70% in the absence of ABCA1 in almost all fractions, most significantly in the Golgi compartments and consequently, the amount of [3H]cholesterol associated with apoA-I in the media also decreased by about 60% (Fig. 6C).



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  		FIGURE 6.
Time course of apoA-I lipidation in the lumens of intracellular fractions and effluxed media following cycloheximide treatment of ABCA1 knock-out cells. Hepatocytes from C57BL/6 ABCA1–/– mice were grown in Williams media for 5 h. Cells were labeled with: A, [3H]choline (10 µCi/ml); B, LDL-[3H]cholesterol (10 µCi/ml) incorporated in LDL (5 µg/ml); and C, [3H]mevalonate (15 µCi/ml) as described under "Experimental Procedures" and treated with cycloheximide (3.55 µl/ml of 100 mM stock) at time 0. Lumenal apoA-I from each intracellular fraction and apoA-I in the effluxed media were immunoprecipitated. The samples were washed three times in PBS and the radioactivity counted. Results presented are the mean ± S.D. of three independent experiments.

 
In conclusion, intracellular phospholipidation of apoA-I was markedly inhibited in the mGolgi and at the plasma membrane of ABCA1-null hepatocytes. As well, LDL-cholesterol lipidation of apoA-I was abolished at the plasma membrane, whereas the lipidation of apoA-I by de novo synthesized cholesterol decreased significantly in ER and Golgi compartments and at the plasma membrane. Therefore the major proportion of the intracellular lipidation of apoA-I is ABCA1 dependent.

Subcellular Interaction between ApoA-I and ABCA1—To further delineate the intracellular sites of ABCA1 lipidation of apoA-I, we investigated the association of ABCA1 and apoA-I in the isolated hepatocyte subcellular fractions. ApoA-I was immunoprecipitated from ER and Golgi fractions using an anti-apoA-I primary antibody and protein G-Sepharose beads. The immunoprecipitated proteins were analyzed by SDS-PAGE and blotted with anti-mouse ABCA1 primary antibodies. ABCA1 clearly interacts with apoA-I in all three intracellular compartments of ABCA1 WT cells (Fig. 7 see three left lanes) but not in ABCA1 knock-out cells (Fig. 7, three right lanes). Control experiments using non-immune IgG did not precipitate immunoreactive ABCA1 (data not shown). These results demonstrate that apoA-I and ABCA1 interact with each other in ER and Golgi compartments, possibly leading to apoA-I lipidation.

Interaction between ApoA-I and ApoB and Secretion of Mixed Lipoproteins—The synthesis and assembly of very low density lipoprotein in hepatocytes is well characterized (24–26) and the acquisition of lipids by apoB begins early in ER and continues throughout the trans-Golgi network. We have shown recently that lipoproteins containing both apoB and apoA-I are secreted by hepatocytes. Here we sought to investigate whether the assembly of these mixed lipoproteins occurred intracellularly or whether apoA-I and apoB proteins associated after secretion.

Either human apoA-I or murine apoB were immunoprecipitated from the lumenal fractions of the pooled ER and Golgi samples. The pulled down fractions were analyzed by Western blots probed for both human apoA-I and murine apoB. When apoA-I in ER or Golgi fractions was immunoprecipitated, no apoB protein was pulled down (Fig. 8A). Similarly, when apoB from ER or Golgi was immunoprecipitated, no apoA-I protein was found (Fig. 8B). Interestingly, when the media samples were immunoprecipitated with either anti-human apoA-I or anti-murine apoB, apoB and apoA-I were, respectively, co-immunoprecipitated (Fig. 8). These results indicate that apoA-I and apoB do not interact in ER and Golgi compartments, but assemble in the last stage of secretion or at the cell surface.



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  		FIGURE 7.
Colocalization of apoA-I and ABCA1 on the membranes of hepatocyte subcellular fractions. Membrane apoA-I was immunoprecipitated from ER, medial Golgi, and distal Golgi fractions using anti-apoA-I and protein G-Sepharose beads. Samples were treated with 1% Triton X-100 for 30 min at 4 °C and centrifuged at 75,000 x g for 30 min at 15 °C. Solubilized proteins were separated by SDS-PAGE and blotted for the presence of ABCA1. Specificity of the anti-ABCA1 antibody was verified in ABCA1 knockout (KO) cells. A representative blot of three independent experiments is shown.

 
Phospholipidation of ApoA-I Occurs Inside ER and Golgi and Is Not an Artifact of Organelle Breakdown and Lysis—Cellular fractionation and separation of intracellular compartments is a complex procedure requiring physical break-up of cells and microsome break-up with sodium carbonate. Although these procedures are conducted rapidly and at 4 or 15 °C as required, some lipids bound to apoA-I may be acquired by the protein artifactually during the fractionation process. To rule out this possibility the following experiment was carried out.

Primary mouse hepatocytes isolated from C57BL/6 WT mice were labeled with [3H]choline. Cells were either infected with adenovirus expressing apoA-I (Fig. 9, bars 2 and 4) or mock infected with Ad-Luc (bars 1 and 3). Prior to cell homogenization, 5 µg of recombinant His-apoA-I (an amount equivalent to that secreted by the cells) (15) was added to mock infected cells. After homogenization, the cells were treated or not with sodium carbonate. Lumenal apoA-I or apoA-I interacting with microsome membranes were immunoprecipitated and pulled down with Protein G-Sepharose, and the radioactivity associated with precipitated apoA-I was measured.



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  		FIGURE 8.
Colocalization of apoA-I and apoB in the lumens of subcellular fractions and media. The lumenal apoA-I from ER and Golgi fractions and media apoA-I were immunoprecipitated (IP) using anti-apoA-I and protein G-Sepharose. The lumenal apoB from the same fractions and media apoB were immunoprecipitated using anti-apoB and protein A-Sepharose. The samples were washed three times in PBS, and the proteins separated by SDS-PAGE and probed for the presence of either apoB (Panel A) or apoA-I (Panel B).

 
In the samples not treated with sodium carbonate, exogenously added His-apoA-I only interacts with the exposed membranes of microsomes, whereas in the samples treated with sodium carbonate, both lumenal newly synthesized apoA-I or exogenously added apoA-I interact with broken microsome membranes. Our data show that the total amount of lipids acquired after organelle carbonate breakdown by His-apoA-I is very minor compared with the total amount of lipids acquired intracellularly by endogenous apoA-I with sodium carbonate treatment (Fig. 9). Significantly more lipids were acquired by endogenously synthesized apoA-I than by exogenously added apoA-I even without addition of sodium carbonate. In conclusion, the lipidation of endogenously synthesized apoA-I measured here occurs intracellularly, in ER and Golgi compartments, and it is not a product of artificial protein-lipid interactions.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here, we have defined the specific intracellular sites of phospholipid and cholesterol lipidation of apoA-I in primary mouse hepatocytes. As well, we have demonstrated the steps at which ABCA1 is present and necessary for lipidation of apoA-I in these intracellular compartments.

We have shown here that apoA-I in the ER undergoes an early phospholipidation, which is independent of ABCA1. The ER is the major site of phospholipid synthesis (27), and the enzymes responsible for lipid synthesis are on the cytosolic side of ER membranes (28, 29). This suggests that the initial lipidation of apoA-I, which we observed, probably occurs early during or immediately after translation. The rapid lipidation of apoA-I that takes place in the ER demonstrates the immediate avidity of de novo synthesized apoA-I for lipid upon translation and transport through the translocon. It argues against the suggestion that apoA-I may exist as a lipid-free protein. We propose that the very high level of apoA-I lipidation with phosphatidylcholine that takes place in the ER is linked both to the high specific activity of newly synthesized phosphatidylcholine on these membranes and to the need or avidity of the newly translated/translocating protein for phospholipids to achieve proper folding. Because apoA-I partially associates with the membrane in the ER (Fig. 3B), a transitional complex with phospholipid may form here. As apoA-I transits from the ER to the Golgi, excess phospholipid may then be transferred or exchanged with other membranes and a stable phospholipidated apoA-I lumenal complex results. As well, exchange of phospholipids with membranes of lower specific activity may partially dilute the label associated with apoA-I in the dGolgi compartment.



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  		FIGURE 9.
Homogenization and carbonate extractions of microsomes do not cause artifactual association of apoA-I and lipids. Hepatocytes were isolated from C57BL/6 ABCA1 WT mice, grown in Williams media for 5 h, and then labeled with [3H]choline (10 µCi/ml) as described under "Experimental Procedures." Cells were either infected with Ad-AI (second and fourth bars) or Ad-Luc (first and third bars). At the time of cell homogenization, the cells with Ad-Luc were treated with 5 µg of His-apoA-I. Half of the cells from each condition were treated with sodium carbonate (third and fourth bars). The lumenal apoA-I was immunoprecipitated using anti-apoA-I and protein G-Sepharose. The samples were washed three times in PBS, the radioactivity counted, and the results expressed as a function of total apoA-I protein. Representative results from one experiment of three independent experiments are shown.

 
The co-immunoprecipitation of apoA-I and ABCA1 in this compartment (Fig. 7) indicates that apoA-I and ABCA1 are both normally folded and able to interact. The existence of an ABCA1-independent lipidation in ER raises the possibility that ABCA1 in the ER may not be fully matured and active, either by lack of phosphorylation (30–33) or not being yet glycosylated (34). The relatively low level of ABCA1 seen here in the ER is in agreement with previous observations that there is some fluorescent tagged ABCA1 in the ER with the main pools in early and late endosomes-lysosomes (22) as well as in the Golgi (23).

We showed that apoA-I is located in all three intracellular compartments (Fig. 3A), mainly in the mGolgi and dGolgi, whereas the ER level remained low, in keeping with its reported rapid transit out of ER (10). In all compartments, apoA-I also partitions between a membrane-bound fraction and lumenal fraction (Fig. 3B), but at 40 min in the chase only lumenal apoA-I is seen in mGolgi and dGolgi, which demonstrates that at that stage apoA-I is sufficiently lipidated to be released from the membranes and form stable soluble lipoproteins. With time the ER compartment emptied and apoA-I transits into mGolgi and dGolgi. After 40 min of chase, apoA-I leaves the mGolgi to accumulate in the media.

In control hepatocytes, while newly synthesized phospholipids are transferred to apoA-I in ER and mGolgi, newly synthesized cholesterol is transferred to apoA-I in the Golgi. However, when LDL-[3H]cholesterol is used to label plasma membrane and recycling endosome, newly synthesized apoA-I does not interact with any of this labeled cholesterol until secreted (Fig. 4). This corroborates earlier results that indicated separate transfers of phospholipids and cholesterol to apoA-I during secretion (16). In ABCA1–/– hepatocytes, the generally decreased lipidation of apoA-I is consistent with earlier studies, a small amount of residual phospholipidated apoA-I is seen in the Golgi that is eventually secreted. Thus, the residual phospholipidation, reported earlier (15), is likely that which takes place in the ER, immediately post-translation (Fig. 6A). There is no intracellular cholesterol-lipidation of apoA-I by LDL-derived cholesterol in ABCA1-null hepatocytes but there is a small residual association of cholesterol with media apoA-I (Fig. 6B). We suggest that this ABCA1-independent transfer of cholesterol to apoA-I is mediated by diffusional transfer from the cell surface compartment to the phospholipidated apoA-I, originating from ER and accumulating in the media (Fig. 6C).

We previously reported the association of apoA-I and apoB in the lipoproteins secreted by murine hepatocytes (16). Early studies of apolipoprotein composition of plasma lipoproteins by Alaupovic et al. (35, 36) showed that very low density lipoprotein from certain hypertriglyceridemic subjects contained apoA-I, but there was no follow up of this observation. Here we showed that in the distal Golgi fractions the lipoproteins containing apoA-I or apoB remain separate and cannot be co-precipitated, but it is clear that during the chase in the course of 40 min the cells secrete lipoproteins that contain both apoA-I and apoB. This suggests that the surface of newly secreted very low density lipoprotein either is not saturated with apolipoproteins or that apoA-I displaces other exchangeable apolipoproteins. Our observations that apoA-I and apoB only associate after secretion could reflect the existence of separate pathways for secretion of these lipoproteins. Earlier studies have shown that the total Golgi of mouse liver contained lipoproteins of the HDL density range that included apoB100, apoB48, and apoA-I (37). However, these experiments did not include immunoprecipitations and provided no evidence of intracellular association. Thus, triglyceride-rich lipoproteins secreted by hepatocytes like those secreted by enterocytes include apoA-I, which after triglyceride hydrolysis by lipases will contribute to the pool of circulating HDL. Our studies have been conducted in murine hepatocytes, which like enterocytes synthesize apoB48. It will be important to verify the same association of apoA-I and apoB in the lipoproteins secreted by isolated human hepatocytes, which only secrete apoB100-containing lipoproteins.

Recent work by Chisholm et al. (12) studied apoA-I secretion from HepG2 cells. These authors found that apoA-I was secreted as both lipid-poor and intracellularly assembled nascent HDL. Whereas HepG2 cells do not secrete very low density lipoprotein-sized but only LDL-sized apoB containing lipoproteins (38), it is unclear if apoA-I/HDL secretion is different compared with primary hepatocytes (39). Chisholm et al. (12) separated the content of ruptured microsomal fractions into top and bottom fractions (d > 1.25, d < 1.25) representing buoyant and non-buoyant particles. On the other hand, we have separated the microsomes into three intracellular compartments providing more detail about localization of apoA-I and the localization of the lipidation steps, but with no description of the extent of buoyancy. Chisholm et al. (12) conclude simply that there is intracellular cholesterol lipidation of apoA-I; we show the specific site of phospholipid and cholesterol lipidation in a relevant primary hepatocyte model.

Thus, in conclusion, apoA-I, newly synthesized in hepatocytes, undergoes an early ABCA1-independent phospholipidation in the ER that is followed by significant phospholipidation in Golgi. ApoA-I acquires some cholesterol in ER and Golgi but the major transfer occurs at the cell surface. These pathways result in the net export of hepatic cholesterol in the HDL fraction. Finally, we have established the formation of apoA-I and apoB containing lipoproteins that are assembled after secretion.


   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

1 To whom correspondence should be addressed: 40 Ruskin St., Ottawa, Ontario K1Y 4W7, Canada. Tel.: 613-761-5254; Fax: 613-761-5281; E-mail: ylmarcel{at}ottawaheart.ca.

2 The abbreviations used are: HDL, high density lipoprotein; ABCA1, ATP-binding cassette transporter A1; apo, apolipoprotein; ER, endoplasmic reticulum; mGolgi, medial Golgi; dGolgi, distal Golgi; Ad-AI, adenovirus expressing apo A-I; Ad-Luc, adenovirus expressing luciferase; GC-MS, gas chromatography-mass spectrometry; LDL, low density lipoprotein; LDL-[3H]cholesterol, [3H]cholesterol incorporated into low density lipoprotein for delivery to cells; WT, wild-type; TEMED, N,N,N',N'-tetramethylethylenediamine; PBS, phosphate-buffered saline; TGN, trans-Golgi network; ManII, mannosidase II. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Khai Tran and Zemin Yao for assistance with intracellular fractionation, Dr. Ross Milne and Dr. Zemin Yao for generous donations of anti-apoB and anti-ManII antibodies, respectively, and Drs. Bassam Haidar, Ruth McPherson, and Ross Milne for critical reading of the manuscript. We thank Kenneth Chan and Dr. Jianjun Li for assistance with GC-MS analysis. We acknowledge the assistance of Karen Paszterko and the animal care technicians at the Heart Institute.



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 DISCUSSION
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