Assembly and Secretion of Very Low Density Lipoproteins Containing Apolipoprotein B48 in Transfected McA-RH7777 Cells

LACK OF EVIDENCE THAT PALMITOYLATION OF APOLIPOPROTEIN B48 IS REQUIRED FOR LIPOPROTEIN SECRETION*

Jelena Vukmiricaabcd, Khai Tranad, Xiquan Liange, Jing Shana, Jane Yuanabf, Brooke A. Miskieg, Robert A. Hegelegh, Marilyn D. Reshe, and Zemin Yaoabij

From the a Lipoprotein and Atherosclerosis Group, University of Ottawa Heart Institute, Ottawa, Canada K1Y 4W7, the b Department of Biochemistry, Microbiology, and Immunology, the i Department of Pathology and Laboratory Medicine, University of Ottawa, Canada K1H 8M5, the e Cell Biology Program, Memorial Sloan-Kettering Cancer Center and Graduate Program in Biochemistry and Structural Biology, Weill Graduate School of Medical Sciences of Cornell University, New York, New York 10021, and the g Robarts Research Institute, London, Ontario, Canada N6A 5K8

Received for publication, November 25, 2002, and in revised form, January 15, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We examined the role of S-linked palmitoylation of human apolipoprotein (apo) B in the assembly and secretion of very low density lipoproteins using recombinant human apoB48. There are four free cysteine residues (Cys1085, Cys1396, Cys1478, and Cys1635) within apoB48 that potentially can be palmitoylated. All four cysteine residues were substituted with serine by site-specific mutagenesis. The mutant protein was expressed in transfected rat hepatoma McA-RH7777 cells. Metabolic labeling of the stably transfected cells with iodopalmitic acid analog showed that the mutant apoB48 lacked palmitoylation. The lack of palmitoylation had little impact on the ability of apoB48 to assemble and secrete very low density lipoproteins or high density lipoproteins. Immunocytochemistry experiments using confocal microscopy failed to reveal any major alterations in the intracellular distribution of the mutant apoB48 at steady state. Pulse-chase analysis combined with subcellular fractionation showed no apparent deficiency in the movement of the mutant apoB48 protein from the endoplasmic reticulum to cis/medial Golgi. However, the mutant apoB48 lacking palmitoylation showed retarded movement toward the distal Golgi and increased association (>2-fold) with the membranes of the secretory compartments. A marginal decrease (by 15-20%) in secretion efficiency as compared with that of wild type apoB48 was also observed. These results suggest that lack of palmitoylation may influence the partitioning of apoB48 between microsomal membranes and microsomal lumen, but it does not compromise the ability of apoB48 to assemble lipoproteins.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hepatic apolipoprotein (apo)1 B100 is synthesized as a single polypeptide chain consisting of 4536 amino acids and is secreted as a major protein constituent of very low density lipoproteins (VLDL). Expression of hepatic apoB100 is regulated primarily at a post-translational level (1, 2). Proper folding of apoB100 and its association with lipids are assisted by interaction with the chaperone-like protein termed microsomal triglyceride transfer protein (3) and by post-translational modifications of the apoB100 polypeptide (4). Mutations within the microsomal triglyceride transfer protein gene are associated with abetalipoproteinemia, a genetic disorder characterized by the absence of lipoproteins containing apoB in the plasma (5). In vitro mutagenesis experiments have shown that the assembly and secretion of lipoproteins containing apoB are markedly affected by abnormal post-translational modification of apoB, including disulfide bond formation (6, 7), asparagine-linked glycosylation (8), and cysteine-linked palmitoylation (9). In most of the in vitro experiments, carboxyl terminally truncated apoB forms were used as a model to test the function of apoB post-translational modifications. The mutant apoB proteins display lowered secretion efficiency, enhanced intracellular degradation, and impaired ability to assemble into lipid-rich lipoproteins (6, 7). However, since the ability of apoB to recruit lipid in forming VLDL is profoundly influenced by apoB length (10), some of the apoB truncates used in the mutagenesis studies were too short to assemble VLDL (6, 9). For example, the apoB truncation mutant used in the palmitoylation experiments was apoB29 (the amino-terminal 29% of the full-length apoB100), which has no ability to form VLDL and can only form small, dense particles resembling that of high density lipoproteins (HDL). Therefore, the functional consequence of palmitoylation of apoB in VLDL assembly and secretion remains unclear.

Palmitoylation of human apoB has been observed in cultured human hepatoblastoma HepG2 cells (11), in plasma LDL (12), and in transfected rat hepatoma McA-RH7777 cells (9). In eukaryotic cells, the 16-carbon fatty acid palmitate is covalently linked to cysteine residues via thioester bonds (reviewed in Refs. 13, 14). There are 25 cysteine residues within apoB100, of which 16 are involved in the formation of eight disulfide bonds (15). One free cysteine residue at the carboxyl terminus of apoB100 is involved in covalent linkage with apolipoprotein (a) (16), and the remaining eight cysteine residues can potentially be modified by palmitic acid. The exact number of palmitic acids that are covalently linked to apoB100 is uncertain (17). Experiments with apoB100 secreted from HepG2 cells showed that there were three moles of palmitic acid per mole of apoB100 (11). One study with apoB29 has mapped a palmitoylation site to Cys1085, which is the only free cysteine residue within apoB29 (9). Palmitoylation is likely catalyzed enzymatically by a cytosolic palmitoyl acyl transferase in cells but can also be achieved non-enzymatically in vitro (12, 18). The mechanism by which the secretory protein apoB100 gets palmitoylated by a cytosolic enzyme is unknown.

In rat hepatic cells, both apoB100 and apoB48 can form VLDL that contains a single copy of apoB100 or apoB48. Kinetic analysis indicates that the assembly of B48-VLDL (19) and B100-VLDL (20) is achieved post-translationally in post-endoplasmic reticulum (ER) compartments. The stepwise assembly of lipids with apoB appears to be initiated when the apoB polypeptide is membrane-bound in the microsomes. Thus, association of apoB with membrane represents an important step in the VLDL assembly dynamics. The mechanisms by which apoB-membrane interaction is achieved or regulated are currently unclear. One possibility is covalent conjugation of fatty acyl chains with the apoB polypeptides. Recent experiments using McA-RH7777 cells transfected with a carboxyl terminally truncated apoB, apoB29, showed that abolishing palmitoylation of apoB29 resulted in impaired lipoprotein assembly and abnormal intracellular distribution of the mutant protein (9). The non-palmitoylated apoB29 proteins were secreted as dense lipoprotein particles and were accumulated in the Golgi apparatus (9). These results are the first indication of functional consequences of apoB polypeptides lacking palmitoylation. However, since apoB29 can only assemble HDL-like lipoproteins, this truncated apoB form did not offer the opportunity to investigate the role of palmitoylation in the assembly and secretion of VLDL. We reasoned that if palmitoylation plays a role in the assembly and secretion of VLDL, then apoB forms longer than apoB29 and, having the ability to assemble VLDL, would be a more suitable model. It has been well documented that the naturally occurring truncated form apoB48 has the ability to form VLDL in the rat liver. In the current study, we used apoB48 to assess the role of apoB palmitoylation in VLDL assembly and secretion.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum were obtained from Sigma. ProMixTM (a mixture of [35S]methionine and [35S]cysteine, 1000 Ci/mmol), protein A-SepharoseTM CL-4B beads and horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G antibody were obtained from Amersham Biosciences. Protease inhibitor mixture and chemiluminescent blotting substrate were obtained from Roche Molecular Biochemicals. Monoclonal antibody 1D1 raised against human apoB was a gift of R. Milne and Y. Marcel (University of Ottawa Heart Institute). Antibody against alpha -mannosidase II was a gift of M. Farquhar (University of California, San Diego, CA). The anti-calnexin and anti-TGN38 antibodies were obtained from StressGen and Affinity BioReagents, respectively.

Preparation of ApoB48 Expression Plasmids Containing Cys-to-Ser Substitution-- The plasmid encoding wild type apoB48 (pB48wt) was prepared using the expression vector pCMV5. The coding sequences encompassing nucleotides 20-6668 of the human apoB cDNA were excised from the published pB48 (21) by digestion with EcoRI and MluI. The EcoRI-MluI fragment was inserted into the polylinker region of pCMV5. The resulting pB48wt plasmid encodes the signal peptide and the amino-terminal 2152 amino acids of human apoB100. The Cys-to-Ser substitution was introduced into pB48wt at Cys1085, Cys1395, Cys1478, and Cys1635, consecutively, using the QuikChangeTM site-directed mutagenesis kit (Stratagene Inc.) (Fig. 1A). Sequences of all primers used for mutagenesis are shown in Table I. The resulting plasmids pB48C1085, pB48C1085-1395, pB48C1085-1478, and pB48C1085-1635 were purified in a CsCl gradient. The integrity of the coding sequence for each apoB construct was authenticated through bidirectional sequencing of the plasmid template using a total of 17 pairs of amplification primers and then in turn using these to prime direct sequencing reactions that were run on an Applied Biosystems Prism 377 automated DNA sequencer.


                              
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Table I
Oligonucleotides used for Cys-to-Ser mutagenesis
The underlined sequences indicate positions of the engineered restrictions sites introduced or altered for diagnosis purposes. XhoI and SacI sites were introduced in the vicinity of Cys1085, Cys1395, and Cys1635, respectively, whereas BamHI was removed near Cys1478.

Cell Culture and Transfection-- McA-RH7777 cells were cultured in DMEM containing 10% fetal bovine serum and 10% horse serum. Transfection of the cells with expression plasmids encoding wild type or mutant forms of apoB48 was achieved using the previously described calcium phosphate precipitation method (22). Expression of the recombinant proteins was verified by immunoblotting of the conditioned media using monoclonal antibody 1D1 (23).

Palmitoylation Assay-- Palmitoylation of recombinant wild type or mutant apoB48 was determined according to the published protocol (9). Briefly, stable cell lines were pulse-labeled for 4 h with [125I]iodopalmitate (100-250 µCi in 5 ml) and chased for 16 h in serum-free DMEM. At the end of chase, the medium was collected and concentrated. The concentrated media were delipidated using chloroform:methanol, and the delipidated samples were dissolved in buffer (4% SDS, 40% glycerol, 65 mM dithiothreitol in 0.5 M Tris-HCl, pH 6.5) prior to PAGE. Incorporation of [125I]iodopalmitate into secreted apoB48 was visualized using phosphorimaging.

Pulse-chase Analysis of ApoB-- Pulse-chase experiments (with [35S]methionine/cysteine) for determination of apoB synthesis and secretion were performed as described previously (7, 20). The results were expressed as percent of the initial counts of 35S-labeled apoB at the end of pulse labeling.

Analysis of ApoB Particles by Rate Flotation Ultracentrifugation-- Cells were continuously labeled with [35S]methionine/cysteine for 2 h in DMEM containing 20% fetal bovine serum and 0.4 mM oleate. The media were fractionated into VLDL1, VLDL2, and other lipoproteins by rate flotation ultracentrifugation in a KBr density gradient as described previously (24). The apoB proteins in each fraction were immunoprecipitated (using an anti-apoB polyclonal antibody that recognized both human and rat apoB) and subjected to PAGE and fluorography. In experiments where secreted apoB mass was determined, cells were incubated for 2 h in DMEM containing 20% fetal bovine serum and 0.4 mM oleate. The media were fractionated in a KBr density gradient as above, and apoB proteins in each fraction were immunoprecipitated using a polyclonal anti-apoB antibody, resolved by SDS-PAGE, and detected by immunoblot analysis using the monoclonal antibody 1D1.

Pulse-chase and Subcellular Fractionation-- Cells were pulse-labeled with [35S]methionine/cysteine for 15-30 min and chased for indicated times as described in figure legends. At the end of chase, cells were homogenized using a ball-bearing homogenizer (25). The postnuclear supernatant was prepared and fractionated into ER, cis/medial Golgi and distal Golgi by ultracentrifugation using a preformed Nycodenz gradient (37,000 rpm, 90 min, 15 °C, Beckman SW41 rotor). A total of 15 fractions (0.8 ml/fraction) were collected, and an aliquot of each fraction was subjected to PAGE and immunoblotted to detect alpha -mannosidase II, calnexin, TGN38, and apoB, respectively (20). In some experiments, the membranes and lumenal contents were isolated from the pooled ER, cis/medial Golgi, and distal Golgi fractions by sodium carbonate treatment followed by ultracentrifugation as described previously (20, 25). The lumenal and membrane-associated apoB proteins were analyzed by PAGE and fluorography as described above.

Immunocytochemistry-- Cells were plated on coverslips and fixed with paraformaldehyde in phosphate-buffered saline and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline as described previously (20). Monoclonal antibody 1D1 was used to probe the recombinant human apoB; ER and Golgi were probed with anti-calnexin and anti-alpha -mannosidase II antibodies, respectively (20). The experiments were performed using both transiently transfected McA-RH7777 cells and individually selected G418-resistant colonies.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of ApoB48 Mutants That Lacked Palmitoylation-- Of the nine free cysteine residues in apoB100, four are located within the amino-terminal 48% of the protein (Fig. 1A, solid ovals). We used apoB48, a naturally occurring form of apoB that can assemble VLDL, to study the role of palmitoylation in VLDL assembly. The four cysteine residues Cys1085, Cys1395, Cys1479, and Cys1635 within apoB48 were substituted with serine residues by site-directed mutagenesis (Fig. 1A, opened ovals), and the resulting cDNA constructs were transfected into McA-RH7777 cells. In stably transfected cell lines, the mutant apoB48 proteins, namely B48C1085, B48C1085-1395, B48C1085-1478, and B48C1085-1635, were secreted with the expected molecular mass and reacted with monoclonal antibody 1D1 that was specific for human apoB (Fig. 1B, bottom panel). Palmitoylation of the apoB proteins was determined by [125I]iodopalmitate labeling. Preliminary experiments with parental McA-RH7777 cells showed that the endogenous rat apoB100 was secreted as palmitoylated proteins (data not shown), which agrees with the previous report (9). [125I]Iodopalmitate labeling experiments showed that B48C1085-1635, in which all free cysteine residues were mutagenized, was expectedly non-palmitoylated (Fig. 1B, top panel). In contrast, palmitoylation occurred in the wild type apoB48 (B48wt) and B48C1085, B48C1085-1395, and B48C1085-1478 mutants containing one or more free cysteine residues. Palmitoylation of the endogenous rat apoB48 was below the limit of detection (Fig. 1B, lane labeled with McA). The intensity of [125I]iodopalmitate labeling of the recombinant mutant apoB48 proteins decreased gradually as the number of mutated cysteine residues increased (Fig. 1B, top panel), suggesting the presence of multiple palmitoylation sites within human apoB48.


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Fig. 1.   Construction and expression of apoB48 mutants. A, the top line represents human apoB100 with free cysteine residues (closed ovals) indicated. The bottom lines depict wild type apoB48 (B48wt) and four mutant forms of apoB48 containing Cys-to-Ser substitutions (open ovals) at the potential palmitoylation sites Cys1085, Cys1395, Cys1479, and Cys1635. B, palmitoylation assay of apoB48. Cells were labeled with [125I]iodopalmitate, and the 125I-labeled apoB48 were concentrated from the conditioned medium and resolved by SDS-PAGE. The labeled apoB proteins were visualized using phosphorimaging (top). An aliquot of the conditioned medium was subjected to immunoblot analysis using monoclonal antibody 1D1 (bottom).

Lack of Palmitoylation in ApoB48 Did Not Impair VLDL Secretion-- As shown previously, apoB48 could form VLDL as well as HDL-like particles in McA-RH7777 cells, and the formation of B48-VLDL was dependent on exogenous oleate (10). The culture media in all subsequent experiments was therefore supplemented with 20% FBS and 0.4 mM oleate to maximize lipogenesis and VLDL assembly/secretion. Having ascertained that B48C1085-1635 was non-palmitoylated, we examined the effect of lack of palmitoylation on the assembly and secretion of VLDL. Cell lines expressing comparable levels of B48wt or B48C1085-1635 were metabolically labeled with [35S]methionine/cysteine, and the conditioned media were fractionated by rate flotation centrifugation to separate VLDL1 (Rf > 100) and VLDL2 (Rf 20-100) from dense lipoproteins (Fig. 2A). Both B48wt and B48C1085-1635 were secreted predominantly (>80%) as HDL-like particles and a small proportion as VLDL. Quantification of 35S-labeled B48wt and B48C1085-1635 showed that the lack of palmitoylation had little impact on the density distribution among different lipoprotein fractions (Fig. 2B). Secretion of VLDL containing endogenous rat 35S-labeled apoB100 was also similar between B48wt- and B48C1085-1635-transfected cells. These results were the first indication that palmitoylation of apoB48 was not essential for the assembly and secretion of VLDL. To confirm that palmitoylation was not required for B48-VLDL secretion, we determined secretion of the apoB48 mass by immunoblotting (Fig. 2C). We also fractionated the medium lipoproteins by ultracentrifugation in a salt gradient that was designed to resolve HDL subfractions (9) (Fig. 2D). In either case (Fig. 2, C and D), there was no apparent difference in the secretion of VLDL or HDL between B48wt- and B48C1085-1635-transfected cells. Furthermore, there was no indication of a requirement of palmitoylation for B48-VLDL secretion from B48C1085-, B48C1085-1395-, or B48C1085-1478-transfected cells (Fig. 2E). Thus, abolishing palmitoylation of B48 was not associated with impairment in B48-VLDL secretion.


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Fig. 2.   Buoyant density of lipoproteins containing apoB48 secreted from transfected cells. A, cells expressing B48wt or B48C1085-1635 were labeled with [35S]methionine/cysteine for 2 h in the presence of 20% serum and 0.4 mM oleate. The medium was collected at the end of labeling and fractionated into VLDL1, VLDL2, and other lipoproteins by ultracentrifugation. Endogenous rat apoB100 (rB100) and recombinant apoB48 were immunoprecipitated from each fraction, and analyzed by SDS-PAGE and fluorography. B, quantification of 35S-labeled apoB48. The bands corresponding to apoB48 (shown in A) were excised, and the associated radioactivity was quantified by scintillation counting. The values of radioactivity associated with 35S-labeled apoB48 in different lipoprotein fractions are expressed as percent of total secreted apoB48. C, immunoblots of B48wt or B48C1085-1635 associated with lipoproteins. Cells expressing B48wt or B48C1085-1635 were cultured in media containing 20% serum and 0.4 mM oleate for 4 h. The conditioned media were fractionated by ultracentrifugation, and the apoB48 proteins in each fraction were detected by immunoblotting using monoclonal antibody 1D1. D, immunoblot of B48wt and B48C1085-1635 associated with secreted lipoproteins in the media that were fractionated by ultracentrifugation in a salt gradient (9). E, the experiments were performed the same as in C, except the that cells used were transfected with mutants containing incomplete Cys-to-Ser substitutions. Data presented in C and E are representatives of five independent experiments using different stable transformants. IDL, intermediate density lipoproteins; LDL, low density lipoproteins.

Lack of Palmitoylation in ApoB48 Resulted in Decreased Secretion Efficiency-- We next contrasted secretion efficiency between B48wt and B48C1085-1635 by pulse-chase experiments (Fig. 3A). In two independent experiments, the secretion efficiency (i.e. the percent of total newly synthesized apoB48 that was secreted during chase) for B48C1085-1635 was reproducibly lower (by 15-25%) than that of B48wt (top panel). Concomitantly, a slightly increased B48C1085-1635 (by 20%) compared with B48wt was retained within the cells during chase (bottom panel). The recovery of total apoB48 (medium + cell) was similar between B48wt and B48C1085-1635, suggesting that the decreased B48C1085-1635 secretion was not accompanied with enhanced intracellular degradation. Similar pulse-chase experiments were also performed with cells expressing B48C1085, B48C1085-1395 or B48C1085-1478 (Fig. 3B). The secretion efficiency of the mutant apoB48 proteins decreased gradually as the number of mutagenized free cysteine residues increased (Fig. 3B, bottom). Once again, the decreased secretion of apoB48 mutants was accompanied with prolonged intracellular retention (Fig. 3B, top). These data indicate that although the ability of apoB48 to assemble VLDL is unimpaired by the lack of palmitoylation, the secretion efficiency of the mutant proteins is somewhat slightly reduced.


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Fig. 3.   Pulse-chase analysis of apoB48 secretion efficiency and post-translational stability. A, stably transfected cells (60-mm dish) expressing B48wt or B48C1085-1635 were incubated in methionine- and cysteine-free media for 30 min and pulse-labeled with [35S]methionine/cysteine (100 µCi/dish) for 30 min. After pulse labeling, the medium was changed to normal media and incubated for up to 3 h (chase). Both pulse and chase media contained 20% serum and 0.4 mM oleate. The media were collected, cells were lysed at the indicated chase times, and the 35S-labeled apoB48 proteins were recovered by immunoprecipitation and subjected to SDS-PAGE and fluorography. Radioactivity associated with secreted apoB48 (top) or cellular apoB48 (bottom) was quantified. Data are expressed as % of initial counts at the end of 30-min pulse labeling and are the average of two independent experiments. The representative fluorograms of 35S-labeled apoB48 in media and cell during chase are shown as insets. B, comparison of cell and medium 35S-labeled apoB48 at the end of the 2-h chase between B48wt- and various mutant-transfected cells. The experiments were performed as described in A, except that they were done only once.

It was reported that mutant apoB29 lacking palmitoylation exhibited an abnormal accumulation within the Golgi apparatus (9). The prolonged intracellular retention of B48C1085-1635 shown by pulse-chase experiments (Fig. 3A) prompted us to examine intracellular distribution of this mutant protein. To this end, we performed indirect double immunofluorescent co-localization experiments (Fig. 4). Confocal images showed that both B48wt and B48C1085-1635 appeared as punctate staining throughout the cells with high intensity near the perinuclear area (Fig. 4, A and B, left panels, labeled with apoB). Merging of fluorescence derived from apoB with that from calnexin (middle panels, labeled with CNX) and alpha -mannosidase II (middle panels, labeled with ManII), respectively, showed substantial co-localization of apoB48 (wild type and mutant) with the ER and the Golgi markers (Fig. 4, right panels, labeled with overlay). There was no discernible difference that could suggest the lack of palmitoylation resulting in abnormal intracellular distribution of B48C1085-1635 at steady state. The immunocytochemistry experiments were performed in both stably and transiently transfected cells. Under no circumstances had different intracellular distribution between B48wt and B48C1085-1635 been observed (data from transiently transfected cells are not shown). The immunocytochemistry data were corroborated by subcellular fractionation experiments in which the microsomes were separated using a Nycodenz gradient. Immunoblot analysis with anti-calnexin, anti-alpha -mannosidase II, and anti-TGN38 antibody was performed to detect ER, cis/medial Golgi, and distal Golgi markers, respectively (Fig. 5A), and antibody 1D1 was used to probe apoB48 (Fig. 5B). Quantification of the intracellular distribution of apoB48 among the Nycodenz fractions showed almost identical profiles between B48C1085-1635 and B48wt (Fig. 5C). These results together indicate that the decreased secretion efficiency of mutant B48C1085-1635 was not associated with abnormal intracellular distribution at steady state.


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Fig. 4.   Immunocytochemistry of apoB48. Cells expressing B48wt or B48C1085-1635 were plated on cover slips and kept in cultured medium containing 20% FBS and 0.4 mM oleate for 2 h. The cells were then fixed and permeabilized. Monoclonal antibody 1D1 was used to probe the recombinant human apoB with goat anti-mouse IgG Alexa Fluro-488 conjugate (green) as secondary antibody. The ER and Golgi were probed with anti-calnexin (CNX) and anti-alpha -mannosidase II (ManII) antibodies, respectively, with Alexa Fluro-594-conjugated anti-rabbit IgG (red) as secondary antibody. The images were captured using the MRC-1024 laser scanning confocal imaging system. Data are representative of two experiments with similar results.


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Fig. 5.   Distribution of apoB48 along the secretory pathway. Cells (100-mm dish, 80% confluent) expressing B48 or B48C1085-1635 were incubated in DMEM containing 20% FBS and 0.4 mM oleate for 2 h. The cells were homogenized, and the postnuclear supernatants were fractionated by Nycodenz gradient ultracentrifugation. Aliquots of each fraction were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and subjected to immunoblotting. A, distribution of marker proteins. The blots were probed with antibodies against TGN-38, calnexin (CNX), or alpha -mannosidase II (ManII), respectively. The intensity of these marker proteins was semiquantified by scanning densitometry. Data are presented as "% of maximum value" in which 100% corresponds to the highest value. B, immunoblots of apoB48 probed with monoclonal antibody 1D1. Bottom, semiquantification of density distribution of B48wt and B48C1085-1635.

Lack of Palmitoylation in ApoB48 Resulted in Enhanced Association with Microsomal Membranes-- To gain kinetic insights into intracellular trafficking of apoB48, we performed pulse (30 min) and chase (45 min) experiments combined with subcellular fractionation (Fig. 6). Preliminary experiments showed that the majority of microsome-associated 35S-labeled apoB48 was present in the lumen (see below). Thus, in this experiment, we monitored trafficking of the lumenal 35S-labeled apoB48 through different subcellular compartments. At the end of the 30-min pulse, the ER lumenal 35S-labeled B48wt and 35S-labeled B48C1085-1635 were predominately associated with HDL-like particles (Fig. 6, A and B, top panels, labeled with ER). Because protein synthesis was not synchronized under the current experimental conditions, some apoB48-HDL particles were found in the lumen of the Golgi apparatus (panels labeled with cis/medial Golgi and distal Golgi) and were released into the media at the end of the 30-min pulse. However, no 35S-labeled apoB48 was associated with VLDL at this time. After the 45-min chase (bottom panels), the 35S-labeled apoB48 exited the ER, became prominent in the distal Golgi, and was secreted into the media. A small proportion of 35S-labeled B48 was associated with VLDL within the distal Golgi lumen, but 35S-labeled B48-VLDL was never observed in the ER lumen. The presence of B48-VLDL, particularly B48-VLDL1, in the distal Golgi lumen, at the end of the 45-min chase is consistent with a post-translational, post-ER process for B48-VLDL assembly (19). Similar to what was observed in continuous labeling experiments (Fig. 2A), secretion of 35S-labeled B48C1085-1635 as VLDL and HDL was also comparable to that of 35S-labeled B48wt at the end of chase (Fig. 6, compare panels labeled with medium), again indicting that the lack of palmitoylation has no effect on the ability of apoB48 to assemble lipoproteins. Notably, the lumenal 35S-labeled B48C1085-1635 presented in different subcellular compartments was markedly lower than 35S-labeled B48wt at the end of both pulse and chase time (Fig. 6, compare panel A with B).


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Fig. 6.   Pulse-chase analysis of intracellular distribution and secretion of apoB48. Cells expressing B48wt (A) or B48C1085-1635 (B) were pulse-labeled with [35S]methionine/cysteine for 30 min as described in Fig 3. The chase was carried on for 45 min. Exogenous oleate (0.4 mM) was present in both pulse and chase. At the end of pulse (top panels) and chase (bottom panels), the cells were homogenized and the postnuclear supernatants were fractioned by Nycodenz gradient ultracentrifugation. The fractions corresponding to ER, cis/medial Golgi, and distal Golgi were pooled, and the lumenal contents of each pooled fraction were released by the sodium carbonate treatment. Lipoproteins in both the lumenal content and media were fractionated into VLDL1, VLDL2, and other lipoproteins as described in Fig. 2. The recombinant apoB48 and endogenous rat apoB100 (rB100) were recovered by immunoprecipitation, subjected to SDS-PAGE, and visualized by fluorography. Repetition of the experiments yielded identical results.

The reduced lumenal B48C1085-1635 during the pulse-chase period (Fig. 6) raised the possibility that the mutant protein might bind to microsomal membranes with high affinity. We hypothesized that the decreased secretion efficiency and prolonged intracellular retention of B48C1085-1635 was attributable to altered membrane association. In the next pulse-chase experiment, we compared membrane-association property together with the trafficking rate between B48wt and B48C1085-1635 (Fig. 7). The pulse time was reduced to 15 min (from the previous 30 min); thus at the beginning of chase the 35S-labeled apoB48 was mainly confined to the ER (see insets of panels labeled with time 0). Moreover, two extra time points, 15-min and 30-min, were added to the chase (in addition to the previous single 45-min point) to monitor the rate of intracellular trafficking of apoB48. As the chase proceeded from 0 to 45 min, the radioactivity associated with apoB48 moved out from the ER lumen and appeared in the distal Golgi lumen, and no apoB48 was accumulated in the cis/medial Golgi (Fig. 7, A and B, left panels). These results, together with the data shown in Fig. 6, indicate that apoB48 was transported mainly in a lumenal HDL-associated form from the ER to distal Golgi, and it passed through cis/medial Golgi rather rapidly.


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Fig. 7.   Comparison of intracellular trafficking between B48wt and B48C1085-1635. Cells expressing B48wt (A) or B48C1085-1635 (B) were pulse-labeled with [35S]methionine/cysteine for 15 min, and were chased for up to 45 min. Exogenous oleate (0.4 mM) was present in both pulse and chase. At each chase time, the cells were homogenized and the postnuclear supernatants were fractionated by Nycodenz gradient ultracentrifugation. The lumenal contents and membranes of the pooled ER, cis/medial Golgi, and distal Golgi fractions were separated by sodium carbonate treatment followed by ultracentrifugation. The 35S-labeled apoB48 proteins present in the respective membranes (insets of right panels) and lumenal contents (insets of left panels) were recovered by immunoprecipitation and subjected to SDS-PAGE/fluorography. Radioactivity associated with 35S-labeled B48wt and 35S-labeled B48C1085-1635 was quantified by scintillation counting. The values were expressed as % of total 35S-labeled B48 (lumen + membrane) at each chase time.

When association of apoB48 with membranes was determined, it became clear that B48C1085-1635 had increased (by at least 2-fold) membrane binding. At the end of the 15-min pulse, 26% of the total ER-associated 35S-labeled B48C1085-1635 was membrane-bound, whereas only 11% of the total 35S-labeled B48wt was membrane-bound. The elevated membrane association of 35S-labeled B48C1085-1635 was observed throughout the entire chase (Fig. 7, A and B, right panels). Thus, the average values of membrane-associated apoB48 in the combined ER, cis/medial Golgi, and distal Golgi fractions from four chase time points (i.e. 0, 15, 30, and 45 min) were 29.8 ± 3.0% for B48C1085-1635 and 10.5 ± 1.3% for B48wt (p < 0.001).

The increased association of B48C1085-1635 with the membranes was accompanied with reduced lumenal form in all subcellular compartments (Fig. 7B, left panel). The percent of total 35S-labeled B48C1085-1635 presented in the lumen of all subcellular compartments was less than that of 35S-labeled B48wt at every chase time point. The average values of lumenal apoB48 in the combined ER, cis/medial Golgi, and distal Golgi fractions from four chase time points were 70.3 ± 3.0% for B48C1085-1635 and 89.5 ± 1.3% for B48wt (p < 0.001). Notably, the proportion of total B48C1085-1635 as lumenal form in the distal Golgi was lower than that of B48wt (compare the left panels between A and B, inverted triangles), whereas the proportion of total B48C1085-1635 as membrane-associated form in the cis/medial Golgi was more pronounced than that of B48wt (compare the right panels between A and B, opened circles). Thus, while B48C1085-1635 exited the ER at a rate similar to that of B48wt, B48C1085-1635 (particularly the membrane-bound form) might have a slightly reduced transit rate between cis/medial Golgi and distal Golgi.

2-Bromopalmitate Inhibited VLDL Secretion-- Consideration was given to the possibility that multiple Cys-to-Ser substitution within apoB48 could have effects that extended beyond loss of palmitoylation. Hence, it was desirable to confirm the mutagenesis data using other approaches. It has been reported that 2-bromopalmitate inhibits palmitoylation (26). We attempted to block apoB palmitoylation using 2-bromopalmitate and found that it indiscriminately inhibited secretion of both B48wt (Fig. 8A) and B48C1085-1635 as VLDL (Fig. 8B) in a dose-dependent manner. Secretion of B48wt or B48C1085-1635 as HDL-like particles was less affected by the treatment (Fig. 8). Secretion of endogenous rat 35S-labeled apoB100 was also inhibited by 2-bromopalmitate treatment (data not shown). The treatment with 2-bromopalmitate had no effect on apoB synthesis as judged by the equal incorporation of 35S-labeled amino acids into B48wt and B48C1085-1635 at different doses of the inhibitor (Fig. 8C). Apparently, 2-bromopalmitate exerts an inhibitory effect on VLDL assembly and secretion independent of apoB palmitoylation.


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Fig. 8.   Effect of 2-bromopalmitate treatment on secretion of apoB48. Cells were preincubated with serum-free medium containing 0.1% BSA and indicated concentrations of 2-bromopalmitate for 7 h and then labeled with [35S]methionine/cysteine for 2 h in the presence of 20% serum, 0.4 mM oleate, and different concentrations of 2-bromopalmitate. At the end of labeling, the 35S-labeled B48wt (A) and 35S-labeled B48C1085-1635 (B) secreted as lipoproteins were fractionated and analyzed by SDS-PAGE and fluorography as described in Fig. 2. C, the cell-associated 35S-labeled B48wt and 35S-labeled B48C1085-1635 at the end of 2-h labeling.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Multiple functional roles have been assigned to post-translational protein palmitoylation. Palmitoylation results in anchoring and targeting otherwise cytosolic proteins to the inner surface of specific membrane domains (27-29). Palmitoylation also governs protein-protein interactions within membranes (12, 18, 30). The reversibility of protein palmitoylation suggests that it may regulate membrane activities. While extensive protein palmitoylation studies have focused on proteins involved in signal transduction, the role of palmitoylation of apoB, one of the rare secretory proteins that can be palmitoylated, is not entirely clear. To date, only one report has suggested that substituting the single free cysteine (Cys1085) with serine in apoB29 results in impaired lipid recruitment and abnormal accumulation of the mutant protein within the Golgi apparatus (9). In the current study, we used the naturally occurring apoB48 as a model to assess the role of apoB palmitoylation in VLDL assembly and secretion. Unexpectedly, results obtained from our biochemical (Fig. 2) and immunocytochemical experiments (Fig. 4) using both transiently and stably transfected cells do not support the notion that lack of palmitoylation in apoB48 results in impaired lipoprotein assembly or secretion. In an attempt to determine the effect of palmitoylation using a chemical inhibitor 2-bromopalmitate, we observed that this compound inhibited VLDL secretion regardless of the status of apoB palmitoylation (Fig. 8). Thus, the use of apoB48 as a model leads us to conclude that apoB palmitoylation within this truncated apoB form does not play a role in VLDL assembly and secretion.

The lack of a gross effect of apoB palmitoylation on VLDL assembly/secretion does not rule out a possible temporal and spatial role that this post-translational modification plays during intracellular apoB trafficking and VLDL assembly. Recent studies with the full-length apoB100 have shown that the VLDL assembly proceeds through membrane-associated apoB100 in the ER and cis/medial Golgi to eventual acquisition of bulk neutral lipids in a compartment close to distal Golgi (20). The physical nature of the apoB100-membrane interaction during VLDL assembly is unclear. One hypothesis is that covalently linked fatty acid may facilitate the interaction between apoB100 and membranes (6). Thus, we performed detailed pulse-chase experiments in conjunction with subcellular fractionation to determine the rate of intracellular trafficking and membrane association of the mutant B48C1085-1635 and contrasted these parameters with those of B48wt. The following two observations are noteworthy.

First, although immunocytochemistry (Fig. 4) and immunoblot analysis (Fig. 5) of the fractionated microsomes failed to detect gross changes in intracellular distribution of the mutant apoB48, two independent pulse-chase experiments reproducibly revealed slightly lowered secretion efficiency and prolonged intracellular retention of B48C1085-1635 (Fig. 3A). The effect of palmitoylation on apoB48 secretion appeared to be a function of the extent of this S-acylation. Thus, the more free cysteine residues are mutagenized, the lower the secretion efficiency and the greater intracellular retention of the mutant apoB48 proteins (Fig. 3B). Furthermore, the low secretion efficiency of B48C1085-1635 was associated with slightly reduced transit rate between cis/medial Golgi and distal Golgi (Fig. 7). Thus, palmitoylation of apoB may exert a subtle effect in fine-tuning the kinetics of intracellular trafficking.

Second, the lack of palmitoylation did not diminish association of apoB48 with the microsomal membranes. Rather, the mutant B48C1085-1635 exhibited enhanced binding to the membranes of the secretory compartments (Fig. 7). These results suggest that the normally observed apoB-membrane interaction is probably mediated by factors other than apoB palmitoylation. As mentioned above, the physical nature of apoB-membrane interaction is currently unclear. However, the present studies with apoB48 revealed a marked distinction between apoB100 and apoB48 in their membrane association during lipoprotein assembly. In the case of apoB100, the newly synthesized proteins were almost exclusively membrane-associated within the ER and only became lumen-borne when they reached distal Golgi as VLDL (20). In contrast, the majority of newly synthesized apoB48 was present in the lumen, while only a small proportion was membrane-associated throughout the secretory compartments (Fig. 7). The difference in membrane association between apoB100 and apoB48 is striking, which may indicate that the carboxyl-terminal half of the apoB100 amino acid sequences governs apoB membrane binding. An important unsolved issue in studies of VLDL assembly is how the membrane-associated lipoprotein precursor particles recruit lipid and eventually "fall-off" the membrane as mature, soluble, lipid-loaded lipoprotein particles that are secretion-competent. Since there are an additional five free cysteine residues located within the carboxyl-terminal half of apoB100 and at least four of which can potentially be palmitoylated, it is of importance to determine further, using the full-length apoB100 as a model, whether or not the fatty acids conjugated onto apoB can act as the link that regulates apoB association/dissociation with the membrane during apoB100-VLDL assembly.

    ACKNOWLEDGEMENTS

We thank J. Vance and R. Milne for a critical reading of the manuscript, M. Farquhar, Y. Marcel, and R. Milne for antibodies used in this work, and H. McBride and J. Ngsee for the guidance and assistance in fluorescence microscopy.

    FOOTNOTES

* This work was supported by Heart and Stroke Foundation of Ontario Grant T-4643 and Canadian Institutes of Health Research Grant MT-15486 (to Z. Y.) and National Institutes of Health Grant GM 57966 (to M. D. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

c Recipient of the Doctoral Research Award from Canadian Institutes of Health Research and Heart & Stroke Foundation of Canada.

d Both authors contributed equally to this work.

f Recipient of the Student Award from Canadian Institutes of Health Research and Heart & Stroke Foundation of Canada.

h Canada Research Chair (Tier 1) in Human Genetics and a Heart and Stroke Foundation of Ontario Career Investigator.

j Scientist of Canadian Institutes of Health Research. To whom correspondence should be addressed. Tel.: 613-798-5555 (ext. 18711); Fax: 613-761-5281; E-mail: zyao@ottawaheart.ca.

Published, JBC Papers in Press, February 11, 2003, DOI 10.1074/jbc.M211995200

    ABBREVIATIONS

The abbreviations used are: apo, apolipoprotein; VLDL, very low density lipoproteins; HDL, high density lipoproteins; ER, endoplasmic reticulum; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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