From the Department of Medical Biochemistry and the Wallenberg Laboratory, University of Göteborg, Sweden and the Division of Metabolic Diseases, Bristol-Myers Squibb Co., Princeton, New Jersey 08543
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ABSTRACT |
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In cells in which the lipoprotein assembly process had been inactivated by brefeldin A (BFA), membrane-associated apoB-100 disappeared without forming lipoproteins or being secreted, indicating that it was degraded. Reactivation of the assembly process by chasing the cells in the absence of BFA, gave rise to a quantitative recovery of the membrane-associated apoB-100 in the very low density lipoprotein (VLDL) fraction in the medium. These results indicate that the membrane-associated apoB-100 can be converted to VLDL.
A new method was developed by which the major amount (88%) of
microsomal apoB-100 but not integral membrane proteins could be
extracted. The major effect of this method was to increase the recovery
of apoB-100 that banded in the LDL and HDL density regions, suggesting
that the membrane-associated form of apoB-100 is partially lipidated.
We also investigated the role of the microsomal triglyceride transfer
protein (MTP) in the assembly of apoB-100 VLDL using a photoactivatable
MTP inhibitor (BMS-192951). This compound strongly inhibited the
assembly and secretion of apoB-100 VLDL when present during the
translation of the protein. To investigate the importance of MTP during
the later stages in the assembly process, the cells were preincubated
with BFA (to reversibly inhibit the assembly of apoB-100 VLDL) and
pulse-labeled (+BFA) and chased (+BFA) for 30 min to obtain full-length
apoB-100 associated with the microsomal membrane. Inhibition of MTP
after the 30-min chase blocked assembly of VLDL. This indicates that
MTP is important for the conversion of full-length apoB-100 into VLDL.
Results from experiments in which a second chase (BFA) was introduced before the inactivation of MTP indicated that only early events in this
conversion of full-length apoB-100 into VLDL were blocked by the MTP
inhibitor. Together these results indicate that there is a
MTP-dependent "window" in the VLDL assembly process that occurs
after the completion of apoB-100 but before the major amount of lipids
is added to the VLDL particle. Thus the assembly of apoB-100 VLDL from
membrane-associated apoB-100 involves an early MTP-dependent phase and a late MTP-independent phase,
during which the major amount of lipid is added.
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INTRODUCTION |
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There exist two forms of apolipoprotein B (apoB),1 apoB-100 and apoB-48 (1, 2). In humans apoB-100 is expressed in the liver, while apoB-48 is synthesized in the intestine (3). In some animals, such as the rat, both proteins are expressed in the liver. When expressed in liver, both apoB-100 and apoB-48 assemble into VLDL particles (4, 5). When expressed in the intestine, they both have the capacity to assemble into chylomicrons (6).
Lipoprotein assembly is a complex process that is not yet understood in all detail. The availability of the McA-RH7777 rat hepatoma cell line, which assembles bona fide VLDL (4), has facilitated studies of this process. In addition, we have recently demonstrated that brefeldin A (BFA) reversibly inhibits the assembly of all apoB-100 lipoproteins, in particular VLDL (7). During BFA treatment, apoB-100 appeared in a non-VLDL form that pelleted with the microsomal membrane after sodium carbonate extraction of the lumenal content of the microsomes (7). Since it was possible to remove the BFA and reactivate the assembly process, we were able to demonstrate that this membrane-associated form of apoB-100 can be a precursor to apoB-100 VLDL (7). Thus it seems that, under certain conditions, the assembly of apoB-100 VLDL involves the following steps: 1) translation and association with the endoplasmic reticulum membrane and 2) conversion of the membrane-associated VLDL precursor into VLDL.
The microsomal triglyceride transfer protein (MTP) catalyzes the transfer of triglycerides, cholesteryl esters, and phospholipids between phospholipid surfaces (8, 9). Mutations that inactivate or abolish MTP give rise to the phenotype of abetalipoproteinemia, an almost total absence of apoB-containing lipoproteins in the plasma (10, 11). Thus MTP is essential for the assembly and secretion of apoB-containing lipoproteins (12, 13). Very recent results indicate that MTP is important for the early events in the assembly of endogenous rat apoB-48 VLDL. However, MTP was not needed for the addition of the major amount of lipid during the later stages of the apoB-48 VLDL assembly (14). Currently, no direct data describing the role of MTP in the early or late phases of apoB-100 assembly have been reported
This report describes the results of studies designed to follow the fate of apoB-100 after it associates with the microsomal membrane. The formation of VLDL from this pool of apoB-100 was elucidated by pulse-chase analysis. The role of MTP in this assembly process was determined using the photoactivatable inhibitor BMS-192951.
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EXPERIMENTAL PROCEDURES |
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Materials-- Eagle's minimum essential medium, nonessential amino acids, glutamine, penicillin, and streptomycin were from ICN Biomedicals (Costa Mesa, CA). Fetal calf serum was from Biochrom KG (Berlin, Germany), and brefeldin A from Epicentre Technologies (Madison, WI). Methionine, fatty acid-free bovine serum albumin, sodium pyruvate, sodium carbonate, sodium bicarbonate, phenylmethylsulfonyl fluoride, pepstatin A, and leupeptin were from Sigma. Polyclonal antibodies to canine Calnexin was from StressGen Biotechnologies Corp (Victoria, Canada). Rabbit immunoglobulin was from DAKO (Glostrup, Denmark). Trasylol® (aprotinin) was from Bayer Leverkusen (Leverkusen, Germany). Immunoprecipitin and Eagle's minimum essential medium without methionine were from Life Technologies, Inc. (Paisley, Scotland). N-Acetyl-Leu-Leu-norleucinal was from Boehringer Mannheim (Mannheim, Germany). Amplify®, [35S]methionine-cysteine mix, and Rainbow® protein molecular weight markers were from Amersham Corp. (Amersham, UK). Ready Safe® was from Beckman (Fullerton, CA). All chemicals used for SDS-polyacrylamide gel electrophoresis, the Silver Stain Plus® kit and alkaline-conjugated goat anti-rabbit Ig were from Bio-Rad. Western Blue®-stabilized substrate for alkaline phosphatase was from Promega (Madison, WI).
Cell Culture-- McA-RH7777 cells were cultured as described earlier (4) in Eagle's minimum essential medium, containing 20% fetal calf serum, 1.6 mM glutamine, 8.0 mM sodium bicarbonate, 1.6 mM sodium pyruvate, 140 mg/ml streptomycin, 140 IU/ml penicillin, and 60 mg/ml nonessential amino acids, in 5% CO2, at 37 °C. The cells were split twice a week and fed every day.
Metabolic Labeling-- The cells were pulse-labeled and chased as described earlier (4). Brefeldin A was dissolved in ethanol and added to the culture medium to a final concentration of 10 µg/ml, unless stated otherwise. Isolation of cells and the microsomal fraction was carried out as described previously (15). The lumenal content of the vesicles was separated from the vesicle membranes by the sodium carbonate method (16), with some modifications as described elsewhere (4, 17). The microsomes were also extracted with a combination of sodium carbonate, 0.025% deoxycholate, and 1.2 M potassium chloride (18). The deoxycholate was dissolved in the high salt solution containing 3 M potassium chloride and 0.02 M Tris-HCl. The final concentration of potassium chloride was 1.2 M and for Tris-HCl 0.008 M. The Tris buffer did not influence the final pH during the extraction. The procedure then followed the procedure for the extraction with sodium carbonate alone. The following protease inhibitors were used: 0.1 mM leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM pepstatin A, 5 mM N-acetyl-Leu-Leu-norleucinal, and 100 kallekrein-inhibitory units aprotinin/ml.
Treatment with the MTP Inhibitor-- A photoactivatable MTP inhibitor, BMS-192951 (14), was used in these studies. The inhibitor (final concentration of 10 µM if not stated otherwise) was dissolved in Me2SO (final concentration, 0.5%) and added directly to the cell cultures. Before photoactivation, the culture dishes were kept in the dark. To activate the inhibitor, the cells were irradiated for 15 min with UV light (365 nm) using a Philips UVA lamp at a distance of 8 cm. The temperature in the cell cultures during this process was measured and found to be 37 °C.
Sucrose Gradient Ultracentrifugation of Lipoproteins-- The lipoproteins present in the microsomal lumen or in the media were separated by sucrose gradient ultracentrifugation as described previously (4).
Immunoprecipitation of ApoB and Electrophoresis-- ApoB was immunoprecipitated from cells, medium, and gradient fractions as described elsewhere (4). Electrophoresis in SDS-polyacrylamide gels, autoradiography, and determination of the radioactivity in the proteins separated in the gels were carried out as described previously (19). To silver stain polyacrylamide gels, we used the Silver Stain Plus® kit (Bio-Rad) following the procedure recommended by the manufacturer.
Immunoblots were carried out on a Trans-Blot®SD (Bio-Rad) as recommended by the manufacturer. The blots were blocked with 5% non-fat dry milk in 20 mM Tris-HCl with 137 mM sodium chloride (TBS) for 1 h. This was followed by a 1-h incubation with the antibody (a rabbit polyclonal antibody against the carboxyl terminus of canine Calnexin (dilution, 1:2000) in TBS with 0.1% Tween 20 (TBS-T) and 5% non-fat dry milk. The blots were washed with TBS-T (twice for 5 min each). To detect bound antibodies, the blots were incubated with an alkaline phosphatase-conjugated goat anti-rabbit Ig (Bio-Rad; dilution, 1:2000) in TBS-T with 5% non-fat dry milk. After a washing twice for 5 min each with TBS-T followed by once for 5 min with TBS, the blots were reacted with Western Blue®-stabilized substrate for alkaline phosphatase as recommended by the manufacturer. ![]() |
RESULTS |
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Turnover of Membrane-associated ApoB-100-- McA-RH7777 cells were pretreated with BFA for 15 min, labeled for 30 min in the presence of BFA, and chased in the presence of BFA for an additional 30 min. After this chase, the major amount of the labeled intracellular apoB-100 was associated with the membrane of the microsomes (i.e. was resistant to carbonate extraction), while no significant amount of radioactive apoB-100 VLDL could be detected in the sodium carbonate extract (i.e. in the lumenal content) of these microsomes (Fig. 1, chase 1). The lack of radiolabeled apoB-100 VLDL in the microsomal lumen was verified by gradient ultracentrifugation of extracts, recovered from the microsomes by the combination of sodium carbonate, 0.025% deoxycholic acid, and 1.2 M potassium chloride (data not shown). This extraction procedure removed the major amount of apoB-100 from the microsomes (see below). Together these results confirm our previous observations (7) that the VLDL assembly is blocked by BFA and that the major amount of intracellular apoB-100 is instead associated with the membrane of the microsomes.
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Effect of Inhibition of MTP during the Early Part of the Assembly of ApoB-100-containing VLDL-- In agreement with previous results (14), we found that inhibition of MTP before labeling of the cells profoundly inhibited the assembly and secretion of radioactive apoB-containing lipoproteins. Compared with the control cells, MTP inhibition (10 µM) resulted in only 7.5% (mean of two experiments) of the apoB-100 VLDL secretion.
Effects of MTP Inhibition on the Conversion of the
Membrane-associated Form of ApoB into VLDL--
In the next
experiment, we investigated the effect of inhibiting MTP on the latter
part of the VLDL assembly process. This experiment was also designed to
delineate a possible time interval during which MTP was important for
this part of the assembly process. In these experiments, we pretreated
the cells with BFA for 15 min, labeled them for 30 min (+BFA), and
chased them for 30 min (+BFA). Under these conditions the VLDL assembly
was inhibited, and full-length apoB-100 was associated with the
microsomal membrane (cf. Fig. 1). We now introduced an
intermediate chase for 0, 15, 30, 60, or 120 min in the absence of BFA.
The MTP inhibitor was photoactivated after the intermediate chase, and
a new chase for 180 min was introduced. This chase was carried out in
the absence of BFA but in the presence of oleic acid. The medium was
then collected and analyzed by gradient ultracentrifugation. With the exception of the addition of the MTP inhibitor, the control cells were
treated in exactly the same way (including the UV irradiation after the
intermediate chase. For details, see legend to Fig. 2A). It should be pointed out
that the VLDL assembly and secretion in BFA-treated cells are restored
during the 180-min chase (BFA with oleic acid), and the intracellular
pool of apoB-100 is converted to VLDL and secreted (cf. Fig.
1) (7).
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Extraction and Gradient Ultracentrifugation of the Membrane-associated ApoB-100-- One problem confounding the interpretation of these results is that a large amount of apoB-100 remained in association with the membrane pellet after sodium carbonate extraction. This raises the possibility that there is selective removal of one or more physical forms of apoB-100, which could lead to a biased result. The nature of apoB-100 that remains associated with the membrane is not clear. To clarify this issue, it is critical to remove and analyze all of the apoB-100 from the membrane fraction during the period when the assembly process is MTP-dependent (i.e. after the intermediate chases of 0 and 15 min). This was accomplished by development of a new extraction method that is based on a combination of sodium carbonate and very small amounts (0.025%) of deoxycholate together with 1.2 M potassium chloride (cf. "Experimental Procedures"). (This procedure is referred to as deoxycholic acid-carbonate extraction.) A comparison of this new technique with the standard carbonate extraction is shown in Fig. 3.
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The Effect of MTP Inhibition on the Assembly of VLDL in Cycloheximide-treated Cells-- Data from the BFA-treated cells indicated that MTP is of importance for VLDL assembly after the completion of apoB-100 and that this MTP dependence could be abolished by a short chase. These experiments relied on the ability to reversibly inhibit VLDL assembly by BFA. To show that the observed MTP effect was also applicable to cells not treated with BFA, we carried out the following experiment (Fig. 5). The cells were pulse-labeled for 30 min and treated with cycloheximide to block translation. We then introduced a chase for 0, 15, or 30 min (+CHX). These chases were followed by MTP inhibition for 15 min. As a control, the cells were treated with only CHX and UV-irradiated without the inhibitor. Finally, the cells were chased for 180 min (+CHX) in the presence of oleic acid, and the secretion of radioactive apoB-100 VLDL was determined. The results indicate that there was a significant inhibition of VLDL assembly when MTP inhibition was introduced after the intermediate chase of 0 min (Fig. 5, 0 min). Recovery of VLDL in the medium was 35% of that observed for the control. This verifies the observation that MTP influences the assembly of apoB-100 VLDL after translation is complete. After an intermediate chase of 15 min (Fig. 5, 15 min) in the presence of CHX, the recovery increased significantly (to 60% of the control). Finally, after an intermediate chase of 30 min (Fig. 5, 30 min), the recovery was 72%, which is in the range of that observed after the corresponding intermediate chase in cells treated with BFA (see Fig. 2). The same amount of apoB-100 radioactivity was present in the cells after the treatment with the MTP inhibitor (i.e. before the 180-min chase) under the three conditions. These results support the observation that MTP influences the apoB-100 VLDL assembly post-translationally and that there is a short time window during which the process is MTP-dependent.
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DISCUSSION |
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Previous results from our group (21), as well as from other investigators (22), have indicated that membrane-associated apoB-100 is sorted to degradation. Recently however, we obtained results which indicated that this form of apoB-100 can also be a precursor to VLDL (7). In these previous experiments we used BFA to reversibly block the assembly of apoB-100-containing lipoproteins, in particular VLDL. It was demonstrated that BFA caused the association of apoB with the endoplasmic reticulum membrane. When the BFA was removed, the cells resumed assembly of apoB-100 VLDL. In this study, we used the same system, but continued to follow the fate of the membrane-associated apoB-100 during a chase in the presence of BFA, under conditions that inhibited the VLDL assembly process. The results showed a clear decrease in the amount of membrane-associated apoB-100. Since no apoB-100 lipoproteins were formed or secreted, these results indicate that the membrane-associated apoB-100 was removed by degradation. Releasing the BFA block, before the membrane-bound apoB-100 was degraded, resulted in formation of apoB-100 VLDL, which was quantitatively recovered in the medium. These results demonstrate that the membrane-associated apoB-100 is a precursor to VLDL. However, when the assembly process is inhibited, this VLDL-precursor will be degraded in the cell. In agreement with our previous results (20), the present observations indicate that the assembly process plays a critical role in the sorting of apoB-100; apoB-100 that is not recruited to the assembly process will gradually be degraded intracellularly.
The physical nature of the membrane-associated apoB-100 is unknown. In this report we demonstrate that the microsomal membranes could be almost completely depleted of apoB-100 by extraction with sodium carbonate together with small amounts of deoxycholate and 1.2 M potassium chloride. The concentrations of deoxycholate are well below the critical micellar concentration and have been shown to remove lumenal proteins and peripheral membrane proteins but not integral membrane proteins (18). In an analogous fashion, we observed that the extraction procedure did not significantly influence recovery of membrane proteins from the membrane pellet during the extraction compared with ordinary sodium carbonate extraction. Of interest was that, although a general increase in the recovery of apoB-100 in all density classes was seen, we observed that the major effect of deoxycholate and high salt was to liberate apoB-100 that banded in the HDL and LDL density ranges. We have previously demonstrated the presence of such forms of apoB-100 lipoproteins in sodium carbonate extracts of the microsomes, and we have suggested that the form that bands in the HDL density range (apoB-100 HDL) may be a precursor to less dense apoB-100-containing lipoproteins (15). Such a precursor role for apoB-100 HDL is supported by the results of the present study (compare Fig. 1). One possibility is that apoB-100 HDL represents a lower percentage of the membrane-associated pool that is released during extraction with carbonate alone, implying that the membrane-associated form of apoB-100 and the carbonate-released dense particles belong to the same pool of apoB-100. This in turn may explain why both the membrane-associated form of apoB-100 (i.e. the carbonate-resistant form) and apoB-100 HDL (i.e. the dense particles extracted by sodium carbonate) appear to be precursors for apoB-100 VLDL (compare Fig. 1).
Previous studies have demonstrated that MTP is essential for the assembly of apoB-containing lipoproteins (12, 13). Moreover, it has been shown that mutations in the MTP gene give rise to the phenotype of abetalipoproteinemia (10, 11). Despite the fact that MTP is characterized in great detail, relatively little is known about the molecular details of the role of this protein in the assembly of the lipoprotein. An interaction with apoB has been demonstrated (23, 24) and suggested to involve the N-terminal globular domain of apoB, which is needed for the action of MTP (25, 26). In a recent study, we demonstrated that MTP is necessary for the early events in the assembly of endogenous apoB-48 VLDL in the McA-RH7777 cells, but does not appear to be essential for the step in which the major amount of lipids was added to the particle (14). In addition, it has recently been shown that inhibition of MTP up to 10 min after a pulse of [35S]methionine blocked the secretion of apoB-100 from HepG2 cells (27). After this time, the MTP inhibitor lost its effect, and apoB secretion was observed. This suggests that, as in the case of apoB-48, MTP also plays a role in the early stages of apoB-100 lipoprotein assembly, but not in the later stages. One drawback with the HepG2 cells is that they do not assemble any substantial amount of bona fide VLDL (20), but rather an LDL-size particle rich in triacylglycerol. The assembly of this particle occurs to a significant degree cotranslationally (28).
As discussed above, a precursor to apoB-100 VLDL appears to be a membrane-associated form of full-length apoB-100 (7). We observed that a photoactivatable MTP inhibitor could block the assembly and secretion of apoB-100 VLDL not only when the inhibitor was photoactivated prior to the labeling of the cells, but also when the photoactivation was carried out after completion of the membrane-associated apoB-100 (a VLDL precursor). This indicates that MTP takes part in the post-translational conversion of membrane-associated apoB-100 to VLDL. Our results also indicate that there is a "window" within which this process is MTP-dependent. This window occurs in BFA-treated cells after translation is complete, but before they have recovered their capacity to assemble and release VLDL particles into the secretory pathway after BFA is withdrawn. This in turn indicates that MTP is not involved in the loading of the major amount of lipid to the particle, but rather in the preparation of apoB-100 for this process. This agrees with our observations that the addition of the major amount of lipid to apoB-48 VLDL occurs in an MTP-independent step (14).
The identification of the window during which the assembly process was MTP-dependent was based on BFA-treated cells. To confirm that this was not due to an artifact introduced by the BFA treatment, an alternate strategy was applied. In this case we used CHX to achieve the necessary manipulations. Qualitatively, the results were nearly identical, showing a distinct post-translational window during which apoB-100 lipoprotein assembly was MTP-dependent. However, since the CHX-treated cells, contrary to BFA-treated cells, continue to assemble and secrete apoB-100 VLDL (4), the exact time window of MTP dependence cannot be compared.
Our results thus indicate that MTP is of importance for the creation of a situation that allows full-length membrane-associated apoB-100 to be assembled into VLDL and secreted. This suggests that an important function of MTP is to prepare apoB-100 for the assembly with the major amount of lipid. The nature of this preparation is a key question that needs to be addressed in future studies; however, possible mechanism can be discussed.
The fact that inhibition of MTP mediated lipid transfer has such a
profound effect on the assembly process during translation of apoB
suggests that MTP is essential for stabilizing the protein during or
immediately after translocation, perhaps by facilitating the folding of
the protein. It has been pointed out that mature apoB contains large
regions of amphipathic structures, in particular long regions of
amphipathic -strands that fold into amphipathic
-sheets (29). The
amphipathic
-sheets, that are unique for apoB, are generally
believed to mediate the irreversible binding between apoB and the
lipoprotein particle (29). It could be speculated that these
amphipathic structures require MTP-mediated lipid transfer to fold
correctly to form a structure capable of receiving the bulk of the core
neutral lipid. In the absence of MTP, these structures would misfold
leading to the degradation of the apoB polypeptide.
The observation that MTP is also needed after the completion of apoB-100 could suggest that portions of the protein remains unfolded after the translation of the protein. There is a possibility that at least part of apoB-100 is post-translationally translocated to the lumenal side of the membrane (20, 30, 31). MTP may participate in such a translocation indirectly by allowing this part of the protein to properly fold as it enters the interior of the endoplasmic reticulum. In this way the protein may acquire a structure that allows it to interact with the downstream VLDL assembly process. It should, however, be kept in mind that the association between apoB-100 and the microsomal membrane is not completely understood. Contradictory results have appeared in the literature concerning the exposure of apoB-100 on the cytosolic side of the endoplasmic reticulum membrane (17, 20, 30-33). An alternative explanation is that apoB-100 is completely translocated cotranslationally (like ordinary secretory proteins) and that the folding of the C terminus occurs post-translationally, involving chaperone proteins in addition to MTP. The observation that the tentative membrane-associated precursor could be extracted under conditions that have been shown not to extract integral membrane proteins (18) (verified in this study) would favor the possibility that apoB-100 is more loosely associated with the membrane than is an integral protein. This does not rule out a transmembrane orientation mediated by chaperones or membrane channels. It should be remembered that the translocation channel Sec 61 has recently been demonstrated to participate not only in the translocation of proteins to the endoplasmic reticulum lumen but also in the retraction of lumenal protein for degradation by proteasomes (see, for example, Cresswell and Hughes (34) for a review and references therein).
Together our data suggest a model for the assembly of apoB-100 VLDL. According to this model, apoB-100 is translated and at least partially translocated into a membrane-associated VLDL precursor form. This process is highly dependent on MTP. The assembly of VLDL from this precursor pool occurs first in an MTP-dependent step during which an immediate VLDL precursor is formed. The final step(s) is MTP-independent and allows the major amount of lipids to be loaded onto the protein.
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ACKNOWLEDGEMENT |
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We thank Anita Magnusson for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by Grant 7142 from the Swedish Medical Research Council and by the Swedish Heart and Lung Foundation, the Swedish Oleo-Margarine Foundation of Nutritional Research, Novo Nordic Foundation, the Loo and Hans Ostermans Foundation, and the Bristol-Myers Squibb Pharmaceutical Research Institute.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.
To whom correspondence should be addressed: Dept. of Medical
Biochemistry, University of Göteborg, Medicinaregatan 9, S-413 90 Göteborg, Sweden. Tel.: 46-31-773 34 85; Fax: 46-31-41 61 08.
1 The abbreviations used are: apoB, apolipoprotein B; BFA, brefeldin A; CHX, cycloheximide; HDL, high density lipoproteins; LDL, low density lipoproteins; MTP, the microsomal triglyceride transfer protein; VLDL, very low density lipoproteins.
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REFERENCES |
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