Abstract
When apolipoprotein B (apoB) is expressed in heterologous cells, it is not secreted but retained and degraded within the endoplasmic reticulum (ER). We have previously characterized carboxyl-terminal truncated forms of apoB expressed in COS cells and have shown that these proteins were readily synthesized but retained within the ER and degraded, if the size of the truncated protein was larger than apoB 29. Below this size, the smaller the size of the apoB truncates, the greater the extent of secretion, although >50% of these smaller proteins were also degraded within the ER. In the present study, we demonstrate that this secretory defect can be overcome by coexpression with microsomal triglyceride transfer protein (MTP); moreover, this complementation is inversely related to the size of apoB. Secretion of apoBs larger than B29 required the coexpression of MTP and, in the presence of MTP, was oleate-responsive. MTP, in the presence or absence of oleate supplementation, had little or no effect on the secretion of the shorter truncates. We discovered, however, that MTP was physically associated with all forms of apoB intracellularly (B13-B41). The association of MTP with apoB 41 was stable to high salt washing, as well as to low pH, suggesting that these interactions may be hydrophobic in nature. In addition to the interaction with MTP, apoB was also found to be associated with calnexin, confirming previous studies, and with proteins bearing the KDEL retention signal. However, studies on overexpression of human calnexin and tunicamycin inhibition of glycosylation showed that interaction with calnexin was not necessary for the formation or secretion of apoB 41-containing lipoproteins; moreover, in the presence of MTP, the association of calnexin with apoB 41 was transient or absent. These data suggest that for apoB to attain a folded state sufficient to escape the quality control of the ER, it needs to obtain neutral lipid (supplied by MTP), as well as its ability to keep it packaged as a rudimentary lipoprotein, dependent on its size being larger than B29.
Highlights
Previously characterized carboxyl-terminal truncated of the endoplasmic reticulum (ER)
These data suggest that for apoB to attain a folded state sufficient to escape the quality control of the ER, it needs to obtain neutral lipid, as well as its ability to keep it packaged as a rudimentary lipoprotein, dependent on its size being larger than B29
Coexpression of apoB with microsomal triglyceride transfer protein (MTP) in COS cells resulted in complementation of the secretory block for apoB truncates that are larger than B29, and the rates of secretion were responsive to oleate supplementation (Fig. 1)
Summary
Reagents—Tissue culture medium, sheep antibodies to apoB, and reagents were obtained as described previously [24]. The cell lysates were cleared of cell debris by centrifugation at 13,000 ϫ g for 15 min at 4 °C; total protein concentrations were determined using a dye-binding assay (Bio-Rad) and immunoprecipitated with the appropriate antibodies, collected by protein A-Sepharose beads and analyzed as described previously [24]. 24 h after transfection, the cells were treated with 0.02% DMSO or 0.02% DMSO and 4 g/ml tunicamycin (final concentrations) This treatment resulted in greater than 90% inhibition of labeled glucosamine incorporation into total cell acidprecipitable material, as well as into immunoprecipitated apoB 41 (data reviewed but not shown). ApoB was either immunoprecipitated when radiolabeled protein was present, as above, or the samples were desalted and concentrated in Centricon 30 filters (Amicon, Beverly, MA) separated by SDS-PAGE, and analyzed by Western blotting, using C1.4 primary antibody. The membranes were blocked in 5% non-fat milk, incubated with primary antibodies at a dilution of 1:1000, washed extensively, incubated with secondary antibodies conjugated with horseradish peroxidase (Bio-Rad) at a dilution of 1:10,000, and washed; the antibody conjugates were detected by chemiluminescence using the Renaissance kit from DuPont NEN as per kit instructions
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