Abstract
We have used an extraction procedure, which released membrane-bound apoB-100, to study the assembly of apoB-48 VLDL (very low density lipoproteins). This procedure released apoB-48, but not integral membrane proteins, from microsomes of McA-RH7777 cells. Upon gradient ultracentrifugation, the extracted apoB-48 migrated in the same position as the dense apoB-48-containing lipoprotein (apoB-48 HDL (high density lipoprotein)) secreted into the medium. Labeling studies with [(3)H]glycerol demonstrated that the HDL-like particle extracted from the microsomes contains both triglycerides and phosphatidylcholine. The estimated molar ratio between triglyceride and phosphatidylcholine was 0.70 +/- 0.09, supporting the possibility that the particle has a neutral lipid core. Pulse-chase experiments indicated that microsomal apoB-48 HDL can either be secreted as apoB-48 HDL or converted to apoB-48 VLDL. These results support the two-step model of VLDL assembly. To determine the size of apoB required to assemble HDL and VLDL, we produced apoB polypeptides of various lengths and followed their ability to assemble VLDL. Small amounts of apoB-40 were associated with VLDL, but most of the nascent chains associated with VLDL ranged from apoB-48 to apoB-100. Thus, efficient VLDL assembly requires apoB chains of at least apoB-48 size. Nascent polypeptides as small as apoB-20 were associated with particles in the HDL density range. Thus, the structural requirements of apoB to form HDL-like first-step particles differ from those to form second-step VLDL. Analysis of proteins in the d < 1.006 g/ml fraction after ultracentrifugation of the luminal content of the cells identified five chaperone proteins: binding protein, protein disulfide isomerase, calcium-binding protein 2, calreticulin, and glucose regulatory protein 94. Thus, intracellular VLDL is associated with a network of chaperones involved in protein folding. Pulse-chase and subcellular fractionation studies showed that apoB-48 VLDL did not accumulate in the rough endoplasmic reticulum. This finding indicates either that the two steps of apoB lipoprotein assembly occur in different compartment or that the assembled VLDL is transferred rapidly out of the rough endoplasmic reticulum.
Highlights
Immunoelectron microscopy studies have shown that apolipoprotein1 B is present in the rough endoplasmic reticulum (ER), but very low density lipoprotein (VLDL)-sized particles are not [1]
ApoB-48 that banded in the high density lipoprotein (HDL) density region migrated in the same position as the apoB-48 extracted from the microsomes (Fig. 1C); we will refer to this form of apoB-48 as intracellular apoB-48 HDL
The released intracellular apoB-48 HDL comigrated with the secreted apoB-48 HDL, and both displayed the characteristics of lipoproteins
Summary
Materials—Eagle’s minimum essential medium, nonessential amino acids, glutamine, penicillin, and streptomycin were obtained from ICN Biomedicals (Costa Mesa, CA). Immunoaffinity chromatography of apoB-containing fractions extracted from the microsomes or present in the culture medium was carried out as described [18]. Phosphatidylcholine and triglycerides were separated as described above, and specific radioactivity was determined (dpm/mg). The lipids were separated as described above, and the spots corresponding to triglycerides and phosphatidylcholine were scraped into scintillation vials; 1 ml of cyclohexane was added, and the radioactivity was determined in the presence of Ready-Safe scintillation mixture. The immunoglobulins were isolated from the hybridoma culture medium and coupled to cyanogen bromide-activated Sepharose 4B as recommended by the manufacturer (Amersham Pharmacia Biotech) and used for immunoadsorption experiments [23] Using this immunoadsorbent, we recovered the 60-kDa protein that reacted with the monoclonal antibody.
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