The assembly and secretion of apolipoprotein B-48-containing very low density lipoproteins in McA-RH7777 cells.
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.
Stillemark
P
P
Department of Medical Biochemistry and the Wallenberg Laboratory for Cardiovascular Research, Göteborg University, Göteborg S-405 30, Sweden.
Borén
J
J
Andersson
M
M
Larsson
T
T
Rustaeus
S
S
Karlsson
K A
KA
Olofsson
S O
SO
D000818:Animals; D053283:Apolipoprotein B-48; D001055:Apolipoproteins B / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D002458:Cell Fractionation; D005990:Glycerol / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008079:Lipoproteins, VLDL / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D008114:Liver Neoplasms, Experimental; D008861:Microsomes / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism*; D008954:Models, Biological; D010713:Phosphatidylcholines / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D014280:Triglycerides / Q000378:metabolism; D014407:Tumor Cells, Cultured
D053283:Apolipoprotein B-48; D001055:Apolipoproteins B; D008079:Lipoproteins, VLDL; D010713:Phosphatidylcholines; D014280:Triglycerides; D005990:Glycerol