Studies on the assembly of apolipoprotein B-100- and B-48-containing very low density lipoproteins in McA-RH7777 cells.

Abstract

The mechanisms by which apolipoprotein B-100 (apoB-100) and apoB-48 assemble lipoproteins have been studied in the McA-RH7777 cell line. After incubation for 2 h with 360 microM oleic acid, the McA-RH7777 cells secreted apoB-100 mainly on very low density lipoprotein (VLDL) particles, as judged from sucrose gradient and sequential ultracentrifugation. ApoB-48 was secreted on both VLDL and on denser, high density lipoprotein (HDL)-like lipoproteins. Both apoB-48 and apoB-100 occurred on VLDL particles in the luminal content of the total microsomal fraction. In addition, both proteins were present on denser particles, in particular, those that banded in the HDL region. The denser particles containing apoB-48 were secreted from the cells, whereas those containing apoB-100 were retained in the cell and degraded. Pulse-chase experiments showed that apoB-100 on VLDL was present in the secretory pathway already after a labeling period as short as 3 min. Thus, this particle was the first apoB-100-containing lipoprotein that could be detected in the microsomal fraction of the cell. The assembly of labeled apoB-100 VLDL was acutely (within min) and completely blocked by cycloheximide, if the cycloheximide was added after the pulse labeling period. If, on the other hand, a 15-min chase was introduced after the labeling period, there was no effect of cycloheximide on the assembly of apoB-100 VLDL. A 15-min chase is enough to allow the pulse-labeled apoB nascent polypeptides to be completed to apoB-100. These results indicated that the assembly of apoB-100-containing VLDL is dependent upon ongoing protein biosynthesis during the time when the nascent polypeptide elongate. After this period, the assembly and secretion of the particles are independent of ongoing protein biosynthesis. The first apoB-48-containing particle seen in the luminal content of the microsomal fraction was the HDL-like particle. Pulse-chase experiments as well as experiments with different lengths of the radioactive pulse indicated that the formation of apoB-48-containing VLDL was delayed compared with the formation of the apoB-48-containing HDL-like particle and also in relation to the assembly of the different apoB-100-containing particles, including VLDL. After a 30-min pulse with [35S]methionine followed by a 120-min chase the secretory pathway of the cell was depleted of almost all lipoproteins except the HDL-like apoB-48-containing particle.(ABSTRACT TRUNCATED AT 400 WORDS)