title: The assembly and secretion of ApoB 100-containing lipoproteins in Hep G2 cells. ApoB 100 is cotranslationally integrated into lipoproteins.
abstract: The possibility that apoB 100 is cotranslationally translocated to the endoplasmic reticulum lumen and integrated into lipoproteins has been investigated. ApoB 100 nascent polypeptides were shown to be secreted from pulse-labeled Hep G2 cells after treatment with puromycin and chase for 1 or 2 h in the presence of puromycin and cycloheximide. These nascent polypeptides banded during sucrose gradient ultracentrifugation between the position of the high (HDL) and the low (LDL) density lipoproteins, revealing an inverse relationship between the length of the polypeptide and the density of the fraction. ApoB 100 occurred in the position of LDL and very low density lipoproteins (VLDL). Electronmicroscopy studies of the apoB-containing particles from the gradient indicated an increase in size with increasing length of the polypeptide. Furthermore, labeling studies indicated that the triglyceride load increased with the length of the polypeptide. An inverse relationship between the size of C-terminally truncated apoB polypeptides and the density of the assembled lipoproteins was also observed in experiments with transfected minigenes coding for apoB 41, apoB 29, and apoB 23. These proteins appeared on HDL particles. Pulse-chase experiments indicated that 80-200-kDa apoB nascent polypeptides on particles with HDL density, with time, were converted into larger polypeptides on lighter particles, to be fully replaced by apoB 100 on LDL-VLDL particles. The formation of these LDL-VLDL particles could be blocked by cycloheximide. Sixty-five percent of pulse-labeled apoB nascent polypeptides present in the microsomal fraction was released by sodium carbonate treatment, and 77% of these polypeptides could be recovered on the immature particles (banding between HDL and LDL) after sucrose gradient ultracentrifugation. Pulse-chase experiments indicated that these nascent polypeptides, on the immature lipoproteins, had the capacity to be precursors for all the apoB 100-containing LDL and VLDL particles formed in the cell. The obtained results indicate that a major portion of the apoB nascent polypeptides in the cell form lipoproteins cotranslationally during the translocation to the lumen of the endoplasmic reticulum.
authors.affiliation: Department of Medical Biochemistry, University of Göteborg, Sweden.
authors.forename: J
authors.initials: J
authors.lastname: Borén
authors.forename: L
authors.initials: L
authors.lastname: Graham
authors.forename: M
authors.initials: M
authors.lastname: Wettesten
authors.forename: J
authors.initials: J
authors.lastname: Scott
authors.forename: A
authors.initials: A
authors.lastname: White
authors.forename: S O
authors.initials: SO
authors.lastname: Olofsson
mesh_terms: D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D003513:Cycloheximide / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008074:Lipoproteins / Q000096:biosynthesis* / Q000302:isolation & purification; D008113:Liver Neoplasms; D008715:Methionine / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D013462:Sulfur Radioisotopes; D014162:Transfection
chemical_list: D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D008074:Lipoproteins; D013462:Sulfur Radioisotopes; D003513:Cycloheximide; D008715:Methionine