Very low density lipoprotein synthesis and secretion. Extrusion of apoprotein B nascent chains through the membrane of the endoplasmic reticulum without protein synthesis.
The hypothesis was tested that chain elongation/translocation during protein synthesis forces the growing polypeptide chain through the membrane of the endoplasmic reticulum into its luminal compartment for secretion. Estrogen-induced chick liver cells which synthesize and secrete the 350,000-dalton apoprotein B of very low density lipoprotein were employed to address this issue. Treatment of [3H]leucine pulse-labeled cells with cycloheximide arrested apoprotein B nascent chains on membrane-bound polysomes without affecting the secretion of completed chains. Puromycin added immediately following the washout of cycloheximide discharged these nascent chains which were subsequently translocated through the membrane and quantitatively secreted into the medium. The size distribution of the secreted apoprotein B nascent chains approximated that of the polysome-bound apoprotein B nascent chains prior to discharge with puromycin. These findings indicate that the translocation of polysome-associated nascent chains through the membrane into the luminal space of the endoplasmic reticulum requires neither protein synthesis nor the attachment of the polypeptide to the ribosome. Since puromycin-discharged nascent chains are readily secreted by these cells, this approach may be used to study the structural requirements for the co- and post-translational events of apoprotein B processing, e.g. glycosylation and assembly of the lipid components of very low density lipoprotein.
D000818:Animals; D001053:Apolipoproteins / Q000378:metabolism*; D002478:Cells, Cultured; D002645:Chickens; D003513:Cycloheximide / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D004958:Estradiol / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008079:Lipoproteins, VLDL / Q000378:metabolism*; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008970:Molecular Weight; D011691:Puromycin / Q000494:pharmacology
D001053:Apolipoproteins; D008079:Lipoproteins, VLDL; D011691:Puromycin; D004958:Estradiol; D003513:Cycloheximide