Intracellular events in the assembly of very-low-density-lipoprotein lipids with apolipoprotein B in isolated rabbit hepatocytes.
Isolated rabbit hepatocytes incorporated [35S]methionine into cellular and secreted apolipoprotein B (apo-B), and [3H]glycerol into cellular and secreted triacylglycerol and phospholipids. Newly synthesized apo-B was incorporated into rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SER), cis-Golgi and trans-Golgi membranes and was preferentially transferred into the lumen of the RER with specific radioactivities ten times those in the membrane. Radiolabelled apo-B did not equilibrate with pre-existing unlabelled apo-B, and pools of different specific radioactivities were established in different subcellular fractions. Only a small fraction of the newly synthesized apo-B was transferred to the Golgi lumen. In pulse-chase experiments, most of the newly synthesized apo-B in the RER membrane and the RER lumen was degraded. [3H]Glycerol was incorporated into triacylglycerol and phospholipids in the lumen of the RER, SER, cis-Golgi and trans-Golgi. However, in contrast with apo-B, all of the radiolabelled lipids in the lumen of the RER, SER and cis-Golgi were transferred to the trans-Golgi lumen or secreted. Analysis of the lipid composition of the lumenal content fractions suggests that, although very-low-density-lipoprotein (VLDL) lipids are present in the endoplasmic reticulum lumen, a large fraction of these is not associated with apo-B. Collectively these observations suggest that assembly of apo-B into complete VLDL is not cotranslational, that most lipids become associated with apo-B late in the endoplasmic reticulum compartment and that the lipids are further modified in the Golgi lumen.
Cartwright
I J
IJ
Department of Molecular Biology and Biotechnology, University of Sheffield, U.K.
Higgins
J A
JA
D000818:Animals; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002458:Cell Fractionation; D002478:Cells, Cultured; D005700:Galactosyltransferases / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D007700:Kinetics; D008079:Lipoproteins, VLDL / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D011817:Rabbits; D013347:Subcellular Fractions / Q000378:metabolism
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D008079:Lipoproteins, VLDL; D008715:Methionine; D009251:NADPH-Ferrihemoprotein Reductase; D005700:Galactosyltransferases