PIETER H. E. GROOT's research while affiliated with The University of Sheffield and other places

We have used a panel of anti-rabbit apolipoprotein B monoclonal antibodies in a competitive ELISA to probe the availability of apoB in rough endoplasmic reticulum, smooth endoplasmic reticulum, cis-enriched Golgi, and trans-enriched Golgi fractions from rabbit liver. The ability of each subcellular fraction to inhibit binding of monoclonal antibody to immobilized low density lipoprotein (LDL)-apoB was determined and compared with the expected inhibition based on the apoB content of the fraction. The vesicles remained closed during ELISA, demonstrated by monitoring loss of radiolabeled secretory proteins from the lumen and by measuring leakage of albumin from the vesicles. In control experiments, vesicles were permeabilized using 0.4% taurocholate. All epitopes of apoB were fully expressed in closed trans-Golgi vesicles, indicating that the membrane-bound apoB is at the cytosolic side of this fraction. In the smooth endoplasmic reticulum the epitopes were expressed between 55 and 70%, suggesting that the two pools of apoB may exist in these membranes. These results suggest that newly synthesized apoB has two possible fates. It may be incorporated into the cytosolic side of the endoplasmic reticulum from where it moves to the cytosolic side of the Golgi membrane, or newly synthesized apoB may be translocated to the lumenal surface of the endoplasmic reticulum membrane followed by assembly with lipids for secretion.

We have investigated the intracellular distribution of apolipoprotein B (apo B) in rabbit liver by immunoblotting, radioimmunoassay (r.i.a.) and enzyme-linked immunoassay (e.l.i.s.a.). Apo B100 was detected in total microsomes, rough microsomes, smooth microsomes, trans-enriched Golgi and cis-enriched Golgi and membrane and cisternal-content subfractions prepared from these fractions. There was also evidence of degradation of apo B100 in the Golgi membrane fractions. The amount of apo B in the subcellular fractions detected by competitive r.i.a. or e.l.i.s.a. ranged from 1.5 micrograms/mg of protein in the rough endoplasmic reticulum to 13 micrograms/mg of protein in the trans-Golgi fraction. Using internal standards (NADPH-cytochrome c reductase for the endoplasmic reticulum and galactosyltransferase for the Golgi membranes) it was calculated that all the apo B of liver is recovered within the secretory compartment, with 63% of the total apo B in the endoplasmic reticulum and the remainder in the Golgi. When the subcellular fractions were separated into membranes and cisternal contents, 60%, 50%, 60% and 30% of the total apo B was recovered in the membrane of the rough microsomes, smooth microsomes, cis-Golgi and trans-Golgi respectively. Using competitive e.l.i.s.a. we found that the membrane-bound form of the apo B was exposed at the cytosolic surface of the intact subcellular fractions. These observations are consistent with a model for assembly of very-low-density lipoproteins (VLDL) in which newly synthesized apo B is incorporated into a membrane-bound pool and a lumenal pool. The membrane-bound pool not used for VLDL assembly may be degraded, possibly in the Golgi region.

We have used a competitive enzyme-linked immunoassay with a panel of monoclonal antibodies to probe the topography of the membrane-bound form of apolipoprotein B (apo B) in rabbit microsomes. All epitopes investigated were found to be expressed at the cytosolic side of the microsomal membrane under conditions in which the vesicles remained sealed. These results indicate that the membrane-associated form of apolipoprotein B is either at the cytosolic side of the endoplasmic reticulum membrane or integrated into the membrane. From this site apo B may be translocated to the lumen for assembly into VLDL or may be degraded.