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J. Biol. Chem., Vol. 265, Issue 17, 10005-10011, Jun, 1990

Apolipoprotein B is both integrated into and translocated across the endoplasmic reticulum membrane. Evidence for two functionally distinct pools

RA Davis, RN Thrift, CC Wu and KE Howell
Cell and Molecular Biology Unit, University of Colorado Medical School, Denver 80262.

Apolipoprotein B (apoB), a protein containing several hydrophobic beta- sheet structures, is essential for the assembly of triglyceride-rich lipoproteins. Previously, we found that only a fraction of de novo synthesized apoB is secreted; the remainder is retained in the endoplasmic reticulum where it is degraded. To understand the basis for these observations, translocation, the first step in the secretory pathway, was examined. Translocation of apoB was determined by its sensitivity to degradation by the exogenous protease, trypsin. In rough microsomes, about half of the apoB was degraded by trypsin. In contrast, in Golgi fractions little (if any) apoB was accessible to trypsin. Essentially all of the apoB that was degraded was membrane bound. Monoclonal IgGs against either the N-terminal or C-terminal halves of apoB were bound to magnetic beads and used to immunoisolate microsomes. In contrast to the specific ability of the IgGs against apoB to isolate microsomes, little or no microsomes were isolated using nonimmune IgG and IgG against albumin. Since microsomes remained intact and oriented right-side out as demonstrated by the inability of trypsin both to degrade albumin and to affect the capacity of the intralumenal enzyme glucose-6-phosphatase to dephosphorylate mannose 6-phosphate, the data suggest that a pool of apoB is exposed on the cytoplasmic surface of the endoplasmic reticulum membrane. To determine if the trypsin-accessible pool of apoB is a transient form, pulse-chase experiments were performed. The results show that the percent of apoB that was trypsin accessible increased during the first 20 min of the chase, suggesting that during this time the trypsin-accessible pool of apoB is not translocated (it does not become trypsin insensitive). Thus, in two in vivo models (cultured cells and rat liver) translocation of apoB is not quantitative. We propose that apoB translocation across the endoplasmic reticulum determines its entry into two functionally distinct pools. The intralumenal trypsin- insensitive pool participates in the assembly of very low density lipoprotein; the trypsin-accessible nontranslocated cytoplasmic pool is shunted into a degradative pathway. Regulated translocation of apoB may provide a unique mechanism with which to determine the rate of very low density lipoprotein assembly/secretion.
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