Translocation efficiency, susceptibility to proteasomal degradation, and lipid responsiveness of apolipoprotein B are determined by the presence of beta sheet domains

Apolipoprotein (apo) B100 is an atypical secretory protein in that its tran slocation across the endoplasmic reticulum membrane is inefficient, resulti ng in the partial translocation and exposure of apoB100 on the cytoplasmic surface of the endoplasmic reticulum. Cytosolic exposure leads to the assoc iation of nascent apoB with heat shock protein 70 and to its predisposition to ubiquitination and proteasomal degradation. The basis for the inefficie nt translocation of apoB100 remains unclear and controversial. To test the hypothesis that beta sheet domains present in apoB100 contribute to its ine fficient translocation, we created human apoB chimeric constructs apoB13,16 and apoB13,13,16, which contain amino-terminal cu globular domains but no beta sheet domains, and apoB13,16,beta, which has an amphipathic beta sheet domain of apoB100 inserted into apoB13,16. These constructs, along with ca rboxyl-terminal truncations of apoB100, apoB34 and apoB42, were used to tra nsfect HepG2 and Chinese hamster ovary cells. In contrast to the lack of ef fect of proteinase K on apoB13,16 and apoB13,13,16, the levels of apoB34, a poB42, and apoB13,16,beta were decreased by 70-85% after proteinase K-induc ed proteolysis in both HepG2 and Chinese hamster ovary cells. Either oleic acid or proteasomal inhibitors (N-acetyl-leucinyl-leucinyl-norleucinal and lactacystin) significantly increased the cell levels of apoB13,16,beta, apo B34, apoB42, and full-length apoB100 but had no effect on the cell levels o f apoB13,16 and apoB13,13,16. When HepG2 cells were incubated with a micros omal triglyceride transfer protein inhibitor, the cellular levels of apoB13 ,16,beta, apoB34, and apoB42 were decreased by 70-80%, whereas the levels o f apoB13,16 and apoB13,13,16 were unaffected. The effects of microsomal tri glyceride transfer protein inhibition were reversed by lactacystin. Our res ults clearly demonstrate that the translocation efficiency, susceptibility to proteasomal degradation, and lipid responsiveness of apoB were determine d by the presence of a lipid binding beta sheet domain. It is possible that beta sheet domains may at least transiently facilitate the interaction of apoB with the lipid bilayer surrounding the translocation channel.