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 translocation across the endoplasmic reticulum membrane is inefficient, resulting in the partial translocation and exposure of apoB100 on the cytoplasmic surface of the endoplasmic reticulum. Cytosolic exposure leads to the association of nascent apoB with heat shock protein 70 and to its predisposition to ubiquitination and proteasomal degradation. The basis for the inefficient translocation of apoB100 remains unclear and controversial. To test the hypothesis that beta sheet domains present in apoB100 contribute to its inefficient translocation, we created human apoB chimeric constructs apoB13,16 and apoB13,13,16, which contain amino-terminal alpha 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 carboxyl-terminal truncations of apoB100, apoB34 and apoB42, were used to transfect HepG2 and Chinese hamster ovary cells. In contrast to the lack of effect of proteinase K on apoB13,16 and apoB13,13,16, the levels of apoB34, apoB42, and apoB13,16,beta were decreased by 70-85% after proteinase K-induced 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, apoB34, apoB42, and full-length apoB100 but had no effect on the cell levels of apoB13,16 and apoB13,13,16. When HepG2 cells were incubated with a microsomal triglyceride transfer protein inhibitor, the cellular levels of apoB13,16,beta, apoB34, and apoB42 were decreased by 70-80%, whereas the levels of apoB13,16 and apoB13,13,16 were unaffected. The effects of microsomal triglyceride transfer protein inhibition were reversed by lactacystin. Our results clearly demonstrate that the translocation efficiency, susceptibility to proteasomal degradation, and lipid responsiveness of apoB were determined 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.
Liang
J
J
Department of Medicine, College of Physicians and Surgeons of Columbia University, New York, New York 10032, USA.
Wu
X
X
Jiang
H
H
Zhou
M
M
Yang
H
H
Angkeow
P
P
Huang
L S
LS
Sturley
S L
SL
Ginsberg
H
H
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D002352:Carrier Proteins; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism*; D019301:Oleic Acid / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D002352:Carrier Proteins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; C106092:microsomal triglyceride transfer protein; D019301:Oleic Acid; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex