The amino-terminal domain of apolipoprotein B does not undergo retrograde translocation from the endoplasmic reticulum to the cytosol. Proteasomal degradation of nascent apolipoprotein B begins at the carboxyl terminus of the protein, while apolipoprotein B is still in its original translocon.
We studied the sequential topology of the NH(2) and COOH termini of apoB during translocation by expressing, in Chinese hamster ovary (CHO) and HepG2 cells, an apoB42 construct with c-Myc and hemagglutinin (HA) tags at 2 and 41% (relative to apoB100) of its amino acid sequence. We conducted similar studies using monoclonal antibodies against the NH(2) and COOH termini of apoB100 in HepG2 cells. After radiolabeling, microsomes were immunoisolated from transfected CHO cells using anti-c-Myc or anti-HA antibodies. Throughout a 60-min chase in the presence of N-acetyl-leucyl-norleucinal, more than 90% of microsomes were isolated by anti-HA antibodies, whereas less than 10% were isolated by anti-c-Myc antibodies. Proteinase K digestion of total microsomes consistently generated two fragments ( approximately 70 and approximately 120 kDa) of apoB42 containing the NH(2) terminus throughout the chase; no fragments containing the COOH terminus were detected. Immunofluorescent studies of transfected CHO cells were consistent with results from the labeling studies. Essentially identical results were obtained from pulse-chase studies in both native and apoB42-transfected HepG2 cells. The present studies support a model in which, in the absence of adequate core lipid synthesis, there is partial translocation of apoB leading to cytosolic exposure, ubiquitination, and proteasomal degradation directly from the original translocation channel.
Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA. jl698@columbia.edu
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S
Liang
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Wu
E A
EA
Fisher
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Ginsberg
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001055:Apolipoproteins B / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D019286:Endopeptidase K / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006388:Hemagglutinins / Q000235:genetics / Q000276:immunology; D006801:Humans; D008861:Microsomes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism*; D019301:Oleic Acid / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D001055:Apolipoproteins B; D006388:Hemagglutinins; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D019301:Oleic Acid; D019286:Endopeptidase K; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex