Co-translational degradation of apolipoprotein B100 by the proteasome is prevented by microsomal triglyceride transfer protein. Synchronized translation studies on HepG2 cells treated with an inhibitor of microsomal triglyceride transfer protein.
We studied the effect of inhibition of microsomal triglyceride transfer protein (MTP) on apolipoprotein (apo) B100 translation and secretion using HepG2 cells. The MTP-mediated lipid transfer activity was reduced using a specific MTP inhibitor. ApoB100 translation was synchronized by treatment with puromycin prior to L-[35S]methionine pulse-chase labeling. During the first 4 min of chase, synthesis of apoB polypeptides the size of 100-200 kDa was insensitive to the inhibitor, suggesting that inhibition of MTP did not affect the initiation of apoB100 translation. After 15 min of chase, the 100-200-kDa species were chased into polypeptides larger than 320 kDa (i.e. apoB65 or 65% of full-length apoB100) in both control and inhibitor-treated cells. However, the amount of these polypeptides decreased (by 36% for apoB65-75, by 64% for apoB75-85, by 76% for apoB85-95, and by 77% for apoB100) upon MTP inhibition. No accumulation of smaller polypeptides was observed, but total immunoprecipitable apoB radioactivity was decreased suggesting that apoB could undergo co-translational degradation when MTP activity was reduced. Inhibitors of the multicatalytic proteinase complex (proteasome) such as lactacystin or MG-115 could prevent apoB co-translational degradation. Nevertheless, MG-115 could not avoid the MTP inhibitor decreasing apoB100 secretion but rather induced the accumulation of secretion-incompetent apoB100 in the cell. These results indicate that MTP activity is required during the elongation of apoB100 polypeptides, particularly at the sequences downstream of carboxyl terminus of apoB65. Co-translational degradation might constitute a more general mechanism of early quality control for large or complex proteins.
Benoist
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F
Laboratoire Glaxo Wellcome, Centre de Recherche, 25 avenue du Quebec, ZA de Courtaboeuf, 91951 Les Ulis cedex, France.
Grand-Perret
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T
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000378:metabolism*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D002478:Cells, Cultured; D053480:Cholesterol Ester Transfer Proteins; D003546:Cysteine Endopeptidases / Q000502:physiology*; D006023:Glycoproteins; D006801:Humans; D009097:Multienzyme Complexes / Q000502:physiology*; D046988:Proteasome Endopeptidase Complex; D014176:Protein Biosynthesis; D014280:Triglycerides / Q000378:metabolism*
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; C506642:CETP protein, human; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D006023:Glycoproteins; D009097:Multienzyme Complexes; D014280:Triglycerides; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex