In vitro reconstitution of assembly of apolipoprotein B48-containing lipoproteins.
Human apolipoprotein B48 (apoB48) and apoB15 (the NH2-terminal 48 and 15% of apoB100, respectively) were translated in vitro from their respective mRNAs using a rabbit reticulocyte lysate and microsomes derived from rat liver or dog pancreas. Synthesis of phosphatidylcholine and triacylglycerols was reconstituted in freshly isolated microsomes by the addition of precursors of these glycerolipids (acylcoenzyme A, glycerol 3-phosphate, and CDP-choline) before, during, or after translation. Assembly of apoB15 and apoB48 with newly synthesized phospholipids and triacylglycerols was favored by active, co-translational lipid synthesis. Moreover, translocation of apoB48 but not B15 into the microsomal lumen was increased in the presence of co-translational lipid synthesis. When apoB48 was translated in vitro, approximately 50% of apoB48 was buoyant at a density of <1.10 g/ml in the lumen of liver microsomes only when lipid synthesis was reconstituted during translation. Microsomal triacylglycerol transfer protein has been proposed to be essential for lipidation and/or translocation of apoB48. However, apoB48 was translocated into the lumen of dog pancreas microsomes in which the activity of the microsomal triacylglycerol transfer protein was not detectable. These data indicate that (i) apoB15 and apoB48 bind newly synthesized phosphatidylcholine during translocation; (ii) apoB48 but not apoB15 associates co-translationally with triacylglycerols; (iii) translocation of apoB48 but not apoB15 is stimulated by lipid synthesis; (iv) assembly of buoyant apoB48-containing lipoproteins can be reconstituted in vitro in the presence of active lipid synthesis; and (v) even in microsomes lacking microsomal triacylglycerol transfer protein activity, apoB48 is translocated into the lumen.
Rusiñol
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Lipid and Lipoprotein Research Group and the Department of Medicine, University of Alberta, Edmonton, Alberta T6G 2S2, Canada.
Jamil
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Vance
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JE
D000818:Animals; D053283:Apolipoprotein B-48; D001055:Apolipoproteins B / Q000378:metabolism*; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002474:Cell-Free System; D004285:Dogs; D006801:Humans; D008055:Lipids / Q000096:biosynthesis; D008074:Lipoproteins / Q000737:chemistry*; D008297:Male; D008861:Microsomes / Q000378:metabolism; D010179:Pancreas / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014280:Triglycerides / Q000378:metabolism
D053283:Apolipoprotein B-48; D001055:Apolipoproteins B; D002352:Carrier Proteins; D008055:Lipids; D008074:Lipoproteins; D010713:Phosphatidylcholines; D012333:RNA, Messenger; D014280:Triglycerides