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 resp ective 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 t he addition of precursors of these glycerolipids (acylcoenzyme A, glyc erol 3-phosphate, and CDP-choline) before, during, or after translatio n. Assembly of apoB15 and apoB48 with newly synthesized phospholipids and triacylglycerols was favored by active, co-translational lipid syn thesis. Moreover, translocation of apoB48 but not B15 into the microso mal lumen was increased in the presence of co-translational lipid synt hesis. When apoB48 was translated in vitro, approximately 50% of apoB4 8 was buoyant at a density of <1.10 g/ml in the lumen of liver microso mes only when lipid synthesis was reconstituted during translation. Mi crosomal triacylglycerol transfer protein has been proposed to be esse ntial for lipidation and/or translocation of apoB48. However, apoB48 w as translocated into the lumen of dog pancreas microsomes in which the activity of the microsomal triacylglycerol transfer protein was not d etectable. These data indicate that (i) apoB15 and apoB48 bind newly s ynthesized phosphatidylcholine during translocation; (ii) apoB48 but n ot apoB15 associates co-translationally with triacylglycerols; (iii) t ranslocation of apoB48 but not apoB15 is stimulated by lipid synthesis ; (iv) assembly of buoyant apoB48-containing lipoproteins can be recon stituted 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.