Abstract
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.
Highlights
Human apolipoprotein1 B100 is an extremely large (4,536 amino acids) hydrophobic protein that is essential for assembly and secretion of VLDL from liver
One unique feature of apoB secretion is that, in contrast to the widely accepted model for secretion of typical secretory proteins, newly synthesized apoB appears to exist in two intracellular populations: a lipoprotein-associated form wholly within the ER lumen, and a membrane-associated form in which portions of apoB are exposed to the cytosol and are susceptible to degradation [7, 23, 25, 26]
ApoB15 and ApoB48 Co-translationally Bind Newly Synthesized Phosphatidylcholine—As a prerequisite for secretion, apoB100 and apolipoprotein B48 (apoB48) are assembled into lipoprotein particles consisting of a hydrophobic core of neutral lipids covered by a surface monolayer of phospholipids, unesterified cholesterol, and apoproteins
Summary
Materials—[35S]Methionine, [14C]CDP-choline, [14C]triolein, and Amplify were purchased from Amersham Canada. and Tran35S-label was from ICN. [3H]Oleoyl-CoA was chemically synthesized [38]. Reisolated membranes were suspended in 100 l of 0.25 M sucrose containing either Tris-HCl (pH 7.5) or 100 mM sodium carbonate (pH 11.5), which releases both luminal contents and loosely bound membrane proteins from microsomes [41]. Translation products were boiled for 2 min in buffer A (1% SDS, 150 mM Tris-HCl (pH 8.0), 150 mM sodium chloride, 10 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). Immunoprecipitated proteins were released from the Sepharose by boiling the beads for 2 min in buffer containing 8 M urea and 2% SDS. Synthesized apoB and -lactamase were isolated from luminal contents by immunoaffinity chromatography under nondenaturing conditions using Sepharose 4B linked covalently to polyclonal antibodies directed against either human apoB100 or -lactamase [1]. Background transfer in the absence of MTP was subtracted to calculate MTP-mediated transfer of triacylglycerol from donor vesicles
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