Identification of domains in apolipoprotein B100 that confer a high requirement for the microsomal triglyceride transfer protein

The microsomal triglyceride transfer protein (MTP) is required for the asse mbly and secretion of apoB-containing lipoproteins, To investigate the role of MTP in lipoprotein assembly, we determined the ability of carboxyl-term inally truncated forms of apoB to be secreted from cells treated with the M TP inhibitor 4'-bromo-3'-methylmetaqualone (Benoist, F., Nicodeme, E., and Grand-Ferret, T. (1996) Eur. J. Biochem. 240, 713-720). In Caco-2 and mhAT3 F cells that produce apoB100 and apoB48, the inhibitor preferentially block ed apoB100 secretion. When the inhibitor was tested on McARH7777 cells stab ly transfected with cDNAs encoding human apoB100, apoB72, apoB53, apoB29, a nd apoB18, the secretion of apoB100, apoB72, and apoB53 was preferentially impaired relative to apoB48 and shorter forms. To delineate the region betw een apoB48 and apoB53 that has a high requirement for MTP, we used puromyci n to generate a range of truncated forms of apoB in HepG2 cells. The secret ion of apoB53 and longer forms of apoB was markedly affected by low concent rations of the MTP inhibitor (similar to 1 mu M), whereas apoB51 and smalle r forms of apoB were only affected at higher concentrations (> 10 mu M). Th e size-related sensitivity to MTP inhibitor was not due to late processing or retention, since the same result was observed when nascent lipoproteins were isolated from the endoplasmic reticulum. The MTP inhibitor did not alt er the density of the secreted lipoproteins, indicating that each apoB poly peptide requires a minimally defined amount of lipid to attain a secretable conformation, Our results suggest that the folding of the domain between a poB51 and apoB53 has a high requirement for lipid, This domain is predicted to form amphipathic alpha-helices and to bind lipid reversibly, It proceed s and is followed by rigid amphipathic beta-sheets that are predicted to as sociate with lipid irreversibly, We speculate that these domains enable apo B to switch from a stable lipid-poor conformation in apoB48 to another lipi d-rich conformation in apoB100 during lipoprotein assembly.