Apolipoprotein B (apoB) is the major protein component of large lipoprotein particles that transport lipids and cholesterol. We have developed a detailed model of the first 1000 residues of apoB using standard sequence alignment programs (ClustalW and MACAW) and the MODELLER6 package for three-dimensional homology modeling. The validity of the apoB model was supported by conservation of disulfide bonds, location of all proline residues in turns and loops, and conservation of the hydrophobic faces of the two C-terminal amphipathic beta-sheets, betaA (residues 600-763) and betaB (residues 780-1000). This model suggests a lipid-pocket mechanism for initiation of lipoprotein particle assembly. In a previous model we suggested that microsomal triglyceride transfer protein might play a structural role in completion of the lipid pocket. We no longer think this likely, but instead propose a hairpin-bridge mechanism for lipid pocket completion. Salt-bridges between four tandem charged residues (717-720) in the turn of the hairpin-bridge and four tandem complementary residues (997-1000) at the C-terminus of the model lock the bridge in the closed position, enabling the deposition of an asymmetric bilayer within the lipid pocket.

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Summary Biogenesis of high density lipoproteins (HDL) is coupled to the transmembrane protein, ATP-binding cassette transporter A1 (ABCA1), which transports phospholipid (PL) from the inner to the outer membrane monolayer. Using a combination of computational and experimental approaches, we show that increased outer lipid monolayer surface density driven by excess PL or by membrane insertion of amphipathic helices, results in pleating of the outer monolayer to form membrane-attached discoidal bilayers. Apolipoprotein (apo)A-I accelerates and stabilizes the pleats. In the absence of apoA-I, pleats collapse to form vesicles. These results mimic cells overexpressing ABCA1 that, in the absence of apoA-I, form and release vesicles. We conclude that the basic driving force for nascent discoidal HDL assembly is a PL pump-induced surface density increase that produces lipid monolayer pleating. We then argue that ABCA1 forms an extracellular reservoir containing an isolated pressurized lipid monolayer decoupled from the transbilayer density buffering of cholesterol.

We previously demonstrated that the N-terminal 1000 amino acid residues of human apolipoprotein (apo) B (designated apoB:1000) are competent to fold into a three-sided lipovitellin-like lipid binding cavity to form the apoB "lipid pocket" without a structural requirement for microsomal triglyceride transfer protein (MTP). Our results established that this primordial apoB-containing particle is phospholipid-rich (Manchekar, M., Richardson, P. E., Forte, T. M., Datta, G., Segrest, J. P., and Dashti, N. Apolipoprotein B (apoB)2 is synthesized primarily in hepatocytes and enterocytes and has a fundamental role in the transport and metabolism of plasma triacylglycerols (TAG) and cholesterol (1, 2). ApoB is a predominant protein component of very low density lipoproteins (VLDL) and intermediate density lipoproteins and is essentially the only apoprotein component of low density lipoproteins (LDL 2 ) (3, 4). ApoB100 (the fulllength protein) is one of the largest monomeric proteins known with 4536 amino acid residues (2). It is expressed primarily in mammalian liver, is an essential structural component for the formation and secretion of VLDL, and serves as a ligand for the LDL receptor (2). ApoB is present as a single molecule per lipoprotein particle (5); and therefore, its concentration in the plasma approximates the number of potential atherogenic lipoprotein particles.The processes involved in the assembly of apoB-containing lipoproteins in the liver are complex and are regulated at multiple levels throughout the secretory pathway. The assembly of apoB-containing lipoproteins occurs co-translationally (1), i.e. while the C-terminal portion is still being synthesized on the ribosome of the endoplasmic reticulum (ER), the N-terminal portion is translocated across the ER and is assembled as a small lipoprotein particle. The addition of lipids to apoB is widely believed to occur in two steps (2, 6, 7). The first step involves the addition of small amounts of lipids to apoB, as it is translated and translocated into the lumen of ER preventing its degradation and formation of a partially lipidated small pre-VLDL particle in the high density lipoprotein (HDL) density range (2,7,8). In the second step, this pre-VLDL particle is believed to acquire the bulk of its core lipids and is converted to bona fide VLDL (2, 7, 9), presumably by fusing with a large, VLDL-sized, apoB-free TAG particle (9). Biochemical studies of VLDL assembly support the concept that the bulk of neutral lipids are added in the second step after apoB translation is completed (10).* This work was supported by the National Institutes of Health Grants HL084685 and PO1 HL34343. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. 2 The abbreviations used are: apoB, apolipoprotein B; apoB:1000, N-terminal 22.05% (residues 1-1000) of the mature protein; BSA, bovine serum albu-

We previously proposed that the N-terminal 1000-residue ␤␣ 1 domain of apolipoprotein B (apoB) forms a bulk lipid pocket homologous to that of lamprey lipovitellin. In support of this "lipid pocket" hypothesis, we demonstrated that apoB:1000 (residues 1-1000) is secreted by a stable transformant of McA-RH7777 cells as a monodisperse particle with high density lipoprotein 3 (HDL 3 ) density. In contrast, apoB:931 (residues 1-931), missing only 69 residues of the sequence homologous to lipovitellin, was secreted as a particle considerably more dense than HDL 3 . In the present study we have determined the stoichiometry of the lipid component of the apoB:931 and apoB:1000 particles. The secreted [ 3 H]-glycerol-labeled apoB:1000 particles, isolated by nondenaturing gradient gel electrophoresis, contained 50 phospholipid (PL) and 11 triacylglycerol (TAG) molecules/particle. In contrast, apoB:931 particles contained only a few molecules of PL and were devoid of TAG. The unlabeled apoB:1000 particles, isolated by immunoaffinity chromatography, contained 56 PL, 8 TAG, and 7 cholesteryl ester molecules/particle. The surface to core lipid ratio of apoB:1000-containing particles was ϳ4:1 and was not affected by oleate supplementation. Although very small amounts of microsomal triglyceride transfer protein (MTP) were associated with apoB:1000 particles, it never approached a 1:1 molar ratio of MTP to apoB. These results support a model in which (i) the first 1000 amino acid residues of apoB are competent to complete the lipid pocket without a structural requirement for MTP; (ii) a portion, or perhaps all, of the amino acid residues between 931 and 1000 of apoB-100 are critical for the formation of a stable, bulk lipid-containing nascent lipoprotein particle, and (iii) the lipid pocket created by the first 1000 residues of apoB-100 is PL-rich, suggesting a small bilayer type organization and has a maximum capacity on the order of 50 molecules of phospholipid.Apolipoprotein (apo) 1 B has a fundamental role in the transport and metabolism of plasma triacylglycerols (TAG) and cholesterol and is synthesized primarily in hepatocytes and enterocytes (1-3). ApoB is present as a single molecule per lipoprotein particle and exists in two forms in humans, apoB-100 and apoB-48. ApoB-100, full-length protein consisting of 4536 amino acid residues (2), is an essential structural component for the formation and secretion of very low density lipoproteins (VLDL), the precursor of low density lipoproteins (LDL), and is expressed primarily in mammalian liver. ApoB-48 (the N-terminal 48% of apoB-100) is produced by a post-transcriptional modification of the apoB mRNA at codon 2153 that converts a glutamine codon to a stop codon (2). ApoB-48 is essential for the formation and secretion of chylomicrons and is expressed in mammalian intestine and in the liver of some non-human mammals (2). The B apolipoproteins are highly insoluble in aqueous solutions and thus remain with the lipoprotein particle throughout their metabolism (4). Because of the ...