A Mechanism of Membrane Neutral Lipid Acquisition by the Microsomal Triglyceride Transfer Protein

Abstract

Chylomicrons (CM)1 and very low density lipoproteins (VLDL) are among the largest macromolecular complexes secreted from eukaryotic cells. The assembly of neutral lipids and phospholipids into CM and VLDL is nucleated around a single molecule of apoB and requires a microsomal triglyceride transfer protein (MTP) complexed to the endoplasmic reticulumresident protein, protein disulfide isomerase (PDI). The function of the MTP-PDI complex is to supply apoB with sufficient lipid to form a soluble lipoprotein. Defects of apoB and MTP cause hypobetalipoproteinemia and abetalipoproteinemia, respectively (1, 2).ApoB and MTP have structural homology with lamprey lipovitellin (LV) (3). LV contains an N-terminal ␤-barrel (amino acids 17-296), an ␣-helical structure (amino acids 297-614), and a C-terminal lipid binding cavity (4). The structural relationship between MTP, apoB, and LV is supported by conservation of the gene and protein structure (3,5). Important features of the quaternary structure of the lamprey LV homodimer are adapted in MTP to form a heterodimer with PDI and to associate with apoB during lipoprotein production (3, 5). The defining difference between MTP, apoB, and LV is related to their C-terminal lipid binding structures, which associate with different amounts of lipid (3). LV binds principally phospholipid with a stoichiometry of ϳ35 molecules/subunit (6). MTP binds 1-5 molecules of lipid (7). ApoB has a long C-terminal extension (ϳ3500 amino acids), which incorporates a large neutral lipid core (8).Here, we have addressed the mechanism by which MTP-PDI acquires neutral lipid from phospholipid bilayers for the assembly of VLDL and CMs. In the absence of a crystal structure for MTP, we derived a homology model to guide mutagenesis and biophysical studies. The experimental data substantiate the overall predictions of the model and provide insights into the mechanism of lipid acquisition and binding. EXPERIMENTAL PROCEDURESModeling-Models were developed on the alignment shown in Fig. 1 and the x-ray crystal structure of lamprey LV (Protein Data Bank accession number 1LLV), refined to an R-value of 0.19 at 2.8 Å resolution (4). The C-sheet was modeled using INSIGHT interactive graphics software and the Homology computer program (Biosym Technologies, San Diego) and the A-sheet with the general purpose modeling program O (9). Models were energy minimized and the quality of the coordinates assessed as described (3).Mutagenesis and Expression Studies-Mutagenesis was performed by a polymerase chain reaction-based strategy (3). All constructs were sequenced before use. Transfections, preparation of cell extracts, and triglyceride transfer activities were performed as described (2, 3).Triglyceride Binding and Fusogenic Activity-Wild-type (WT) and mutant MTP-PDI complexes were purified as described (10). Donor small unilamellar vesicles (SUVs) were prepared as described (2), purified to homogeneity (11), and incubated with MTP (w/w 70:1) for 2 h at 37°C. Lipid-protein complexes were separated on a Sepharose CL-4B...

Microsomal Triglyceride Transfer Protein Promotes the Secretion of Xenopus laevis Vitellogenin A1

“…The abetalipoproteinemia missense mutation (N780Y) has been reported to accumulate to wild type levels; however, it displays only background triglyceride transfer activity (14,22). As observed in Fig.…”

A Drosophila Microsomal Triglyceride Transfer Protein Homolog Promotes the Assembly and Secretion of Human Apolipoprotein B

“…Despite these functional similarities, the human and Drosophila proteins displayed different susceptibilities to two inhibitors of vertebrate MTP. Furthermore, the Drosophila protein displayed low vesicle-based TG transfer activity, possibly due to divergence of a predicted hydrophobic alpha-helical peptide thought critical for efficient lipid acquisition and transfer by vertebrate MTP (45). We conclude, based on these criteria, that CG9342 is an ortholog of vertebrate MTP but may possess only a subset of vertebrate MTP functions.…”

“…One of these peptides (peptide A) is predicted to associate with membrane surfaces at an oblique angle, destabilizing the bilayer and facilitating lipid acquisition (45). Interestingly, however, this peptide sequence, which is highly conserved in all vertebrate species, appears divergent in both Drosophila and Anopheles (Fig.…”

“…Transfer-A structural feature of human MTP is the presence of two hydrophobic peptides important for membrane binding and lipid transfer activity (45). One of these peptides (peptide A) is predicted to associate with membrane surfaces at an oblique angle, destabilizing the bilayer and facilitating lipid acquisition (45).…”

Interfacial properties of amphipathic β strand consensus peptides of apolipoprotein B at oil/water interfaces

“…The N-terminal domain up to apoB-20.5 contains ␣ helices and amphipathic ␤ sheets and is essential for the secretion of apoB (3,(15)(16)(17). It has high homology to MTP and lipovitellins (18)(19)(20). The X-ray crystal structure of lamprey lipovitellin (21) suggests that this homologous region of apoB could form part of a "lipid pocket" domain (18,19), but homology to apoB stops at apoB-20.5 (18), and MTP-poor C-127 cells secreting apoB-17 (18) and apoB-20.5 (M. Carraway and H. Herscovitz, personal communication) are secreted with only a very small amount of surface lipids.…”