title: Folding of the amino-terminal domain of apolipoprotein B initiates microsomal triglyceride transfer protein-dependent lipid transfer to nascent very low density lipoprotein.
abstract: The initial assembly of apolipoprotein B100 (apoB) into lipoprotein particles occurs cotranslationally. To examine steps required to initiate this process, the intracellular folding and assembly of the amino-terminal 28% of apoB (apoB28) was examined using several criteria including nonreducing gel electrophoresis, sensitivity to dithiothreitol (DTT)-mediated reduction, and buoyant density gradient centrifugation. In hepatoma cells, after a 1-min pulse with radiolabeled amino acids, labeled apoB28 migrated during gel electrophoresis in the folded position and was resistant to reduction in vivo with 2 mM DTT. A similar rate and extent of folding was observed in Chinese hamster ovary cells, a microsomal triglyceride transfer protein (MTP)-negative cell line that can neither lipidate nor efficiently secrete apoB28. Amino-terminal folding of apoB28 was essential for its subsequent intracellular lipidation as apoB28 synthesized in hepatoma cells under reducing conditions remained lipid poor (d > 1.25 g/ml) and was retained intracellularly. Upon DTT removal, reduced apoB28 underwent a process of rapid (t1/2 approximately 2 min) post-translational folding followed by a slower process of MTP-dependent lipidation. As with the cotranslational assembly pathway, post-translational lipidation of apoB28 displayed a strict dependence upon amino-terminal folding. We conclude that: 1) folding of the amino-terminal disulfide bonded domain of apoB is achieved prior to the completion of translation and is independent of MTP and events associated with buoyant lipoprotein formation and 2) domain-specific folding of apoBs amino-terminal region is required to initiate MTP-dependent lipid transfer to nascent apoB in the hepatic endoplasmic reticulum.
authors.affiliation: Department of Comparative Medicine, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27157-1040, USA.
authors.forename: M F
authors.initials: MF
authors.lastname: Ingram
authors.forename: G S
authors.initials: GS
authors.lastname: Shelness
mesh_terms: D000818:Animals; D053299:Apolipoprotein B-100; D053283:Apolipoprotein B-48; D001055:Apolipoproteins B / Q000737:chemistry* / Q000378:metabolism; D001693:Biological Transport, Active; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism*; D053480:Cholesterol Ester Transfer Proteins; D002788:Cholesterol Esters / Q000378:metabolism*; D006224:Cricetinae; D006023:Glycoproteins / Q000378:metabolism*; D008079:Lipoproteins, VLDL / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D014280:Triglycerides / Q000378:metabolism*
chemical_list: D053299:Apolipoprotein B-100; D053283:Apolipoprotein B-48; D001055:Apolipoproteins B; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D002788:Cholesterol Esters; D006023:Glycoproteins; D008079:Lipoproteins, VLDL; D010446:Peptide Fragments; D014280:Triglycerides