FOLDING OF THE AMINO-TERMINAL DOMAIN OF APOLIPOPROTEIN-B INITIATES MICROSOMAL TRIGLYCERIDE TRANSFER PROTEIN-DEPENDENT LIPID TRANSFER A TO NASCENT VERY-LOW-DENSITY LIPOPROTEIN

The initial assembly of apolipoprotein B100 (apoB) into lipoprotein pa rticles occurs cotranslationally. To examine steps required to initiat e this process, the intracellular folding and assembly of the amino-te rminal 28% of apoB (apoB28) was examined using several criteria includ ing nonreducing gel electrophoresis, sensitivity to dithiothreitol (DT T)-mediated reduction, and buoyant density gradient centrifugation, In hepatoma cells, after a 1-min pulse with radiolabeled amino acids, la beled apoB28 migrated during gel electrophoresis in the folded positio n 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 f olding of apoB28 was essential for its subsequent intracellular lipida tion 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 (t(1/2) approximate to 2 min) posttranslational folding followed by a slower process of MTP-dependent lipidation. As with the cotranslational assem bly pathway, post-translational lipidation of apoB28 displayed a stric t dependence upon amino-terminal folding. We conclude that: 1) folding of the aminoterminal disulfide bonded domain of apoB is achieved prio r to the completion of translation and is independent of MTP and event s associated with buoyant lipoprotein formation and 2) domain specific folding of apoBs amino-terminal region is required to initiate MTP-de pendent lipid transfer to nascent apoB in the hepatic endoplasmic reti culum.