Folding of the Amino-terminal Domain of Apolipoprotein B Initiates Microsomal Triglyceride Transfer Protein-dependent Lipid Transfer to Nascent Very Low Density Lipoprotein

Michael F Ingram,Gregory S Shelness

doi:10.1074/jbc.272.15.10279

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.

Highlights

In addition to the protein and lipid biosynthetic capacities present in most cell types, the biogenesis of very low density lipoprotein requires at least two dedicated gene products: apoB, the major protein component of very low density lipoprotein [1,2,3,4,5,6], and the microsomal triglyceride transfer protein (MTP),1 a soluble lipid transfer protein localized to the ER of hepato
ApoB28F: A Model System for Examination of Early Stages of Hepatic Very Low Density Lipoprotein Assembly—Because of the cotranslational kinetics of lipoprotein assembly, steps required to initiate small particles formed by the amino-terminal ϳ25% of apoB may be the same as those required for apoB48 and apoB100-containing lipoproteins
We investigated whether an epitope-tagged form of apoB28 could serve as an appropriate model system for analyzing the relationship between apoB folding and subsequent steps required for the initiation of lipoprotein particle assembly

Summary

Introduction

In addition to the protein and lipid biosynthetic capacities present in most cell types, the biogenesis of very low density lipoprotein requires at least two dedicated gene products: apoB, the major protein component of very low density lipoprotein [1,2,3,4,5,6], and the microsomal triglyceride transfer protein (MTP),1 a soluble lipid transfer protein localized to the ER of hepato-. Since proteins labeled during the 1-min pulse were, by definition, nascent polypeptides at the time that [35S]Met/ Cys was added to the cells, and it is estimated that ϳ3– 4 min is required to synthesize apoB28F (ϳ300 – 400 amino acids per minute [37]), these results indicate that the disulfide bonds in apoB28F progressed to the DTT resistant form either before or shortly after (within 1 min) translation was completed.

Results

Conclusion