Abstract

Microsomal triglyceride (TG) transfer protein (MTP) is an endoplasmic reticulum lumenal protein consisting of a 97-kDa subunit and protein disulfide isomerase. It is believed that MTP delivers TG to nascent apoB molecules during the assembly of lipoprotein particles in the secretory pathway. Although in vitro studies have established the mechanism of TG transfer between donor and acceptor membranes, the mechanism of action of MTP in vivo remains unknown. The present studies were undertaken to examine whether or not the transfer of TG to nascent apoB in the endoplasmic reticulum involves the physical interaction between MTP and apoB. HepG2 cells were labeled with [3H]leucine, lysed in a nondenaturing homogenizing buffer, and immunoprecipitated with anti-MTP antiserum. We found that labeled apoB and protein disulfide isomerase were co-immunoprecipitated by this procedure. In addition, we were able to detect the 97-kDa subunit of MTP in these immunoprecipitates by immunoblot. The association of MTP and apoB, as assessed in pulse-labeled cells by co-immunoprecipitation, was transient; apoB was prominent on fluorgraphy at 10 min of chase but minimal thereafter. Oleic acid treatment, which protects apoB from rapid intracellular degradation by increasing TG availability, increased both the degree and the duration of association between MTP and apoB dramatically. Inhibition of TG synthesis by Triacsin D, on the other hand, significantly decreased the MTP-apoB binding. N-Acetyl-leucyl-leucyl-norleucinal, a cysteine protease inhibitor, which directly protects apoB from rapid intracellular degradation but does not affect TG synthesis, increased the interaction between MTP and apoB only slightly, although it did prolong it. Our results suggest that direct interaction between MTP and apoB occurs during the assembly of apoB-containing lipoproteins in HepG2 cells.

Highlights

  • Synthesized triglyceride (TG)1 may be the most important [13,14,15,16,17]

  • Our results suggest that direct interaction between microsomal TG transfer protein (MTP) and apoB occurs during the assembly of apoB-containing lipoproteins in HepG2 cells

  • Despite the large body of evidence demonstrating the importance of hepatic lipids in the assembly and secretion of apoBcontaining lipoproteins, TG synthesis appears to be normal, and lipid droplets accumulate, in livers of patients with abetalipoproteinemia [18], a condition characterized by the absence of apoB secretion

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Summary

Introduction

Synthesized triglyceride (TG)1 may be the most important [13,14,15,16,17]. when lipid availability is increased, rapid intracellular degradation of newly synthesized apoB is inhibited; inhibition of lipid synthesis, on the other hand, results in enhanced degradation of apoB. HepG2 cells were labeled with [3H]leucine, lysed in a nondenaturing homogenizing buffer, and immunoprecipitated with anti-MTP antiserum. Our results suggest that direct interaction between MTP and apoB occurs during the assembly of apoB-containing lipoproteins in HepG2 cells.

Results
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