Abstract
Although microsomal triglyceride transfer protein (MTP) and newly synthesized triglyceride (TG) are critical for co-translational targeting of apolipoprotein B (apoB100) to lipoprotein assembly in hepatoma cell lines, their roles in the later stages of lipoprotein assembly remain unclear. Using N-acetyl-Leu-Leu-norleucinal to prevent proteasomal degradation, HepG2 cells were radiolabeled and chased for 0-90 min (chase I). The medium was changed and cells chased for another 150 min (chase II) in the absence (control) or presence of Pfizer MTP inhibitor CP-10447 (CP). As chase I was extended, inhibition of apoB100 secretion by CP during chase II decreased from 75.9% to only 15% of control (no CP during chase II). Additional studies were conducted in which chase I was either 0 or 90 min, and chase II was in the presence of [(3)H]glycerol and either BSA (control), CP (inhibits both MTP activity and TG synthesis),BMS-1976360-1) (BMS) (inhibits only MTP activity), or triacsin C (TC) (inhibits only TG synthesis). When chase I was 0 min, CP, BMS, and TC reduced apoB100 secretion during chase II by 75.3, 73.9, and 53.9%. However, when chase I was 90 min, those agents reduced apoB100 secretion during chase II by only 16.0, 19.2, and 13.9%. Of note, all three inhibited secretion of newly synthesized TG during chase II by 80, 80, and 40%, whether chase I was 0 or 90 min. In both HepG2 cells and McA-RH7777 cells, if chase I was at least 60 min, inhibition of TG synthesis and/or MTP activity did not affect the density of secreted apoB100-lipoproteins under basal conditions. Oleic acid increased secretion of TG-enriched apoB100-lipoproteins similarly in the absence or presence of either of CP, BMS, or TC. We conclude that neither MTP nor newly synthesized TG is necessary for the later stages of apoB100-lipoprotein assembly and secretion in either HepG2 or McA-RH7777 cells.
Highlights
The assembly of apolipoprotein B100 (apoB100)-lipoproteins requires the initial translocation of apoB100 across the endoplasmic reticulum (ER) membrane, during which apoB100 interacts with a number of molecular chaperones, including Hsp70 in the cytosol, calnexin in the ER membrane, and microsomal triglyceride transfer protein (MTP) in the ER lumen (9 –13)
Despite being physically close to Sec61 as determined by cross-linking studies, and being significantly ubiquitinated, apoB100 could be secreted rapidly after stimulation of cellular lipid synthesis with oleic acid. In those studies, we observed that, after a 1–2-h chase period, apoB100 secretion appeared to be independent of MTP (22)
On the basis of these data, in the present study, we conducted additional experiments to 1) confirm our prior observation that apoB100 secretion could be independent of MTP activity, 2) determine the role of TG synthesis in the MTP-independent secretion of apoB100, and 3) characterize the type of lipoprotein assembled and secreted when secretion is MTP-independent
Summary
The assembly of apoB100-lipoproteins requires the initial translocation of apoB100 across the ER membrane, during which apoB100 interacts with a number of molecular chaperones, including Hsp70 in the cytosol, calnexin in the ER membrane, and microsomal triglyceride transfer protein (MTP) in the ER lumen (9 –13). In both HepG2 cells and McA-RH7777 cells, if chase I was at least 60 min, inhibition of TG synthesis and/or MTP activity did not affect the density of secreted apoB100-lipoproteins under basal conditions.
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