Proteasome-mediated Degradation of Apolipoprotein B Targets Both Nascent Peptides Cotranslationally Before Translocation and Full-length Apolipoprotein B After Translocation into the Endoplasmic Reticulum

Abstract

A major portion of newly synthesized apolipoprotein B (apoB) is degraded intracellularly. This degradation has been demonstrated to be mediated largely by the ubiquitin-proteasome pathway. We examined whether nascent apoB polypeptides or full-length apoB is selectively retrotranslocated from the endoplasmic reticulum into the cytosol for degradation. Herein, we found that full-length apoB as well as partial-length apoB peptides are ubiquitinated in HepG2 cells, and ubiquitination is an exclusively cytosolic process. Calnexin, which binds specifically to glycoproteins, has been postulated to promote apoB folding and complete translocation; we found that ubiquitinated apoB is bound to calnexin, suggesting that ubiquitinated apoB is glycosylated. In addition to calnexin binding, we have other pieces of evidence that the full-length intracellular ubiquitinated apoB is glycosylated, because (i) it binds to concanavalin A, and (ii) glycan can be demonstrated in the fulllength ubiquitinated apoB by a chemical detection method involving oxidation of adjacent hydroxyl groups in the glycan moiety. Because glycosylation occurs inside the endoplasmic reticulum, the full-length glycosylated apoB must have been retrotranslocated into the cytosol for ubiquitination and proteasome-mediated degradation. Next we synchronized translation in HepG2 cells by puromycin treatment. A pulse-chase experiment using [35 S]methionine labeling of intracellular apoB in these synchronized cells demonstrated that nascent partial-length apoB peptides are also ubiquitinated cotranslationally. We conclude that the ubiquitin proteasome-mediated degradation of apoB targets both nascent peptides cotranslationally before translocation as well as full-length apoB after its translocation into the endoplasmic reticulum.

Inhibition of Microsomal Triglyceride Transfer Protein: Another Mechanism for Drug–Induced Steatosis in Mice

“…to be ubiquitinated and to undergo proteasomal degradation, 4 while yet another part may undergo endoluminal proteolysis (Fig. 7).…”

“…Although fully lipidated Apo B molecules follow vesicular flow to be secreted, in contrast, part of the incompletely lipidated Apo B molecules fail to translocate completely into the ER lumen and/or undergo retrotranslocation to the cytosol to be ubiquitinated and then to be digested by the proteasome. 4 We have shown previously that several drugs that can cause hepatic steatosis in humans inhibit hepatic fatty acid oxidation in mice. 5 Indeed, amineptine, 6 amiodarone, 7,8 doxycycline, 9 pirprofen, 10 tetracycline, 11 and tianeptine 12 have been shown to inhibit mitochondrial fatty acid ␤-oxidation.…”

The Assembly and Secretion of Apolipoprotein B-48-containing Very Low Density Lipoproteins in McA-RH7777 Cells

“…the protein is retracted through the translocation channel) (13). However, there is strong evidence that the degradation involves nascent as well as full-length apoB chains (14,15), suggesting that this proteasomal degradation may also involve other pathways (for reviews, see Refs. 16 and 17).…”

Studies on Degradative Mechanisms Mediating Post-translational Fragmentation of Apolipoprotein B and the Generation of the 70-kDa Fragment

“…More specifically, efficient translocation of newly synthesized apoB molecules across the membrane of the endoplasmic reticulum (ER) is believed to be an important event that contributes to the formation of secretion competent apoB-containing lipoproteins (5)(6)(7)(8). Inefficient translocation has been suggested to lead to the formation of a pool of membrane-associated apoB that becomes ubiquitinated (12)(13)(14)(15)(16)(17)(18)(19)(20)22) and ultimately destined for intracellular degradation (6, 8 -11). It is evident that co-translational degradation of membrane-associated apoB is mediated by the cytosolic proteasome based on its sensitivity to proteasome inhibitors such as ALLN, lactacystin, and MG132 (9,(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22).…”

“…Inefficient translocation has been suggested to lead to the formation of a pool of membrane-associated apoB that becomes ubiquitinated (12-20, 22) and ultimately destined for intracellular degradation (6, 8 -11). It is evident that co-translational degradation of membrane-associated apoB is mediated by the cytosolic proteasome based on its sensitivity to proteasome inhibitors such as ALLN, lactacystin, and MG132 (9,[12][13][14][15][16][17][18][19][20][21][22].…”