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J Biol Chem. 1998 Oct 16;273(42):27225-30.

Proteasome-mediated degradation of apolipoprotein B targets both nascent peptides cotranslationally before translocation and full-length apolipoprotein B after translocation into the endoplasmic reticulum.

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1
Departments of Medicine and Cell Biology, Division of Endocrinology, Baylor College of Medicine, Houston, Texas 77030, USA.

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 full-length 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 [35S]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.

PMID:
9765244
[Indexed for MEDLINE]
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