Cytosolic components are required for proteasomal degradation of newly synthesized apolipoprotein B in permeabilized HepG2 cells

Nobuhiro Sakata, J. Daniel Stoops, Joseph L. Dixon

Research output: Contribution to journalArticlepeer-review

15 Scopus citations

Abstract

Recent studies have proposed that post-translational degradation of apolipoprotein B100 (apoB) involves the cytosolic ubiquitin-proteasome pathway. In this study, immunocytochemistry indicated that endoplasmic reticulum (ER)-associated proteasome molecules were concentrated in perinuclear regions of digitonin-permeabilized HepG2 cells. Signals produced by antibodies that recognize both α- and β-subunits of the proteasome co- localized in the ER with specific domains of apoB. The mechanism of apoB degradation in the ER by the ubiquitin-proteasome pathway was studied using pulsechase labeling and digitonin-permeabilized cells, ApoB in permeabilized cells incubated at 37 °C in buffer alone was relatively stable. When permeabilized cells were incubated with both exogenous ATP and rabbit reticulocyte lysate (RRL) as a source of ubiquitin-proteasome factors, >50% of [3H]apoB was degraded in 30 min. The degradation of apoB in the intact ER of permeabilized cells was much more rapid than that of extracted [3H]apoB incubated with RRL and ATP in vitro. The degradation of apoB was reduced by clasto-lactacystin β-lactone, a potent proteasome inhibitor, and by ubiquitin K48R mutant protein, an inhibitor of polyubiquitination. ApoB in HepG2 cells was ubiquitinated, and polyubiquitination of apoB was stimulated by incubation of permeabilized cells with RRL. These results suggest that newly synthesized apoB in the ER is accessible to the cytoplasmic ubiquitin- proteasome pathway and that factors in RRL stimulate polyubiquitination of apoB, leading to rapid degradation of apoB in permeabilized cells.

Original languageEnglish (US)
Pages (from-to)17068-17074
Number of pages7
JournalJournal of Biological Chemistry
Volume274
Issue number24
DOIs
StatePublished - Jun 11 1999
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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