The
ATPase associated with various cellular activities (
AAA-ATPase) p97 (p97) has been implicated in the retrotranslocation of target
proteins for delivery to the cytosolic
proteasome during endoplasmic reticulum-associated degradation (ERAD).
Apolipoprotein B-100 (apoB-100) is an ERAD substrate in liver cells, including the human
hepatoma, HepG2. We studied the potential role of p97 in the ERAD of
apoB-100 in HepG2 cells using cell permeabilization, coimmunoprecipitation, and gene silencing. Degradation was abolished when HepG2 cytosol was removed by
digitonin permeabilization, and treatment of intact cells with the
proteasome inhibitor MG132 caused accumulation of ubiquitinated
apoB protein in the cytosol. Cross-linking of intact cells with the
thiol-cleavable agent
dithiobis(succinimidylpropionate) (DSP), as well as nondenaturing immunoprecipitation, demonstrated an interaction between p97 and intracellular
apoB. Small interfering
ribonucleic acid (
siRNA)-mediated reduction of p97
protein increased the intracellular levels of newly synthesized
apoB-100, predominantly because of a decrease in the turnover of newly synthesized
apoB-100 protein. However, although the posttranslational degradation of newly synthesized
apoB-100 was delayed by p97 knockdown, secretion of
apoB-100 was not affected. Knockdown of p97 also impaired the release of
apoB-100 and polyubiquitinated
apoB into the cytosol. In summary, our results suggest that retrotranslocation and proteasomal degradation of
apoB-100 can be dissociated in HepG2 cells, and that the
AAA-ATPase p97 is involved in the removal of full-length
apoB from the biosynthetic pathway to the cytosolic
proteasome.