The AAA-ATPase p97 facilitates degradation of apolipoprotein B by the ubiquitin-proteasome pathway.
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.
Fisher
Eric A
EA
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada.
Lapierre
Louis R
LR
Junkins
Robert D
RD
McLeod
Roger S
RS
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D053299:Apolipoprotein B-100 / Q000378:metabolism*; D045744:Cell Line, Tumor; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D009687:Nuclear Proteins / Q000378:metabolism*; D010539:Permeability; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport / Q000187:drug effects; D025801:Ubiquitin / Q000378:metabolism*
D053299:Apolipoprotein B-100; D004791:Enzyme Inhibitors; D007976:Leupeptins; D009687:Nuclear Proteins; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases; C404942:p97 ATPase; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde