The AAA-ATPase p97 facilitates degradation of apolipoprotein B by the ubiquitin-proteasome pathway

Eric A. Fisher, Louis R. Lapierre, Robert D. Junkins, and Roger S. McLeod

Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 1X5

Corresponding Author: rmcleod2{at}dal.ca

The ATPase associated with various cellular activities (AAA-ATPase) p97 has been implicated in the retro-translocation of target proteins for delivery to the cytosolic proteasome during endoplasmic reticulum-associated degradation (ERAD). Apolipoprotein (apo) B-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, co-immunoprecipitation 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. Crosslinking of intact cells with the thiol-cleavable agent dithiobis (succinimidylpropionate) (DSP), as well as non-denaturing immunoprecipitation, demonstrated an interaction between p97 and intracellular apoB. siRNA-mediated reduction of p97 protein increased the intracellular levels of newly synthesized apoB-100, predominantly due to a decrease in the turnover of newly synthesized apoB-100 protein. However, while the post-translational 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 retro-translocation 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.

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