J. Lipid Res.
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Originally published In Press as doi:10.1194/jlr.M800108-JLR200 on June 11, 2008

Papers In Press, published online ahead of print October 1, 2008
J. Lipid Res., doi:10.1194/jlr.M800108-JLR200
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Journal of Lipid Research, Vol. 49, 2149-2160, October 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

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

Eric A. Fisher1, Louis R. Lapierre1, Robert D. Junkins and Roger S. McLeod2

Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada

boxs The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of two figures.

Published, JLR Papers in Press, June 11, 2008.

This work was supported by the Canadian Institutes of Health Research, Grant MOP-67073, and by graduate scholarships from the Nova Scotia Health Research Foundation and the Walter C. Sumner Foundation (L.R.L.).

1 E. Fisher and L. Lapierre contributed equally to this work.

2 To whom correspondence should be addressed. e-mail: rmcleod2{at}dal.ca

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.

Supplementary key words retrotranslocation • endoplasmic reticulum-associated degradation • ERAD • permeabilization • proteolysis


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