Dissecting the roles of p97 AAA ATPase and the 26S proteasome in endoplasmic reticulum (ER)‐extraction of CYP3A during degradation

Human liver CYP3A4 metabolizes >50% of all endo‐ and xenobiotics. Hepatic CYP3A4 stabilization/turnover determines its functional content and thus is clinically relevant. CYP3A4 and its rat orthologs (CYPs 3A) incur ubiquitin dependent ER‐associated degradation (ERAD). We have shown in yeast and rat hepatocytes that the p97 AAA‐ATPase complex is involved in extraction of ER‐anchored CYPs 3A into the cytosol. However, given that the CYP3A bulk is in the cytosol and a significant 26S proteasomal fraction is ER‐associated, it was plausible that 26S proteasomal AAA ATPases were also involved in CYP3A extraction. To determine the relative roles of p97 and 26S proteasome, we used lentiviral shRNA mediated knockdown of rat hepatic p97, and MG262 and hemin as probes. 90% p97 knockdown nearly abolished membrane extraction of CYP3A, resulting in a marked accumulation of functionally active parent CYP3A and its ubiquitinated species that remained firmly ER‐tethered. Little CYP3A was extracted into the cytosol, even after MG262‐proteasomal inhibition. Furthermore hemin, a known proteasomal AAA ATPase inhibitor, failed to further increase CYP3A ER‐accumulation, although the extent of CYP3A ubiquitination was considerably enhanced in both cases. These findings reveal that p97 AAA ATPase rather than the 26S proteasome ATPases is obligatory in CYP3A ER‐extraction. Supported by NIH grants DK26506, GM44037 and DK26743.