Cytochrome P450, CYP3A4, is the dominant human liver endoplasmic reticulum (ER) hemoprotein enzyme, responsible for the metabolism of over 60% of clinically relevant drugs. We have previously shown that mechanism-based suicide inactivation of CYP3A4 and its rat liver ER orthologs, CYPs 3A, via heme-modification of their protein moieties, results in their ubiquitin (Ub)-dependent 26S proteasomal degradation (Korsmeyer et al. (1999) Arch. Biochem. Biophys. 365, 31; Wang et al. (1999) Arch. Biochem. Biophys. 365, 45). This is not surprising given that the heme-modified CYP3A proteins are structurally damaged. To determine whether the turnover of the native enzyme similarly recruited this pathway, we heterologously expressed this protein in wild-type Saccharomyces cerevisiae and mutant strains (hrd1Delta, hrd2-1, and hrd3Delta) previously shown to be deficient in the Ub-dependent 26S proteasomal degradation of the polytopic ER protein 3-hydroxy-3-methylglutaryl-CoA reductase (isoform Hmg2p), the rate-limiting enzyme in sterol biosynthesis, as well as in strains deficient in ER-associated Ub-conjugating enzymes, Ubc6p and/or Ubc7p (Hampton et al. (1996) Mol. Biol. Cell 7, 2029; Hampton and Bhakta (1997) Proc. Natl. Acad. Sci. USA 94, 12,944). Our findings reveal that in common with the degradation of Hmg2p, that of native CYP3A4 also requires Hrd2p (a subunit of the 19S cap complex of the 26S proteasome) and Ubc7p, and to a much lesser extent Hrd3p, a component of the ER-associated Ub-ligase complex. In contrast to Hmg2p-degradation, that of native CYP3A4 does not appear to absolutely require Hrd1p, another component of the ER-associated Ub-ligase complex. Furthermore, studies in a S. cerevisiae pep4Delta strain proven to be deficient in the vacuolar degradation of carboxypeptidase Y indicated that CYP3A4 degradation is also largely independent of vacuolar (lysosomal) proteolytic function. The degradation of two other native ER proteins, Sec61p and Sec63p, normal components of the ER translocon, were also examined in parallel and found to be stabilized to some extent in HRD2- and UBC7-deficient strains. Together these findings attest to the remarkable mechanistic diversity in the normal degradation of ER proteins.
Copyright 2001 Academic Press.