Ubiquitin-dependent 26S proteasomal pathway: A role in the degradation of native human liver CYP3A4 expressed in Saccharomyces cerevisiae?

Cytochrome P450, CYP3A4, is the dominant human liver endoplasmic reticulum (ER) hemoprotein enzyme, responsible for the metabolism of over 60% of clin ically relevant drugs. We have previously shown that mechanism-based suicid e inactivation of CYP3A4 and its rat liver ER orthologs, CYPs 3A, via hemem odification of their protein moieties, results in their ubiquitin (Ub)-depe ndent 26S proteasomal degradation (Korsmeyer et al. (1999) Arch. Biochem. B iophys. 365, 31; Wang et al. (1999) Arch. Biochem. Biophys. 365, 45). This is not surprising given that the heme-modified CYP3A proteins are structura lly damaged. To determine whether the turnover of the native enzyme similar ly recruited this pathway, we heterologously expressed this protein in wild -type Saccharomyces cerevisiae and mutant strains (hrd1 Delta, hrd2-1, and hrd3 Delta) previously shown to be deficient in the Ub-dependent 26S protea somal degradation of the polytopic ER protein 3-hydroxy-3-methylglutaryl-Co A reductase (isoform Hmg2p), the rate-limiting enzyme in sterol biosynthesi s, 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 rev eal that in common with the degradation of Hmg2p, that of native CYP3A4 als o requires Hrd2p (a subunit of the 19S cap complex of the 26S proteasome) a nd Ubc7p, and to a much lesser extent Hrd3p, a component of the ER-associat ed Ub-ligase complex. In contrast to Hmg2p-degradation, that of native CYP3 A4 does not appear to absolutely require Hrd1p, another component of the ER -associated Ub-ligase complex. Furthermore, studies in a S. cerevisiae pep4 Delta strain proven to be deficient in the vacuolar degradation of carboxy peptidase Y indicated that CYP3A4 degradation is also largely independent o f vacuolar (lysosomal) proteolytic function. The degradation of two other n ative ER proteins, Sec61p and Sec63p, normal components of the ER transloco n, 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. (C) 2001 Academic Press.