The monotopic, endoplasmic reticulum (ER)-anchored cytochromes P450 (P450s) undergo variable proteolytic turnover. CYP3A4, the dominant human liver drug-metabolizing enzyme, is degraded via a ubiquitin (Ub)-dependent 26S proteasomal pathway after heterologous expression in Saccharomyces cerevisiae. This turnover involves the Ub-conjugating enzyme Ubc7p and the 19S proteasomal subunit Hrd2p but is independent of Hrd1p/Hrd3p, a major Ub-ligase (E3) involved in ER protein degradation. We now show that CYP3A4 ERAD also involves the Ubc7p-ER anchor Cue1p, because CYP3A4 is significantly stabilized at the stationary growth phase in Cue1p-deficient yeast. To determine whether the other major Ub-ligase Doa10p or Rsp5p involved in ER protein degradation functions in CYP3A4 ERAD, wild type and Doa10p- or Rsp5p-deficient yeast strains were also similarly examined. No appreciable CYP3A4 stabilization was detected in either Doa10p- or Rsp5p-deficient yeast, thereby excluding these E3s and revealing that CYP3A4 ERAD involves a novel or yet to be identified E3. Similar studies also revealed that the Cdc48p-Ufd1p-Hrd4p complex, responsible for the translocation of polyubiquitinated ER proteins was critical for CYP3A4 ERAD. We previously reported that grafting of the C-terminal (CT) CYP3A4 heptapeptide onto the CYP2B1 C terminus switched its proteolytic susceptibility from predominantly vacuolar to proteasomal degradation. To determine the relevance of this CT heptapeptide to CYP3A4 ERAD, CYP3A4 degradation after CT heptapeptide-deletion (CYP3A4DeltaCT) was similarly examined in yeast. These findings revealed that CYP3A4DeltaCT was also degraded by Ubc7p-26S proteasomal pathway, thereby indicating that this CT heptapeptide is not critical for CYP3A4 proteasomal degradation. Thus, unlike CYP2B1, CYP3A4 harbors additional/multiple structural degrons for its recruitment into the Ubproteasomal pathway.
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