Endoplasmic reticulum-associated degradation of cytochrome P450 CYP3A4 in Saccharomyces cerevisiae: further characterization of cellular participants and structural determinants.
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.
Dept. of Cellular and Molecular Pharmacology, Box 2280, University of California-San Francisco, CA 94143-2280, USA.
Mingxiang
M
Liao
Saadia
S
Faouzi
Andrey
A
Karyakin
Maria Almira
MA
Correia
D000251:Adenosine Triphosphatases; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport; D006801:Humans; D008099:Liver / Q000201:enzymology; D008565:Membrane Proteins / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism; D029543:Nucleocytoplasmic Transport Proteins; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D000074405:Valosin Containing Protein; D033921:Vesicular Transport Proteins
C109280:CUE1 protein, S cerevisiae; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D008565:Membrane Proteins; D018832:Molecular Chaperones; C104361:NPL4 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D029543:Nucleocytoplasmic Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; C444652:UFD1 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D003577:Cytochrome P-450 Enzyme System; C465260:Cyp3a2 protein, rat; D051544:Cytochrome P-450 CYP3A; D043743:Ubiquitin-Protein Ligase Complexes; C091362:SSM4 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases; C000614177:CDC48 protein, S cerevisiae; D000074405:Valosin Containing Protein; C469287:RSP5 protein, S cerevisiae