Vacuolar Degradation of Rat Liver CYP2B1 inSaccharomyces cerevisiae: Further Validation of the Yeast Model and Structural Implications for the Degradation of Mammalian Endoplasmic Reticulum P450 Proteins

Abstract

Mammalian hepatic cytochromes P450 (P450s) are endoplasmic reticulum (ER)-anchored hemoproteins with highly variable half-lives. CYP3A4, the dominant human liver drug-metabolizing enzyme, and its rat liver orthologs undergo ubiquitin (Ub)-dependent 26S proteasomal degradation after suicide inactivation or after heterologous expression in Saccharomyces cerevisiae. In contrast, rat liver CYP2C11 is degraded by the vacuolar "lysosomal" pathway when similarly expressed in yeast. The structural determinants that commit P450s to proteasomal or lysosomal degradation are unknown. To further validate S. cerevisiae as a model for exploring mammalian P450 turnover, the degradation of phenobarbital-inducible liver CYP2B1, an enzyme reportedly degraded via the rat hepatic autophagic-lysosomal pathway, was examined in a yeast strain (pep4delta) deficient in vacuolar degradation and its isogenic wild-type control (PEP4). Although CYP2B1 was equivalently expressed in both strains during early logarithmic growth, its degradation was retarded in pep4delta strain, remaining at a level 5-fold higher than that in PEP4 yeast when monitored at the stationary phase. No comparable CYP2B1 stabilization was detected in yeast genetically deficient in the ER Ub-conjugating enzyme Ubc6p or Ubc7p or defective in 19S proteasomal subunit Hrd2p. Thus, as in the rat liver, CYP2B1 is a target of vacuolar/lysosomal rather than proteasomal degradation in yeast, thereby further validating this model for mammalian P450 turnover. It is intriguing that a chimeric protein, CYP2B1-3A4CT, with the CYP3A4 C-terminal heptapeptide grafted onto the CYP2B1 C terminus, was proteasomally degraded after similar expression. Such diversion of CYP2B1 from its predominantly vacuolar degradation suggests that the CYP3A4 heptapeptide could either actively signal its proteasomal degradation or block its vacuolar proteolysis.