Physiological role of the N-terminal processed P4501A1 targeted to mitochondria in erythromycin metabolism and reversal of erythromycin-mediated inhibition of mitochondrial protein synthesis

Recently, we showed that the major species of beta-naphthoflavone-inducible rat liver mitochondrial P450MT2 consists of N-terminal truncated microsoma l P4501A1 (+33/1A1) and that the truncated enzyme exhibits different substr ate specificity as compared with intact P4501A1. The results of the present study show that P450MT2 targeted to COS cell mitochondria by transient tra nsfection of P4501A1 cDNA is localized inside the mitochondrial inner membr ane in a membrane-extrinsic orientation. Go-expression with wild type P4501 A1 and adrenodoxin (Adx) cDNAs resulted in 5-7-fold higher erythromycin N-d emethylation (ERND) in the mitochondrial fraction but minimal changes in th e microsomal fraction of transfected cells. Erythromycin, a potent inhibito r of bacterial and mitochondrial protein synthesis, caused 8-12-fold higher accumulation of CYP1A1 mRNA, preferential accumulation of P450MT2, and 5-6 -fold higher ERND activity in the mitochondrial compartment of rat C6 gliom a cells. Consistent with the increased mitochondrial ERND activity, cc-expr ession with P4501A1 and Adx in COS cells rendered complete protection again st erythromycin-mediated mitochondrial translation inhibition. Mutations th at specifically affect the mitochondrial targeting of P4501A1 also abolishe d protection against mitochondrial translation inhibition. These results fo r the first time suggest a physiological function for the xenobiotic induci ble cytochrome P4501A1 against drug-mediated mitochondrial toxicity.