In the past, CYP1A1 protein was known to be located in the endoplasmic reticulum (ER; microsomes). More recently, CYP1A1 was shown also to be targeted to the inner mitochondrial membrane; mitochondrial import is dependent on NH₂-terminal processing that exposes a cryptic targeting signal. It is interesting that microsomal and mitochondrial CYP1A1 enzymes exhibit different substrate specificities, electron donors, and inducer properties. To understand the physiological functions of microsomal versus mitochondrial CYP1A1, we have generated three knock-in lines by altering the CYP1A1 NH₂ terminus. Cyp1a1(mtt/mtt) mice encode an NH₂-terminal 31-amino acid-truncated protein, deleting the ER-targeting signal and exposing the cryptic mitochondrial-targeting signal. Cyp1a1(mtp/mtp) mice encode a protein carrying L7N and L17N mutations; this mutant lacks the signal recognition particle (SRP)-binding site and subsequent ER-targeting, but requires proteolysis by a cytosolic peptidase for mitochondrial import. Cyp1a1(mc/mc) mice encode a microsomal protein having R34D and K39I mutations, which abolish the mitochondrial targeting signal. After dioxin or β-naphthoflavone treatment of these mouse lines, the CYP1A1 protein was shown to be located in the mitochondria of the Cyp1a1(mtp/mtp) and Cyp1a1(mtt/mtt) lines and in microsomes of the Cyp1a1(mc/mc) line. To test for differences in function, we compared the response to dietary benzo[a]pyrene (BaP). After 18 days of daily oral BaP, wild-type and Cyp1a1(mc/mc) mice were completely protected, whereas Cyp1a1(-/-) and Cyp1a1(mtp/mtp) mice showed striking toxicity and compensatory up-regulation of CYP1A2 and CYP1B1 mRNA in several tissues. Our data support the likelihood that it is the microsomal rather than mitochondrial CYP1A1 enzyme that protects against oral BaP toxicity.

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