An unusual TOM20/TOM22 bypass mechanism for the mitochondrial targeting of cytochrome P450 proteins containing N-terminal chimeric signals

Previously we showed that xenobiotic-inducible cytochrome P450 (CYP) proteins are bimodally targeted to the endoplasmic reticulum and mitochondria. In the present study, we investigated the mechanism of delivery of chimeric signal-containing CYP proteins to the peripheral and channel-forming mitochondrial outer membrane translocases (TOMs). CYP+33/1A1 and CYP2B1 did not require peripheral TOM70, TOM20, or TOM22 for translocation through the channel-forming TOM40 protein. In contrast, CYP+5/1A1 and CYP2E1 were able to bypass TOM20 and TOM22 but required TOM70. CYP27, which contains a canonical cleavable mitochondrial signal, required all of the peripheral TOMs for its mitochondrial translocation. We investigated the underlying mechanisms of bypass of peripheral TOMs by CYPs with chimeric signals. The results suggested that interaction of CYPs with Hsp70, a cytosolic chaperone involved in the mitochondrial import, alone was sufficient for the recognition of chimeric signals by peripheral TOMs. However, sequential interaction of chimeric signal-containing CYPs with Hsp70 and Hsp90 resulted in the bypass of peripheral TOMs, whereas CYP27 interacted only with Hsp70 and was not able to bypass peripheral TOMs. Our results also show that delivery of chimeric signal-containing client proteins by Hsp90 required the cytosol-exposed N-terminal 143 amino acids of TOM40. TOM40 devoid of this domain was unable to bind CYP proteins. These results suggest that, compared with the unimodal mitochondria-targeting signals, the chimeric mitochondria-targeting signals are highly evolved and dynamic in nature.