Mitochondrial Targeting of Cytochrome P450 Proteins Containing NH2-terminal Chimeric Signals Involves an Unusual TOM20/TOM22 Bypass Mechanism

Hindupur K Anandatheerthavarada,Naresh Babu V Sepuri,Narayan G Avadhani

doi:10.1074/jbc.m109.007492

Hindupur K Anandatheerthavarada, Naresh Babu V Sepuri + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m109.007492

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Abstract

Previously we showed that xenobiotic inducible cytochrome P450 (CYP) proteins are bimodally targeted to the endoplasmic reticulum and mitochondria. In this 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 CYP27A1 interacted only with Hsp70 and was not able to bypass peripheral TOMs. Our results also show that delivery of a chimeric signal containing client protein by Hsp90 required the cytosol-exposed NH(2)-terminal 143 amino acids of TOM40. TOM40 devoid of this domain was unable to import CYP proteins. These results suggest that compared with the unimodal mitochondrial targeting signals, the chimeric mitochondrial targeting signals are highly evolved and dynamic in nature.

Highlights

Grant GM-34883. □S The on-line version of this article contains supplemental Fig. 1. 1 To whom correspondence should be addressed: Dept. of Animal Biology, somes, and mitochondria, vary markedly in terms of amino acid sequence, hydrophobicity, and secondary structure and interact with distinctly different sets of carrier proteins, receptors, and protein translocator complexes [3,4,5,6,7,8]
We have shown previously that mitochondrial import of these cytochrome P450 (CYP) is dependent on transmembrane potential and energy (14, 16 –18)
The results of in vitro import showed that nearly intact CYP2B1 protein was imported and rendered resistant to trypsin treatment, CYP2E1 and CYPϩ5/1A1 were not imported into Pronase-treated mitochondria (Fig. 1C)

Summary

Noncanonical Mitochondrial Import Signals of CYP Proteins

Proteins with canonical mitochondrial targeting signals bind to the outer membrane protein import complex, TOM, as the critical initial step in mitochondrial protein targeting [13, 25,26,27,28]. The role of these peripheral TOMs in the mitochondrial import of proteins containing chimeric signals remains unclear. We investigated the requirement for peripheral TOM proteins for the translocation of CYPMT2a (ϩ5/1A1), CYPMT2b (ϩ33/1A1) (the NH2-terminal truncated forms of CYP1A1), CYPMT4, and CYPMT5 (phosphorylated forms of CYP2B1 and CYP2E1, respectively) that contain NH2-terminal chimeric signals. Our results suggest that differential interaction of chimeric signals with Hsp and Hsp might be the key discriminating factor in the TOM20/TOM22 bypass mechanism of mitochondrial protein targeting

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DISCUSSION