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J. Biol. Chem., Vol. 281, Issue 41, 30834-30847, October 13, 2006

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Role of Protein Kinase C-mediated Protein Phosphorylation in Mitochondrial Translocation of Mouse CYP1A1, Which Contains a Non-canonical Targeting Signal^*

Venkata Ramesh Dasari¹, Hindupur K. Anandatheerthavarada¹, Marie-Anne Robin², Ettickan Boopathi, Gopa Biswas, Ji-Kang Fang, Daniel W. Nebert, and Narayan G. Avadhani³

From the Department of Animal Biology and the Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and the Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati Medical Center, P. O. Box 670056, Cincinnati, Ohio 45267-0056

A large number of mitochondrial proteins lack canonical mitochondrial-targeting signals. The bimodal transport of cytochromes P450 (CYPs) to endoplasmic reticulum and mitochondria (MT), reported previously by us, likely represents one mode of non-canonical protein targeting to MT. Herein, we have studied the mechanism of mouse MT-CYP1A1 targeting to gain insight into the regulatory features and evolutionary conservation of bimodal targeting mechanism. Mouse MT-CYP1A1 consists of two NH₂-terminal-truncated molecular species, +91A1 and +331A1. Mutations Pro-2 Leu and Tyr-5 Leu, which increase the signal recognition particle (SRP) binding, diminished MT targeting of the protein in intact cells. By contrast, mutations Leu-7 Asn and Leu-17 Asn, which decreased SRP-binding affinity, enhanced MT targeting, thus suggesting that SRP binding is an important regulatory step that modulates bimodal targeting. Protein kinase C (PKC)-mediated phosphorylation of nascent chains at Thr-35 vastly decreased affinity for SRP binding suggesting an important regulatory step. In support of these results, COS cell transfection experiments show that phosphomimetic mutation Thr-35 Asp or induced cellular PKC caused increased CYP1A1 targeting to MT and correspondingly lower levels to the endoplasmic reticulum. Results suggest evolutionary conservation of chimeric signals and bimodal targeting of CYP1A1 in different species. The mouse MT-CYP1A1 is an extrinsic membrane protein, which exhibited high FDX1 plus FDXR-mediated N-demethylation of a number of tricyclic antidepressants, pain killers, anti-psychotics, and narcotics that are poor substrates for microsomal CYP1A1.

Received for publication, October 3, 2005 , and in revised form, August 7, 2006.

^* This work was supported by National Institutes of Health Grants GM-34883 (to N. G. A.) and ES-08147 (to D. W. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ Both authors contributed equally to this work.

² Present address: INSERM, Unite 481, Faculte de Medecine Xavier Bichat, 750118 Paris, France.

³ To whom correspondence should be addressed: Dept. of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce St., Philadelphia, PA 19104. Tel.: 215-898-8819; Fax: 215-573-6651; E-mail: narayan{at}vet.upenn.edu.

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