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© The Rockefeller University Press, 0021-9525/1997//589 $5.00
The Journal of Cell Biology, Volume 139, Number 3, , 1997 589-599

Article

Targeting of NH2-terminal–processed Microsomal Protein to Mitochondria: A Novel Pathway for the Biogenesis of Hepatic Mitochondrial P450MT2



Sankar Addya, Hindupur K. Anandatheerthavarada, Gopa Biswas, Shripad V. Bhagwat, Jayati Mullick, and Narayan G. Avadhani

Laboratories of Biochemistry, Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Cytochrome P4501A1 is a hepatic, microsomal membrane–bound enzyme that is highly induced by various xenobiotic agents. Two NH2-terminal truncated forms of this P450, termed P450MT2a and MT2b, are also found localized in mitochondria from β-naphthoflavone–induced livers. In this paper, we demonstrate that P4501A1 has a chimeric NH2-terminal signal that facilitates the targeting of the protein to both the ER and mitochondria. The NH2-terminal 30–amino acid stretch of P4501A1 is thought to provide signals for ER membrane insertion and also stop transfer. The present study provides evidence that a sequence motif immediately COOH-terminal (residues 33–44) to the transmembrane domain functions as a mitochondrial targeting signal under both in vivo and in vitro conditions, and that the positively charged residues at positions 34 and 39 are critical for mitochondrial targeting. Results suggest that 25% of P4501A1 nascent chains, which escape ER membrane insertion, are processed by a liver cytosolic endoprotease. We postulate that the NH2-terminal proteolytic cleavage activates a cryptic mitochondrial targeting signal. Immunofluorescence microscopy showed that a portion of transiently expressed P4501A1 is colocalized with the mitochondrial-specific marker protein cytochrome oxidase subunit I. The mitochondrial-associated MT2a and MT2b are localized within the inner membrane compartment, as tested by resistance to limited proteolysis in both intact mitochondria and mitoplasts. Our results therefore describe a novel mechanism whereby proteins with chimeric signal sequence are targeted to the ER as well as to the mitochondria.

Abbreviations used in this paper: Adx, adrenodoxin; BNF, β-napthoflavone; COX, cytochrome c oxidase; DHFR, dihydrofolate reductase; PVDF, polyvinyldifluoride; P450, cytochrome P450; P450 reductase, NADPH cytochrome P450 reductase; SRP, signal recognition particle.

We are thankful to Drs. Michael Waterman, Byron Kemper, Michael Atchison, and Steven Liebhaber for critically reading the manuscript. We also thank Drs. Reid Gilmore, Michael Waterman, Frank Gonzalez, and Gordon Shore for generously providing some of the reagents and expression plasmids used in this study, and Dr. Lee Peechy and Ms. Gladys Gray-Board for the use of their confocal microscopy facility. Finally, the suggestions and help of members of the Avadhani laboratory, particularly those of Drs. C. Vijayasarathy and Nibedita Lenka on many aspects of this work, are gratefully acknowledged.

This research was supported in part by National Institutes of Health (NIH) grants GM-34883 and CA-22762, and by a grant from the Council for Tobacco Research. The microscopy facility is supported by the NIH core grant RR-2483.

Address all correspondence to Narayan G. Avadhani, University of Pennsylvania, Philadelphia, PA 19104. Tel.: (215) 898-8819; Fax: (215) 898-9923; E-mail: narayan{at}vet.upenn.edu

Drs. Addya and Anandatheerthavarada contributed equally to this work.


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