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

Cytochrome P4501A1 is a hepatic, microsomal membrane–bound enzyme that is highly induced by various xenobiotic agents. Two NH 2 -terminal truncated forms of this P450, termed P450MT2a and MT2b, are also found localized in mitochondria from b -naphthoflavone–induced livers. In this paper, we demonstrate that P4501A1 has a chimeric NH 2 -terminal signal that facilitates the targeting of the protein to both the ER and mitochondria. The NH 2 -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 NH 2 -terminal proteolytic cleavage activates a cryptic mitochondrial targeting signal. Immunofluorescence microscopy showed that a portion of transiently expressed P4501A1 is colocalized with the mitochondrialspecific 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. P rotein targeting to the mitochondrial and ER compartments follow distinct pathways and involve different primary translation sites, targeting signals, and transport machinery (Schatz and Dobberstein, 1996). A majority of proteins targeted to the ER reach their destination through a cotranslational mechanism that requires the association of the NH 2 -terminal signal sequence with signal recognition particle (SRP) 1 and association of the translation complex with the ER membrane (Walter et al., 1981; Gilmore et al., 1982; and see Isenman et al., 1995 for a recent review). Exceptions to the cotranslational mechanisms that do not follow the SRP pathway have also been reported for a limited number of proteins targeted to the ER (Andersson et al., 1983; Wickner and Lodish, 1985). The membrane topology and mechanisms of targeting of the hepatic P450 isoenzymes that remain bound to the ER have been studied extensively (Bar-Nun et al., 1980; Black and Coon, 1982; Sakaguchi et al., 1987; Monier et al., 1988; Szcesna-Skorupa et al., 1988); it is generally believed that the enzyme is anchored through a single transmembrane domain with most of the catalytic domains facing the cytosolic side of the membrane (Monier et al., 1988; Szczesna-Skorupa and Kemper, 1993). Various studies (Fujii-Kuriyama et al., 1979; Bar-Nun et al., 1980; Sakaguchi et al., 1987; Monier et al., 1988; Szczesna-Skorupa et al., 1988) have shown that the NH 2 -terminal hydrophobic sequence functions as an unclipped signal for targeting to the ER through the cotranslational SRP pathway. In particular, the NH 2 -terminal 25–30 hydrophobic residues have been shown to provide the signal for membrane insertion and stop transfer, in addition to serving as the transmembrane anchor (Sakaguchi et al., 1987; Monier et al., 1988; Szczesna-Skorupa et al., 1988). Mitochondrial protein transport, on the other hand, occurs through a posttranslational mechanism and involves a complex series of interactions of the protein with various cytosolic Address all correspondence to Narayan G. Avadhani, University of Pennsylvania, Philadelphia, PA 19104. Tel.: (215) 898-8819; Fax: (215) 8989923; E-mail: [email protected] Drs. Addya and Anandatheerthavarada contributed equally to this work. 1. Abbreviations used in this paper : Adx, adrenodoxin; BNF, b -napthoflavone; COX, cytochrome c oxidase; DHFR, dihydrofolate reductase; PVDF, polyvinyldifluoride; P450, cytochrome P450; P450 reductase, NADPH cytochrome P450 reductase; SRP, signal recognition particle. on Jne 9, 2017 D ow nladed fom Published November 3, 1997