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J. Cell Biol.,
Volume 139, Number 3, November 3, 1997 589-599
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 
-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.
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