Dual targeting property of the N-terminal signal sequence of P4501A1. Targeting of heterologous proteins to endoplasmic reticulum and mitochondria

• Published in: The Journal of biological chemistry 1999-08, Vol.274 (34), p.24014-24022
• Main Authors: Bhagwat, S V, Biswas, G, Anandatheerthavarada, H K, Addya, S, Pandak, W, Avadhani, N G
• Format: Article
• Language: English
• Subjects: Animals; Biological Transport; Cholestanetriol 26-Monooxygenase; COS Cells; Cytochrome P-450 CYP1A1 - chemistry; Cytochrome P-450 CYP1A1 - metabolism; Cytochrome P-450 Enzyme System - metabolism; Endoplasmic Reticulum - metabolism; Immunohistochemistry; Mitochondria - metabolism; Protein Sorting Signals - metabolism; Rats; Recombinant Fusion Proteins - metabolism; Steroid Hydroxylases - metabolism; Tetrahydrofolate Dehydrogenase - metabolism
• ISSN: 0021-9258
• DOI: 10.1074/jbc.274.34.24014

Recent studies from our laboratory showed that the beta-naphthoflavone-inducible cytochrome P4501A1 is targeted to both the endoplasmic reticulum (ER) and mitochondria. In the present study, we have further investigated the ability of the N-terminal signal sequence (residues 1-44) of P4501A1 to target heterologous proteins, dihydrofolate reductase, and the mature portion of the rat P450c27 to the two subcellular compartments. In vitro transport and in vivo expression experiments show that N-terminally fused 1-44 signal sequence of P4501A1 targets heterologous proteins to both the ER and mitochondria, whereas the 33-44 sequence strictly functions as a mitochondrial targeting signal. Site-specific mutations show that positively charged residues at the 34th and 39th positions are critical for mitochondrial targeting. Cholesterol 27-hydroxylase activity of the ER-associated 1-44/1A1-CYP27 fusion protein can be reconstituted with cytochrome P450 reductase, but the mitochondrial associated fusion protein is functional with adrenodoxin + adrenodoxin reductase. Consistent with these differences, the fusion protein in the two organelle compartments exhibited distinctly different membrane topology. The results on the chimeric nature of the N-terminal signal of P4501A1 coupled with interaction with different electron transport proteins suggest a co-evolutionary nature of some of the xenobiotic inducible microsomal and mitochondrial P450s.