Critical Region in the Extension Peptide for the Import of Cytochrome P-450 (SCC) Precursor into Mitochondria

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In order to establish the role of the extension peptide of the precursor of P-450 (SCC), a mitochondrial inner membrane protein, in the import into the organella, three deletion mutants of the precursor, in which the deletions were in the mature portion, were constructed. These mutant precursors were imported into mitochondria in vitro as efficiently as the original precursor, indicating that the extension peptide contains sufficient information for the import of the precursor into mitochondria. To investigate which portion of the extension peptide contains the mitochondrial targeting signal, various lengths of the amino-terminal portion of the extension peptide of P-450 (SCC) precursor were fused to the mature portion of adrenodoxin. The fusion proteins consisting of 44 and 19 amino-terminal amino acids and mature adrenodoxin were imported into mitochondria, whereas those containing 14, 7, and 2 amino-terminal amino acid residues were not. The importance of the amino-terminal portion of the extension peptide was confirmed by the deletion from the amino-terminal end of a fusion protein consisting of the amino-terminal 44 amino acid residues of P-450 (SCC) precursor and mature adrenodoxin, SCC44RAd. The amino-terminal deletions abolished the import of the fusion proteins into mitochondria. Substitution of all of the three basic amino acids, Arg (4), Arg (9), and Lys (14) in the extension peptide of SCC44RAd to Ser or Thr inhibited the binding of the fusion protein to mitochondria as well as its import. These results indicated that the amino-terminal 19 amino acid residues of the extension peptide of P-450 (SCC) precursor contain sufficient information for the import of the precursor into mitochondria, and that the basic amino acids in that particular portion of the extension peptide are essential for the binding of the precursor to mitochondria.