Different structures in the amino-terminal domain of the ornithine transcarbamylase leader peptide are involved in mitochondrial import and carboxyl-terminal cleavage.

The cytoplasmic precursor of mitochondrial ornithine transcarbamylase (carbamoyl-phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.2) contains an amino-terminal leader peptide of 32 amino acids. Secondary structure and helical-wheel analyses predict that the extreme amino-terminal domain (residues 1-15) forms an alpha-helix. To test this thesis, leucine residues at positions 2, 5, 8, and 9 were systematically replaced by either helix-breaking glycine residues or by helix-preserving alanine residues. Triple substitutions of glycine for leucine in positions 2, 5, and 9 or 5, 8, and 9 abolished the uptake of the rat precursor by intact mitochondria, whereas similar alanine substitutions had much less effect. Theoretical computations predicted that the decreased helical stability of the Gly-5,8,9 substitution could be significantly increased by replacing a serine in position with phenylalanine. The introduction of Phe-3, indeed, restored the mitochondrial uptake of the mutant precursor. These results lend strong support to the hypothesis that an alpha-helix is present at the leader's amino terminus during the import of the precursor by mitochondria. Although the precursors with the triply-substituted leaders were impaired with respect to import, they were still cleaved readily by a protease found in a mitochondrial matrix fraction. Substitution of glycine or alanine for all four leucine residues, however, rendered the leader uncleavable at the carboxyl-terminal cleavage site. These results suggest that the structure of the amino-terminal domain is important for recognition of the carboxyl-terminal cleavage sites by the matrix proteases.