Processing peptidase of Neurospora mitochondria. Two-step cleavage of imported ATPase subunit 9.
Subunit 9 (dicyclohexylcarbodiimide binding protein, 'proteolipid') of the mitochondrial F1F0-ATPase is a nuclearly coded protein in Neurospora crassa. It is synthesized on free cytoplasmic ribosomes as a larger precursor with an NH2-terminal peptide extension. The peptide extension is cleaved off after transport of the protein into the mitochondria. A processing activity referred to as processing peptidase that cleaves the precursor to subunit 9 and other mitochondrial proteins is described and characterized using a cell-free system. Precursor synthesized in vitro was incubated with extracts of mitochondria. Processing peptidase required Mn2+ for its activity. Localization studies suggested that it is a soluble component of the mitochondrial matrix. The precursor was cleaved in two sequential steps via an intermediate-sized polypeptide. The intermediate form in the processing of subunit 9 was also seen in vivo and upon import of the precursor into isolated mitochondria in vitro. The two cleavage sites in the precursor molecule were determined. The data indicate that: the correct NH2-terminus of the mature protein was generated, the NH2-terminal amino acid of the intermediate-sized polypeptide is isoleucine in position -31. The cleavage sites show similarity of primary structure. It is concluded that processing peptidase removes the peptide extension from the precursor to subunit 9 (and probably other precursors) after translocation of these polypeptides (or the NH2-terminal part of these polypeptides) into the matrix space of mitochondria.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000595:Amino Acid Sequence; D046911:Macromolecular Substances; D008345:Manganese / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D010446:Peptide Fragments / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D046911:Macromolecular Substances; D010446:Peptide Fragments; D011498:Protein Precursors; D008345:Manganese; D010447:Peptide Hydrolases; D000251:Adenosine Triphosphatases; D015032:Zinc