Processing peptidase of Neurospora mitochondria. Two-step cleavage of imported ATPase subunit 9

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Abstract
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: (a) the correct NH2‐terminus of the mature protein was generated, (b) 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.