CHARACTERIZATION OF THE MITOCHONDRIAL PROCESSING PEPTIDASE OF NEUROSPORA-CRASSA

The mitochondrial processing peptidase (MPP) of Neurospora crassa is c onstituted by an alpha- and a beta-subunit. We have purified alpha-MPP after expression in Escherichia coli while beta-MPP was purified from mitochondria. A fusion protein between precytochrome b(2) and mouse d ihydrofolate reductase was expressed in E. coli, and the purified prot ein was used as substrate for MPP. Both subunits of MPP are required f or processing. MPP removes the matrix targeting signal of cytochrome b (2) by a single cut, and the resulting presequence peptide is 31 amino acid residues in length. It acts as a competitive inhibitor of proces sing but has a similar to 30-fold lower affinity for MPP than the prep rotein. Competition assays show that MPP recognizes the COOH-terminal portion of the presequence of cytochrome b(2) rather than the NH2-term inal part which has the potential to form an amphiphilic helix. Substi tution of arginine in position -2 of the matrix targeting sequence of cytochrome b(2) prevents processing but not import of a chimeric precu rsor. Substitution of the tyrosyl residue in position +1 also prevents processing, indicating that MPP interacts with sequences COOH-termina l to the cleavage site. Noncleavable preprotein is still recognized by MPP. Our data suggest that processing peptidase and import machinery recognize distinct structural elements in preproteins which, however, can be overlapping.