Characte~zation of the ~ I T O C ~ O N ~ a ~ Processing Peptidase of Neurospora Crassa"

The mitochondrial processing peptidase (MPP) of Neurospora: crwsu is constituted by an aand a &subunit. We have purified a"PP after expression in Escherichia coli while f3-MPP was purified from mitochondria. A fusion protein between precytochrome b2 and mouse dihydrofolate reductase was expressed in E. coli, and the purified protein was used as substrate for MPP. Both subunits of MPP are required for processing. MPP removes the matrix targeting signal of cytochrome bz by a single cut, and the resulting presequence peptide is 31 amino acid residues in length. It acts as a competitive inhibitor of processing but has a -30-fold lower affinity for MPP than the preprotein. Competition assays show that MPP recognizes the COOH-terminal portion of the presequence of cytochrome bz rather than the NH2-terminal part which has the potential to form an amphiphilic helix. Substitution of arginine in position -2 of the matrix targeting sequence of cytochrome b2 prevents processing but not import of a chimeric precursor. Substitution of the tyrosyl residue in position +1 also prevents processing, indicating that MPP interacts with sequences COOH-terminal 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.