Characterization of the mitochondrial processing peptidase of Neurospora crassa

The mitochondrial processing peptidase (MPP) of Neurospora crassa
is constituted 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
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 b2 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
approximately 30-fold lower affinity for MPP than the preprotein.
Competition assays show that MPP recognizes the COOH- terminal
portion of the presequence of cytochrome b2 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.
Non-cleavable 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.