Mutational Analysis of Both Subunits from Rat Mitochondrial Processing Peptidase

Rat liver mitochondrial processing peptidase (MPP) is the primary peptidase that cleaves leader peptides from nuclearly encoded mitochondrial proteins following their transport from the cytosol to the mitochondrial matrix. This enzyme consists of two nonidentical subunits that have overall similarity to each other and share certain amino acid motifs. These include the putative metal-ion binding HFLEH motif in the β-subunit and the HFLEK motif of the α-subunit, as well as a possibly helical amino acid stretch bearing a high concentration of negatively charged residues about 70 amino acids downstream of these motifs in both subunits. In order to achieve a better understanding of the role of certain amino acids in rat MPP, we performed site-directed mutagenesis on both of its subunits. Our results show that whereas both histidines and the glutamate of the HFLEH motif in the β-subunit are crucial for MPP function, this holds true only for the glutamate in the related HFLEK motif in the α-subunit. In addition, functionally important negatively charged residues in the region 70 amino acids downstream occur only in the β-subunit and not in the α-subunit. This indicates a functional asymmetry between the subunits, with the β-subunit containing a majority of residues participating in the active center.