Abstract
Mitochondrial processing peptidase, a metalloendopeptidase consisting of alpha- and beta-subunits, specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off amino-terminal extension peptides. The alpha-subunit has a characteristic glycine-rich segment in the middle portion. To elucidate the role of the region in processing functions of the enzyme, deletion or site-directed mutations were introduced, and effects on kinetic parameters and substrate binding of the enzyme were analyzed. Deletion of three residues of the region, Phe(289) to Ala(291), led to a dramatic reduction in processing activity to practically zero. Mutation of Phe(289), Lys(296), and Met(298) to alanine resulted in a decrease in the activity, but these mutations had no apparent effect on interactions between the two subunits, indicating that reduction in processing activity is not due to structural disruption at the interface interacting with the beta-subunit. Although the mutant enzymes, Phe289Ala, Lys296Ala, and Met298Ala, had an approximate 10-fold less affinity for substrate peptides than did that of the wild type, the deletion mutant, delta 289-291, showed an extremely low affinity. Thus, shortening of the glycine-rich stretch led to a dramatic reduction of interaction between the enzyme and substrate peptides and cleavage reaction, whereas mutation of each amino acid in this region seemed to affect primarily the cleavage reaction.
Highlights
Most mitochondrial proteins encoded in the nucleus are synthesized as precursor proteins with extension peptides at the amino termini and are targeted to the mitochondria
Crystal structures of the bc1 complex from bovine and chicken heart mitochondria have been reported (39 – 41); one can expect that mitochondrial processing peptidase (MPP) is similar in structure to the complex
Our present observations indicate that the glycine-rich segment in the middle portion of ␣-MPP, which is characteristic of this subunit, is important for MPP functions
Summary
Most mitochondrial proteins encoded in the nucleus are synthesized as precursor proteins with extension peptides at the amino termini and are targeted to the mitochondria. The middle portion of core 2, which corresponds to the glycine-rich region of ␣-MPP conserved among many species, is located at the face toward the core 1 in the cavity These findings suggest the possible involvement of segment characteristics of ␣-MPP in dimer formation, substrate recognition, or catalytic reaction of MPP. Deletion of some amino acids in the segment led to an almost complete loss of the activity, without affecting formation of a stable heterodimeric complex From these results, together with a simulation model calculated from the reported crystallographic data on the core proteins (38 – 40), the possible role of the glycine-rich segment in the processing of precursor proteins by MPP is discussed
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