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Substrate recognition by mitochondrial processing peptidase toward the malate dehydrogenase precursor
K Shimokata1, T Nishio, M C Song
1Department of Chemistry, Kyushu University, Fukuoka.
Abstract:
Mitochondrial processing peptidase (MPP) cleaves the extension peptides of precursor proteins newly imported into the mitochondria. Using synthetic oligopeptides modeled on the extension peptide of malate dehydrogenase, the critical elements of the substrate for the processing of MPP were determined [Niidome, T., Kitada, S., Shimokata, K., Ogishima, T., and Ito, A. (1994) J. Biol. Chem. 269, 24719-24722; Ogishima, T., Niidome, T., Shimokata, K., Kitada, S., and Ito, A. (1995) ibid. 270, 30322-30326]. In the present study, we constructed mutant precursors and compared the processing reaction with that of the peptide substrates to confirm the validity of use of peptide substrates. In both cases, the arginine residue presents at a proximal (-2) position relative to the processing site proved to be important for the processing. The distal arginine residue at position 7 was replaceable with alanine with no significant loss in cleavage efficiency if the precursor protein contained two consecutive arginine residues at a proximal position, although the arginine residue at a position 7 was indispensable in the model peptide. The proline residue, lying between the distal and proximal arginine residues, which is assumed to break a continuous alpha-helix region in the extension peptide, was needed for the processing. This peptidase has a preference for aromatic amino acids at the P1' site. These results were essentially the same as those obtained with model peptides except for the role of the distal arginine. We also found that amino acids at P2' and P3' sites had some effects on the processing. Thus we concluded that an effective combination of model peptides with precursor proteins is needed for the studies on MPP responsible substrate-recognition mechanisms.
Insights
Mitochondrial processing peptidase (MPP) substrate recognition is clarified. Arginine residues and proline at specific positions are crucial for MPP cleavage, with some flexibility in precursor proteins.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Processing
Background:
- Mitochondrial processing peptidase (MPP) removes extension peptides from proteins imported into mitochondria.
- Previous studies used model peptides to identify MPP substrate requirements.
Purpose of the Study:
- To validate the use of model peptides for studying MPP substrate recognition.
- To investigate the role of specific amino acid residues in precursor proteins during MPP processing.
Main Methods:
- Construction and processing of mutant precursor proteins.
- Comparison of processing efficiency between mutant precursors and synthetic peptide substrates.
- Analysis of amino acid requirements at proximal and distal positions relative to the cleavage site.
Main Results:
- Both proximal and distal arginine residues are important for MPP processing, with some redundancy in precursor proteins.
- A proline residue between arginine residues is essential for processing, likely by disrupting alpha-helical structure.
- MPP shows a preference for aromatic amino acids at the P1' site.
- Amino acids at P2' and P3' also influence processing efficiency.
Conclusions:
- Model peptides are useful but not entirely sufficient for understanding MPP substrate recognition.
- Precursor protein structure and specific amino acid combinations, particularly arginine and proline, dictate MPP cleavage.
- A combined approach using both model peptides and precursor proteins is necessary for comprehensive studies on MPP substrate-specificity.