SUBSTRATE RECOGNITION BY MITOCHONDRIAL PROCESSING PEPTIDASE TOWARD THE MALATE-DEHYDROGENASE PRECURSOR

Mitochondrial processing peptidase (MPP) cleaves the extension peptide s of precursor proteins newly imported into the mitochondria. Using sy nthetic oligopeptides modeled on the extension peptide of malate dehyd rogenase, 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 substra tes to confirm the validity of use of peptide substrates. In both case s, 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 wit h no significant loss in cleavage efficiency if the precursor protein contained two consecutive arginine residues at a proximal position, al though the arginine residue at a position 7 was indispensable in the m odel peptide. The proline residue, lying between the distal and proxim al arginine residues, which is assumed to break a continuous ct-helix region in the extension peptide, was needed for the processing. This p eptidase has a preference for aromatic amino acids at the P-1' site.(2 ) These results were essentially the same as those obtained with model peptides except for the role of the distal arginine. We also found th at amino acids at P-2' and P-3' sites had some effects on the processi ng. Thus we concluded that an effective combination of model peptides with precursor proteins is needed for the studies on MPP responsible s ubstrate-recognition mechanisms.