Abstract
Mitochondrial processing peptidase is a heterodimer consisting of alpha-mitochondrial processing peptidase (alpha-MPP) and beta-MPP. We investigated the role of alpha-MPP in substrate recognition using a recombinant yeast MPP. Disruption of amino acid residues between 10 and 129 of the alpha-MPP did not essentially impair binding activity with beta-MPP and processing activity, whereas truncation of the C-terminal 41 amino acids led to a significant loss of binding and processing activity. Several acidic amino acids in the region conserved among the enzymes from various species were mutated to asparagine or glutamine, and effects on processing of the precursors were analyzed. Glu353 is required for processing of malate dehydrogenase, aspartate aminotransferase, and adrenodoxin precursors. Glu377 and Asp378 are needed only for the processing of aspartate aminotransferase and adrenodoxin precursors, both of which have a longer extension peptide than the others studied. However, processing of the yeast alpha-MPP precursor, which has a short extension peptide of nine amino acids, was not affected by these mutations. Thus, effects of substitution of acidic amino acids on the processing differed with the precursor protein and depended on length of the extension peptides. alpha-MPP may function as a substrate-recognizing subunit by interacting mainly with basic amino acids at a region distal to the cleavage site in precursors with a longer extension peptide.
Highlights
Tidases, the pitrilysin family, which includes Escherichia coli pitrilysin, the insulin-degrading enzymes from mammals and insects, and the N-arginine dibasic convertase from the rat [15]
Studies using radiolabeled precursors and synthetic peptides as a substrate demonstrated that an arginine residue at Ϫ2 position and basic amino acid residues distal to the cleavage site are essential for specific cleavage by mitochondrial processing peptidase (MPP) [23,24,25,26]
Expression and Purification of Recombinant Yeast MPP—To reconstitute an active MPP, cDNAs for mature ␣- and -MPP were inserted into pET-3d expression vector, and two proteins were co-expressed in E. coli BL21 cells
Summary
Tidases, the pitrilysin family, which includes Escherichia coli pitrilysin ( called protease III), the insulin-degrading enzymes from mammals and insects, and the N-arginine dibasic convertase from the rat [15]. Except the ␣-MPPs, have a metal binding motif, His-Xaa-Xaa-Glu-His. The ␣- and -subunits of the MPP are homologous to core 2 and core 1 proteins, respectively, of the mitochondrial ubiquinol-cytochrome c oxidoreductase (bc complex), a component of the respiratory chain [16]. Studies using radiolabeled precursors and synthetic peptides as a substrate demonstrated that an arginine residue at Ϫ2 position and basic amino acid residues distal to the cleavage site are essential for specific cleavage by MPP [23,24,25,26]. We report here that acidic amino acids in the C-terminal portion of ␣-MPP are involved in substrate recognition and are mainly responsible for binding of distal basic amino acids of longer extension peptides
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