Determination of the Cleavage Site of the Presequence by Mitochondrial Processing Peptidase on the Substrate Binding Scaffold and the Multiple Subsites inside a Molecular Cavity

Abstract

Mitochondrial processing peptidase (MPP) recognizes a large variety of basic presequences of mitochondrial preproteins and cleaves the single site, often including arginine, at the ؊2 position (P 2 ). To elucidate the recognition and specific processing of the preproteins by MPP, we mutated to alanines at acidic residues conserved in a large internal cavity formed by the MPP subunits, ␣-MPP and ␤-MPP, and analyzed the processing efficiencies for various preproteins. We report here that alanine mutations at a subsite in rat ␤-MPP interacting with the P 2 arginine cause a shift in the processing site to the C-terminal side of the preprotein. Because of reduced interactions with the P 2 arginine, the mutated enzymes recognize not only the N-terminal authentic cleavage site with P 2 arginine but also the potential C-terminal cleavage site without a P 2 arginine. In fact, it competitively cleaves the two sites of the preprotein. Moreover, the acidified site of ␣-MPP, which binds to the distal basic site in the long presequence, recognized the authentic P 2 arginine as the distal site in compensation for ionic interaction at the proximal site in the mutant MPP. Thus, MPP seems to scan the presequence from ␤-to ␣-MPP on the substrate binding scaffold inside the MPP cavity and finds the distal and P 2 arginines on the multiple subsites on both MPP subunits. A possible mechanism for substrate recognition and cleavage is discussed here based on the notable character of a subsite-deficient mutant of MPP in which the substrate specificity is altered.Nuclear-encoded mitochondrial proteins generally carry Nterminal transport signal sequences (presequences) to target into mitochondria (1, 2), and inside the organelle, the presequences are removed by the mitochondrial processing peptidase (MPP) 1 (3-5). The presequences vary in length and sequence, but each is specifically recognized during protein transport and the maturation event by transport receptors and MPP, respectively. Because protease processing is irreversible, the maturation step should be highly regulated and specifically catalyzed by MPP for the biosynthesis of mitochondria.MPP consists of two structurally related subunits, ␣-and ␤-MPP (6, 7). The ␤-MPPs have a zinc binding motif, HXXEHX 76 E, in which histidine and the final glutamate residues participate in metal binding, and the first glutamate residue is involved in water activation for the hydrolysis of the peptide bond (8). Although ␤-MPP has an active site containing a zinc ion, the recombinant subunit alone has no apparent peptidase activity (9 -12). ␣-MPPs are mainly responsible for the binding of the basic site in the long presequence; a preprotein was cross-linked to ␣-MPP (13), and acidic amino acids in the C-terminal domain are mainly required for binding (14). The cooperative function of the subunits is involved not only in cleavage but also in the high affinity interaction to the presequence (15). Thus, ␣-MPP regulates the catalytic activity of ␤-MPP and also participates in substrate binding.Mammal...

Identification of proteins associated with the yeast mitochondrial RNA polymerase by tandem affinity purification

“…Although mt targeting peptides and their cleavage sites have been successfully predicted by similarity to known consensus motifs in a number of proteins (Fujiwara et al , 1997; Kitada et al , 2003), the reliability of such predictions is limited because MPP recognize many leader peptides with different primary and secondary structures, and can function at sites as far away as 100 residues from the N-terminus of the precursor protein (Graf et al , 1988; Ito, 1999). In previous work it had been suggested that the first 29 N-terminal aa residues in Rpo41p are likely to comprise a mt targeting sequence (Masters et al , 1987), and it was shown that fusion of this signal to various N-terminal deletion variants of Rpo41p was sufficient to direct delivery of the fusion variants to the mitochondria (Wang and Shadel, 1999).…”

Antisense RNA Inhibition of the β Subunit of theDictyostelium discoideumMitochondrial Processing Peptidase Induces the Expression of Mitochondrial Proteins

“…In both cases, an arginine residue is present at position -2 relative to the cleavage site, but the former site is more probable, because it agrees with the tendency of the MPP cleavage site to have hydrophobic (Phe-19) and polar uncharged (Ser-20) residues at positions +1 and +2 respectively. 14,15) All of the prediction programs PSORT II, Mitoprot, TargetP, and Predotar predict that Dd-MPP is destined for the mitochondria. Some predict the cleavage site after Ser-20, and others after Pro-26.…”

Reductive Evolution of the Mitochondrial Processing Peptidases of the Unicellular Parasites Trichomonas vaginalis and Giardia intestinalis

“…The part of the substrate-binding cavity formed by βMPP thus displays an evenly distributed negative charge to accommodate both proximal and distal positively charged residues of mitochondrial presequences. The αMPP interacts only with the distal positive residues of longer (>20 amino acid residues) mitochondrial presequences [9],[10].…”

“…Their most prominent common feature is the presence of a cleavage motif, which determines the peptide bond to be cleaved by the processing peptidase. The cleavage motif includes a positively charged residue, typically arginine, at the -2 or -3 position from the cleavage site (P 2 or P 3 ), which is followed by hydrophobic (P 1 ′) and hydrophilic (P 2 ′, P 3 ′) residues [9]. Mutational analyses indicate that the P 2 (P 3 ) arginine plays a key role in the recognition of the processing site by MPP and interacts with the glutamate of the βMPP active site [9].…”

“…The cleavage motif includes a positively charged residue, typically arginine, at the -2 or -3 position from the cleavage site (P 2 or P 3 ), which is followed by hydrophobic (P 1 ′) and hydrophilic (P 2 ′, P 3 ′) residues [9]. Mutational analyses indicate that the P 2 (P 3 ) arginine plays a key role in the recognition of the processing site by MPP and interacts with the glutamate of the βMPP active site [9]. In addition, there are one or more basic amino acid residue(s) N-terminally distal from the processing site that bind to acidic residues of the MPP cavity and stabilize the substrate-MPP complex [10].…”