Mutational Analysis of Both Subunits from Rat Mitochondrial Processing Peptidase

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...

Section: Selection Of Acidic Amino Acid Residuesmentioning

confidence: 99%

“…So far, mutation analyses for MPP subunits have been reported (8,14,31), and the target residues are usually acidic amino acids because MPP recognizes basic sites on the presequences. MPP has a large polar cavity where a variety of presequences can bind; thus, acidic binding sites should be located inside the cavity.…”

mentioning

confidence: 99%

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

Kitada

Yamasaki

Kojima

et al. 2003

“…5). The core I proteins from man, beef, and yeast exhibit some sequence similarity to ␤-MPP subunits from different organisms, but they have an incomplete inverse zinc-binding motif, which was shown to be essential for the proteolytic activity of MPP (34,35) (Fig. 5A).…”

Section: Discussionmentioning

confidence: 99%

New Insights into the Co-evolution of Cytochrome cReductase and the Mitochondrial Processing Peptidase

Brumme¹,

Kruft²,

Schmitz³

et al. 1998

The mitochondrial processing peptidase (MPP) is a heterodimeric enzyme that forms part of the cytochrome c reductase complex from higher plants. Mitochondria from mammals and yeast contain two homologous enzymes: (i) an active MPP within the mitochondrial matrix and (ii) an inactive MPP within the cytochrome c reductase complex. To elucidate the evolution of MPP, the cytochrome c reductase complexes from lower plants were isolated and tested for processing activity. Mitochondria were prepared from the staghorn fern Platycerium bifurcatum, from the horsetail Equisetum arvense, and from the colorless algae Polytomella, and cytochrome c reductase complexes were purified by a micro-isolation procedure based on Blue-native polyacrylamide gel electrophoresis and electroelution. This is the first report on the subunit composition of a respiratory enzyme complex from a fern or a horsetail. The cytochrome c reductase complexes from P. bifurcatum and E. arvense are shown to efficiently process mitochondrial precursor proteins, whereas the enzyme complex from Polytomella lacks proteolytic activity. An evolutionary model is suggested that assumes a correlation between the presence of an active MPP within the cytochrome c reductase complex and the occurrence of chloroplasts.

“…In addition, amino acid residues and regions in the ␤-MPP seem to be functionally important, as deduced from mutation studies (37,38).…”

mentioning

confidence: 99%

Glutamate Residues Required for Substrate Binding and Cleavage Activity in Mitochondrial Processing Peptidase

Kitada

Kojima

Shimokata

et al. 1998

Mitochondrial processing peptidase, a metalloendopeptidase consisting of ␣-and ␤-subunits, specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off N-terminal extension peptides. The enzyme requires the basic amino acid residues in the extension peptides for effective and specific cleavage. To elucidate the mechanism involved in the molecular recognition of substrate by the enzyme, several glutamates around the active site of the rat ␤-subunit, which has a putative metal-binding motif, H 56 XXEH 60 , were mutated to alanines or aspartates, and effects on kinetic parameters, metal binding, and substrate binding of the enzyme were analyzed. None of mutant proteins analyzed was impaired in dimer formation with the ␣-subunit. Most nuclear-encoded mitochondrial proteins are synthesized on cytoplasmic ribosomes as larger precursors with Nterminal extension peptides for targeting into mitochondria (1-3). During or after import of the precursors into mitochondria, the extension peptides are proteolytically removed by three types of processing peptidases. Mitochondrial processing peptidase (MPP 1 ; EC 3.4.24.64) (4 -8) generally cleaves off a large part of the extension peptide, including the mitochondrial matrix-targeting sequence as the initial step of the processing. Many precursors are converted into the mature forms by the one-step processing. The second enzyme is mitochondrial intermediate peptidase, which catalyzes second-step cleavage in the two-step processing of some precursor proteins (9, 10). These precursors are first cleaved by MPP, and then the residual octapeptides are removed by the mitochondrial intermediate peptidase. The last enzyme is inner membrane protease I, which processes the signal sequence for inner membrane and intermembrane space (11). The last two peptidases sequentially act after cleavage of the matrix targeting sequences by MPP. Thus, MPP plays an important role in proteolytical processing of precursors in mitochondria. MPP has been purified from mitochondria of Neurospora crassa (12), yeast Saccharomyces cerevisiae (13), rat liver (14), and a few plants (15,16). The enzymes, except for those of the plant, are soluble proteins in the mitochondrial matrix and consist of ␣ and ␤ subunits. The subunits from yeast and rat liver form a stable heterodimer, whereas Neurospora subunits could be separated from each other by gel filtration. Processing activity of the enzymes is sensitive to metal chelators, and the lost activity is restored by divalent metal ions (4 -7). MPP specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off the extension peptides at single sites (14,17,18). Contrary to the strict substrate specificity of the enzyme, amino acid sequences of the extension peptides are wide in length and poor in similarity (1). Many experimental observations have indicated that basic amino acid residues in the extension peptides were required for the effective processing by . A consensus sequence of the processing signals has...