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Results for “Mitochondrial Processing Peptidase”

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At least 19 recordsLinked to original sources

PubMed1995

Conversion of a nonprocessed mitochondrial precursor protein into one that is processed by the mitochondrial processing peptidase.

Mitochondrial processing peptidase (MPP) cleaves the signal sequence from a variety of mitochondrial precursor proteins. A subset of mitochondrial proteins, including rhodanese and 3-oxoacyl-CoA thiolase, are imported into the matrix space, yet are not processed. Rhodanese signal peptide and translated protein were recognized by MPP, as both were inhibitors of processing. The signal peptide of precursor aldehyde dehydrogenase consists of a helix-linker-helix motif but when the RGP linker is removed, processing no longer occurs (Thornton, K., Wang, Y., Weiner, H., and Gorenstein, D. G. (1993) J. Biol. Chem. 268, 19906-19914). Disruption of the helical signal sequence of rhodanese by the addition of the RGP linker did not allow cleavage to occur. However, addition of a putative cleavage site allowed the protein to be processed. The same cleavage site was added to 3-oxoacyl-CoA thiolase, but this protein was still not processed. Thiolase and linker-deleted aldehyde dehydrogenase signal peptides were poor inhibitors of MPP. It can be concluded that both a processing site and the structure surrounding this site are important for MPP recognition.

Acetyl-CoA C-Acyltransferase↗

PubMed1998

Cooperative formation of a substrate binding pocket by alpha- and beta-subunits of mitochondrial processing peptidase.

Mitochondrial processing peptidase (MPP) specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off N-terminal extension peptides. The enzyme is a metalloprotease and forms a heterodimer consisting of structurally related alpha- and beta-subunits. To investigate the responsibility of MPP subunits for substrate recognition, we monitored interaction of the fluorescent-labeled peptide substrates with the MPP and its subunits. The specific binding of the peptide to the MPP was confirmed by findings of the direct participation of arginine residues in the binding, which are located at position -2 and the position distal to the cleavage site and are essential for the cleavage reaction. MPP bound the substrate peptides with high affinity only in the dimeric complex, and each subunit monomer had about a 30-fold less affinity than the complex. The individual subunit required arginines at different positions in the peptide for binding, although their affinities were much lower than that of MPP. Fluorescence quenching analysis showed that the peptide bound to MPP was buried in the enzyme. Thus, both subunits of MPP might be required for formation of a substrate binding pocket with multiple subsites lying across them.

Amino Acid Sequence↗

PubMed2001

Electrostatic recognition of matrix targeting signal by mitochondrial processing peptidase.

Mitochondrial processing peptidase (MPP), a metalloendopeptidase consisting of alpha and beta subunits, specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off the N-terminal basic matrix targeting signals (MTS). Basic residues in MTS and acidic sites in MPP are required for effective processing. To elucidate whether the enzyme recognizes the MTS through electrostatic interaction, we investigated the effects of various salts on MPP activity. Decreases in the activity depended on the ionic strength and increases in the Michaelis constant value correlated clearly with the ionic strength, indicating a lower affinity of the enzyme for the substrate. Direct determination of the affinity between MPP and a MTS peptide using surface plasmon resonance showed a decrease in the association rate with high salt and that dissociation constant values were decreased. The effect of salt on the processing activity towards a variety of precursors was confirmed using five precursors with different sequences and lengths of MTS. Thus, we propose that electrostatic interactions are indispensable for the association between various MTS and MPP.

Amino Acid Sequence↗

PubMed1998

Glutamate residues required for substrate binding and cleavage activity in mitochondrial processing peptidase.

Mitochondrial processing peptidase, a metalloendopeptidase consisting of alpha- and beta-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 beta-subunit, which has a putative metal-binding motif, H56XXEH60, 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 alpha-subunit. Mutation of glutamates at positions 79, 129, and 136, in addition to an active-site glutamate at position 59, resulted in a marked decrease in cleavage efficiency. Together with sequence alignment data, glutamate 136 appears to be involved in metal binding. Glutamate 129 is mostly responsible for the catalysis, as there was a considerable decrease in kcat value by the mutation. Mutation of glutamate 79 led to decrease in kcat value and increase in Km values. Substrate binding experiments using an environmentally sensitive fluorescence probe attached to the peptide showed that the mutation caused a remarkable environmental change at the binding site to the N-terminal region of the substrate peptide and decreased binding of the peptide, thereby suggesting that glutamate 79 participates primarily in substrate binding. Thus, some glutamate residues required for substrate binding and cleavage activity have been identified.

Amino Acid Sequence↗

PubMed2000

A proposed common structure of substrates bound to mitochondrial processing peptidase.

Mitochondrial processing peptidase (MPP), a metalloendopeptidase consisting of alpha- and beta-subunits, specifically cleaves off the N-terminal presequence of the mitochondrial protein precursor. Structural information of the substrate bound to MPP was obtained using fluorescence resonance energy transfer (FRET) measurement. A series of the peptide substrates, which have distal arginine residues required for effective cleavage at positions -7, -10, -14, and -17 from the cleavage site, were synthesized and covalently labeled with 7-diethyl aminocoumarin-3-carboxylic acid at the N termini and N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD) at position +4, as fluorescent donor and acceptor, respectively. When the peptides were bound to MPP, substantially the same distances were obtained between the two probes, irrespective of the length of the intervening sequence between the two probes. When 7-diethylamino-3-(4'-maleimidyl phenyl)-4-methyl coumarin was introduced into a single cysteine residue in beta-MPP as a donor and IANBD was coupled either at the N terminus or the +4 position of the peptide substrate as an acceptor, intermolecular FRET measurements also demonstrated that distances of the donor-acceptor pair were essentially the same among the peptides with different lengths of intervening sequences. The results indicate that the N-terminal portion and the portion around the cleavage site of the presequence interact with specific sites in the MPP molecule, irrespective of the length of the intervening sequence between the two portions, suggesting the structure of the intervening sequence is flexible when bound to the MPP.

Amino Acid Sequence↗

PubMedDate not supplied

The mitochondrial processing peptidase.

The mitochondrial processing peptidase (MPP) is a heterodimeric enzyme which plays an essential role in mitochondrial protein import. It cleaves off the N-terminal targeting signals of nuclear encoded mitochondrial proteins upon their transport into the organelle. In mammals and yeast the enzyme is localized in the mitochondrial matrix while in plants it is integrated into a protein complex of the respiratory chain. As the activity of MPP is essential for the viability of eukaryotic cells it is conceivable that inhibitors of MPP which are specific for the soluble enzyme only present in fungi and animals may work as fungicides or insecticides.

Animals↗

PubMed1998

New insights into the co-evolution of cytochrome c reductase and the mitochondrial processing peptidase.

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.

Amino Acid Sequence↗

PubMed1996

Evidences for adenine nucleotide binding in the subunits of Neurospora mitochondrial processing peptidase.

The mitochondrial processing peptidase (MPP) of Neurospora crassa consists of two subunits termed alpha-and beta-MPP. Here we present spectroscopic and chromatographic studies indicative of adenine nucleotide binding in the two MPP subunits. ADP was identified as the cofactor of alpha-MPP and ATP as the cofactor of beta-MPP. The nucleotides are not covalently bound and exert strong control on the conformational and functional properties of the subunits. The ADP cofactor of alpha-MPP seems to be of utmost importance for the proteolytic activity because no processing of the precursor protein was registered in the assay containing alpha-MPP depleted of ADP and native beta-MPP. On the contrary, with native alpha-MPP and depleted of ATP beta-MPP almost 100% processing activity could be measured. Very strong increase of the intensity and significant changes in the shape and maximum position of the protein fluorescence spectra were observed after removal of the adenine cofactors of alpha- and of beta-MPP.

Adenine Nucleotides↗

PubMed1994

A helical element in the C-terminal domain of the N. plumbaginifolia F1 beta presequence is important for recognition by the mitochondrial processing peptidase.

The mitochondrial processing peptidase (MPP) in lower eucaryots and mammals is a matrix enzyme, whereas MPP in plants constitutes an integral part of the bc1 complex of the respiratory chain. The isolated spinach leaf bc1 complex catalyzes cleavage of the precursor of Nicotiana plumbaginifolia F1 beta subunit of the ATP synthase, resulting in a production of mature protein and a presequence that consists of 54 amino acids. A synthetic peptide derived from the C-terminal part of the presequence, containing 17 amino acids with a helical structural element, p-F1 beta(38-54), was an efficient inhibitor of the processing, whereas a peptide derived from the N-terminal part of the presequence, p-F1 beta(1-18), was much less effective. ATIII, a helical peptide derived from antithrombin III, was not recognized by MPP. Synthetic peptides corresponding to 4, 6, and 11 amino acids of the C terminus of the presequence, p-F1 beta(51-54), p-F1 beta(49-54), and p-F1 beta(44-54) were almost completely inert. Competition studies show that MPP recognizes the C-terminal domain of the presequence rather than the N-terminal domain. Furthermore, the alpha-helical element of the C-terminal domain is shown to be required for the recognition event.

Amino Acid Sequence↗

PubMed1996

Studies on protein processing for membrane-bound spinach leaf mitochondrial processing peptidase integrated into the cytochrome bc1 complex and the soluble rat liver matrix mitochondrial processing peptidase.

The plant mitochondrial processing peptidase (MPP) that catalyses the cleavage of the presequences from precursor proteins during or after protein import is a membrane-bound enzyme that constitutes an integral part of the bc1 complex of the respiratory chain. In contrast, MPP from mammals is soluble in the matrix space and does not form part of the respiratory chain. In the present study, we have compared the substrate specificity of the isolated spinach leaf bc1/MPP with rat liver MPP using synthetic signal peptides and different mitochondrial precursor proteins. Inhibition studies of processing with synthetic peptides showed a similar inhibition pattern for plant and rat MPP activity. A peptide derived from the presequence of rat liver mitochondrial aldehyde dehydrogenase (ALDH) was a potent inhibitor of the spinach and rat MPP. Two nonprocessed signal peptides, rhodanese and linker-deleted ALDH (a form of ALDH that lacks the RGP linker connecting two helices in the presequence) had lower inhibitory effects towards each protease. The signal peptide from thiolase, another nonprocessed protein, had little inhibitory effect on MPP. Peptides derived from presequence of the plant Nicotiana plumbaginifolia F1 beta also showed a similar inhibitory pattern with rat MPP as with spinach MPP processing. In-vitro synthesised precursors of plant N. plumbaginifolia F1 beta and rat liver ALDH were cleaved to mature form by both spinach and rat MPP. However, the efficiency of processing was higher with the homologous precursor. Linker-deleted ALDH, rhodanese, and thiolase were not processed by the mammalian or plant MPP. However, both forms of MPP cleaved a mutated form of rhodanese that possesses a typical MPP cleavage motif, RXY S. Addition of the same cleavage motif to thiolase did not result in processing by either MPP. These results show that similar higher-order structural elements upstream from the cleavage site are important for processing by both the membrane-bound plant and the soluble mammalian MPP.

Acetyl-CoA C-Acetyltransferase↗

PubMed1996

Mutational analysis of both subunits from rat mitochondrial processing peptidase.

Rat liver mitochondrial processing peptidase (MPP) is the primary peptidase that cleaves leader peptides from nuclearly encoded mitochondrial proteins following their transport from the cytosol to the mitochondrial matrix. This enzyme consists of two nonidentical subunits that have overall similarity to each other and share certain amino acid motifs. These include the putative metal-ion binding HFLEH motif in the beta-subunit and the HFLEK motif of the alpha-subunit, as well as a possibly helical amino acid stretch bearing a high concentration of negatively charged residues about 70 amino acids downstream of these motifs in both subunits. In order to achieve a better understanding of the role of certain amino acids in rat MPP, we performed site-directed mutagenesis on both of its subunits. Our results show that whereas both histidines and the glutamate of the HFLEH motif in the beta-subunit are crucial for MPP function, this holds true only for the glutamate in the related HFLEK motif in the alpha-subunit. In addition, functionally important negatively charged residues in the region 70 amino acids downstream occur only in the beta-subunit and not in the alpha-subunit. This indicates a functional asymmetry between the subunits, with the beta-subunit containing a majority of residues participating in the active center.

Amino Acid Sequence↗

PubMed2002

Protein import and processing reconstituted with isolated rat liver mitochondria and recombinant mitochondrial processing peptidase.

Most mitochondrial proteins are synthesized in the cytoplasm as larger precursors carrying N-terminal matrix-targeting presequences, and are subsequently transported to the mitochondria. The presequence mediates the interaction between the precursor polypeptide and components of the mitochondrial protein import machinery, a complex apparatus that is responsible for translocation of the precursor across the two mitochondrial membranes. Once the precursor has reached the mitochondrial matrix, the presequence is removed by the general mitochondrial processing peptidase (MPP). Some precursors undergo additional processing steps carried out by specialized processing peptidases. For most mitochondrial proteins, however, cleavage by MPP is the step that precedes folding and assembly into the native form. We describe methods to isolate import-competent mitochondria from rat liver and to perform import reactions with precursor proteins synthesized in vitro by coupled transcription-translation. We also describe methods to perform in vitro processing reactions of mitochondrial precursors by recombinant MPP and to identify the cleavage sites used by this enzyme.

Animals↗

PubMed1998

Role of alpha-subunit of mitochondrial processing peptidase in substrate recognition.

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.

Amino Acid Sequence↗

PubMed2002

Timing and structural consideration for the processing of mitochondrial matrix space proteins by the mitochondrial processing peptidase (MPP).

Most mitochondrial matrix space proteins are synthesized as a precursor protein, and the N-terminal extension of amino acids that served as the leader sequence is removed after import by the action of a metalloprotease called mitochondrial processing peptidase (MPP). The crystal structure of MPP has been solved very recently, and it has been shown that synthetic leader peptides bind with MPP in an extended conformation. However, it is not known how MPP recognizes hundreds of leader peptides with different primary and secondary structures or when during import the leader is removed. Here we took advantage of the fact that the structure of the leader from rat liver aldehyde dehydrogenase has been determined by 2D-NMR to possess two helical portions separated by a three amino acid (RGP) linker. When the linker was deleted, the leader formed one long continuous helix that can target a protein to the matrix space but is not removed by the action of MPP. Repeats of two and three leaders were fused to the precursor protein to determine the stage of import at which processing occurs, if MPP could function as an endo peptidase, and if it would process if the cleavage site was part of a helix. Native or linker deleted constructs were used. Import into isolated yeast mitochondria or processing with recombinantly expressed MPP was performed. It was concluded that processing did not occur as the precursor was just entering the matrix space, but most likely coincided with the folding of the protein. Further, finding that hydrolysis could not take place if the processing site was part of a stable helix is consistent with the crystal structure of MPP. Lastly, it was found that MPP could function at sites as far as 108 residues from the N terminus of the precursor protein, but its ability to process decreases exponentially as the distance increases.

Aldehyde Dehydrogenase↗

PubMed1994

Characterization of the mitochondrial processing peptidase of Neurospora crassa.

The mitochondrial processing peptidase (MPP) of Neurospora crassa is constituted by an alpha- and a beta-subunit. We have purified alpha-MPP after expression in Escherichia coli while beta-MPP was purified from mitochondria. A fusion protein between precytochrome b2 and mouse dihydrofolate reductase was expressed in E. coli, and the purified protein was used as substrate for MPP. Both subunits of MPP are required for processing. MPP removes the matrix targeting signal of cytochrome b2 by a single cut, and the resulting presequence peptide is 31 amino acid residues in length. It acts as a competitive inhibitor of processing but has a approximately 30-fold lower affinity for MPP than the preprotein. Competition assays show that MPP recognizes the COOH-terminal portion of the presequence of cytochrome b2 rather than the NH2-terminal part which has the potential to form an amphiphilic helix. Substitution of arginine in position -2 of the matrix targeting sequence of cytochrome b2 prevents processing but not import of a chimeric precursor. Substitution of the tyrosyl residue in position +1 also prevents processing, indicating that MPP interacts with sequences COOH-terminal to the cleavage site. Non-cleavable preprotein is still recognized by MPP. Our data suggest that processing peptidase and import machinery recognize distinct structural elements in preproteins which, however, can be overlapping.

Base Sequence↗

PubMed2002

Mitochondrial processing peptidases.

Three peptidases are responsible for the proteolytic processing of both nuclearly and mitochondrially encoded precursor polypeptides targeted to the various subcompartments of the mitochondria. Mitochondrial processing peptidase (MPP) cleaves the vast majority of mitochondrial proteins, while inner membrane peptidase (IMP) and mitochondrial intermediate peptidase (MIP) process specific subsets of precursor polypeptides. All three enzymes are structurally and functionally conserved across species, and their human homologues begin to be recognized as potential players in mitochondrial disease.

Amino Acid Sequence↗

PubMed1999

Complementation between mitochondrial processing peptidase (MPP) subunits from different species.

Mitochondrial processing peptidase (MPP), a dimer of nonidentical subunits, is the primary peptidase responsible for the removal of leader peptides from nuclearly encoded mitochondrial proteins. Alignments of the alpha and beta subunits of MPP (alpha- and beta-MPP) from different species show strong protein sequence similarity in certain regions, including a highly negatively charged region as well as a domain containing a putative metal ion binding site. In this report, we describe experiments in which we combine the subunits of MPP from yeast, rat, and Neurospora crassa, both in vivo and in vitro and mesure the resultant processing activity. For in vivo complementation, we used the temperature sensitive mif1 and mif2 yeast mutants, which lack MPP activity at the nonpermissive temperature (37 degrees C). We found that the defective alpha-MPP of mif2 cannot be substituted for by the alpha-MPP from rat or Neurospora. On the other hand, the beta-MPP from rat and Neurospora can fully substitute for the defective beta-MPP in the mif1 mutant. These results were confirmed in in vitro experiments in which individually expressed subunits were combined. Only combinations of the alpha-MPP from yeast with the beta-MPP from rat or Neurospora produced active MPP.

Animals↗

PubMed1997

Substrate recognition by mitochondrial processing peptidase toward the malate dehydrogenase precursor.

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.

Amino Acid Sequence↗