Spatial orientation of mitochondrial processing peptidase and a preprotein revealed by fluorescence resonance energy transfer

Mitochondrial processing peptidase (MPP), which is composed of heterodimeric alpha-MPP and beta-MPP subunits. It specifically recognizes mitochondrial preproteins and removes their basic N-terminal signal prepeptides. In order to elucidate the spatial orientation of the preproteins toward MPP, which has been missed by crystal structures of a yeast MPP including a synthetic prepeptide in its acidic proteolytic chamber, we analysed the fluorescence resonance energy transfer (FRET) between EGFP fused to a yeast aconitase presequence (preEGFP) and regiospecific 7-dietylamino-3-(4'-maleimidyl phenyl)-4-methyl coumarin (CPM)-labelled yeast MPPs. FRET efficiencies of 65 and 55% were observed between the EGFP chromophore and CPM-Ser(84) and -Lys(156) of beta-MPP, respectively, leading to calculated distances between the molecules of 48 and 50 A, respectively. Considering the FRET results and the structural validity based on the crystal structure of the MPP-presequence complex, a plausible model of preEGFP associated with MPP was constructed in silico. The modelled structure indicated that amino acid residues on the C-terminal side of the cleavage site in the preprotein were orientated tail out from the large cavity of MPP and interacted with the glycine-rich loop of alpha-MPP. Thus, MPP orientates preproteins at the specific cleft between the catalytic domain and the flexible glycine-rich loop which seems to pinch the extended polypeptide.     

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

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, alpha-MPP and beta-MPP, and analyzed the processing efficiencies for various preproteins. We report here that alanine mutations at a subsite in rat beta-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 alpha-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 beta- to alpha-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.     

A protein from a parasitic microorganism, Rickettsia prowazekii, can cleave the signal sequences of proteins targeting mitochondria

The obligate intracellular parasitic bacteria rickettsiae are more closely related to mitochondria than any other microbes investigated to date. A rickettsial putative peptidase (RPP) was found to resemble the alpha and beta subunits of mitochondrial processing peptidase (MPP), which cleaves the transport signal sequences of mitochondrial preproteins. RPP showed completely conserved zinc-binding and catalytic residues compared with beta-MPP but barely contained any of the glycine-rich loop region characteristic of alpha-MPP. When the biochemical activity of RPP purified from a recombinant source was analyzed, RPP specifically hydrolyzed basic peptides and presequence peptides with frequent cleavage at their MPP-processing sites. Moreover, RPP appeared to activate yeast beta-MPP so that it processed preproteins with shorter presequences. Thus, RPP behaves as a bifunctional protein that could act as a basic peptide peptidase and a somewhat regulatory protein for other protein activities in rickettsiae. These are the first biological and enzymological studies to report that a protein from a parasitic microorganism can cleave the signal sequences of proteins targeted to mitochondria.     

The processing peptidase of yeast mitochondria: the two co-operating components MPP and PEP are structurally related.

Two proteins co-operate in the proteolytic cleavage of mitochondrial precursor proteins: the mitochondrial processing peptidase (MPP) and the processing enhancing protein (PEP). In order to understand the structure and function of this novel peptidase, we have isolated mutants of Saccharomyces cerevisiae which were temperature sensitive in the processing of mitochondrial precursor proteins. Here we report on the mif2 mutation which is deficient in MPP. Mitochondria from the mif2 mutant were able to import precursor proteins, but not to cleave the presequences. The MPP gene was isolated. MPP is a hydrophilic protein consisting of 482 amino acids. Notably, MPP exhibits remarkable sequence similarity to PEP. We speculate that PEP and MPP have a common origin and have evolved into two components with different but mutually complementing functions in processing of precursor proteins.     

Mutagenesis and computer modelling approach to study determinants for recognition of signal peptides by the mitochondrial processing peptidase

Determinants for the recognition of a mitochondrial presequence by the mitochondrial processing peptidase (MPP) have been investigated using mutagenesis and bioinformatics approaches. All plant mitochondrial presequences with a cleavage site that was confirmed by experimental studies can be grouped into three classes. Two major classes contain an arginine residue at position -2 or -3, and the third class does not have any conserved arginines. Sequence logos revealed loosely conserved cleavage motifs for the first two classes but no significant amino acid conservation for the third class. Investigation of processing determinants for a class III precursor, Nicotiana plumbaginifolia F1beta precursor of ATP synthase (pF1beta), was performed using a series of pF1beta presequence mutants and mutant presequence peptides derived from the C-terminal portion of the presequence. Replacement of -2 Gln by Arg inhibited processing, whereas replacement of either the most proximally located -5 Arg or -15 Arg by Leu had only a low inhibitory effect. The C-terminal portion of the pF1beta presequence forms a helix-turn-helix structure. Mutations disturbing or prolonging the helical element upstream of the cleavage site inhibited processing significantly. Structural models of potato MPP and the C-terminal pF1beta presequence peptide were built by homology modelling and empirical conformational energy search methods, respectively. Molecular docking of the pF1beta presequence peptide to the MPP model suggested binding of the peptide to the negatively charged binding cleft formed by the alpha-MPP and beta-MPP subunits in close proximity to the H111XXE114H115X(116-190)E191 proteolytic active site on beta-MPP. Our results show for the first time that the amino acid at the -2 position, even if not an arginine, as well as structural properties of the C-terminal portion of the presequence are important determinants for the processing of a class III precursor by MPP.     

Identification and reverse genetic analysis of mitochondrial processing peptidase and the core protein of the cytochrome bc1 complex of Caenorhabditis elegans, a model parasitic nematode

Mitochondria could be a good target for anti-parasitic drugs. The alpha and beta subunits of mitochondrial processing peptidase (MPP) and the core subunits of the cytochrome bc1 complex, UCR-1 and UCR-2, are homologous to one another and are important for mitochondrial functions. However, our knowledge of these proteins in nematodes is very limited. Caenorhabditis elegans, a free-living nematode, has six genes coding for proteins homologous to these subunits. On primary structure comparison, and immunochemical and enzymological analyses, the gene products were assigned as follows: Y71G12B.24, alpha-MPP; ZC410.2, beta-MPP; F56D2.1, UCR-1; VW06B3R.1, T10B10.2; and T24C4.1, UCR-2. The primary structures of beta-MPP and UCR-1 from Brugia malayi, a parasitic nematode causing human filariasis, were deduced from their cDNA structures. Phylogenetic analysis showed that the UCR-1s from both C. elegans and B. malayi were less related to mammalian UCR-1s than to MPPs from various organisms. MPP and the bc1 complex are essential for the life cycle of C. elegans, because their reverse genetic inhibition is lethal. This suggests the possibility that these proteins are also essential for the viability of B. malayi and other parasitic nematodes, and are potential targets for anti-parasitic agents.     

Matrix processing peptidase of mitochondria. Structure-function relationships

The mitochondrial processing peptidase (MPP) and the processing enhancing protein (PEP) cooperate in the proteolytic cleavage of matrix targeting sequences from nuclear-encoded mitochondrial precursor proteins. We have determined the cDNA sequence of Neurospora MPP after expression cloning. MPP appears to contain two domains of approximately equal size which are separated by a loop-like sequence. Considerable structural similarity exists to the recently sequenced yeast MPP as well as to Neurospora and yeast PEP. Four cysteine residues are conserved in Neurospora and yeast MPP. Inactivation of MPP can be achieved by using sulfhydryl reagents. MPP (but not PEP) depends on the presence of divalent metal ions for activity. Both MPP and PEP are synthesized as precursors containing matrix targeting signals which are processed during import into mitochondria by the mature forms of MPP and PEP.     

Substrate binding changes conformation of the alpha-, but not the beta-subunit of mitochondrial processing peptidase

Lifetime analysis of tryptophan fluorescence of the mitochondrial processing peptidase (MPP) from Saccharomyces cerevisiae clearly proved that substrate binding evoked a conformational change of the alpha-subunit while presence of substrate influenced neither the lifetime components nor the average lifetime of the tryptophan excited state of the beta-MPP subunit. Interestingly, lifetime analysis of tryptophan fluorescence decay of the alpha-MPP subunit revealed about 11% of steady-state fractional intensity due to the long-lived lifetime component, indicating that at least one tryptophan residue is partly buried at the hydrophobic microenvironment. Computer modeling, however, predicted none of three tryptophans, which the alpha-subunit contains, as deeply buried in the protein matrix. We conclude this as a consequence of a possible dimeric (oligomeric) structure.     

Antisense RNA inhibition of the beta subunit of the Dictyostelium discoideum mitochondrial processing peptidase induces the expression of mitochondrial proteins

We cloned and characterized a cDNA encoding the Dictyostelium discoideum beta subunit of mitochondrial processing peptidase (Ddbeta-MPP). Western blot analysis of the mitochondrial subfractions revealed that Ddbeta-MPP is located in the mitochondrial matrix and membrane, whereas Dd(alpha)-MPP, another subunit of DdMPP, is located only in the matrix. Although expression of Ddbeta-MPP mRNA is down-regulated during early development, the level of the Ddbeta-MPP protein is constant throughout the Dictyostelium life cycle. In a transformant expressing the antisense RNA of the beta-MPP gene, unexpectedly, the beta-MPP protein increased about 1.8-fold relative to the wild type, and its mRNA increased 4.5-fold. Expression of other mitochondrial proteins, alpha-MPP and Cox IV, was also induced. These results suggest that antisense RNA inhibition of the beta-MPP gene induces gene expression of mitochondrial proteins, presumably in a retrograde signaling manner. This is the pathway of the transfer of information from the mitochondria to the nucleus.     

Amino-terminal octapeptides function as recognition signals for the mitochondrial intermediate peptidase.

We have shown previously that cleavage of a number of precursors by the mitochondrial processing peptidase (MPP) requires an intermediate octapeptide (FXXSXXXX) between the MPP cleavage site and the mature protein amino terminus. We show now that these octapeptides, present at the amino termini of the intermediates, direct recognition of these substrates by the mitochondrial intermediate peptidase (MIP), leading to formation of mature proteins. Synthetic peptides, corresponding to the intermediate octapeptides of human ornithine transcarbamylase (OTC) and of Neurospora cytochrome c reductase Fe/S subunit (Fe/S), inhibit the processing activity of purified rat liver MIP in vitro, without affecting MPP activity; this indicates that the octapeptides can be recognized by MIP independent of the presence of the corresponding mature proteins and interact with a site that is crucial for MIP activity. MIP activity is not inhibited by a peptide lacking the amino-terminal hydrophobic residue, while substitution of such a residue by a polar amino acid causes a 10-fold reduction in the efficiency of MIP inhibition. To analyze the requirements for removal of the octapeptide from the intermediate proteins by MIP, artificial intermediates were synthesized and subjected to in vitro processing by purified MIP. The octapeptide can be cleaved by MIP only when the amino-terminal hydrophobic residue is also the amino terminus of the intermediate. Further, when the OTC octapeptide is joined to the mature amino terminus of another twice-cleaved precursor (pFe/S; rat malate dehydrogenase, pMDH), the chimeric intermediate is cleaved by MIP to the corresponding mature-sized protein. When the OTC octapeptide is joined to the mature amino terminus of a once-cleaved precursor (yeast F1-beta-ATPase, pF1-beta), however, this intermediate is not cleaved by MIP; rather, it is processed by MPP to mature-sized F1-beta. Therefore, amino-terminal octapeptides can be cleaved by MIP only within the structural context of twice-cleaved precursors.     

Mitochondrial protein import: identification of processing peptidase and of PEP, a processing enhancing protein

Transport of nuclear-encoded precursor proteins into mitochondria includes proteolytic cleavage of amino-terminal targeting sequences in the mitochondrial matrix. We have isolated the processing activity from Neurospora crassa. The final preparation (enriched ca. 10,000-fold over cell extracts) consists of two proteins, the matrix processing peptidase (MPP, 57 kd) and a processing enhancing protein (PEP, 52 kd). The two components were isolated as monomers. PEP is about 15-fold more abundant in mitochondria than MPP. It is partly associated with the inner membrane, while MPP is soluble in the matrix. MPP alone has a low processing activity whereas PEP alone has no apparent activity. Upon recombining both, full processing activity is restored. Our data indicate that MPP contains the catalytic site and that PEP has an enhancing function. The mitochondrial processing enzyme appears to represent a new type of "signal peptidase," different from the bacterial leader peptidase and the signal peptidase of the endoplasmic reticulum.     

Yeast and human frataxin are processed to mature form in two sequential steps by the mitochondrial processing peptidase.

Frataxin is a nuclear-encoded mitochondrial protein which is deficient in Friedreich's ataxia, a hereditary neurodegenerative disease. Yeast mutants lacking the yeast frataxin homologue (Yfh1p) show iron accumulation in mitochondria and increased sensitivity to oxidative stress, suggesting that frataxin plays a critical role in mitochondrial iron homeostasis and free radical toxicity. Both Yfh1p and frataxin are synthesized as larger precursor molecules that, upon import into mitochondria, are subject to two proteolytic cleavages, yielding an intermediate and a mature size form. A recent study found that recombinant rat mitochondrial processing peptidase (MPP) cleaves the mouse frataxin precursor to the intermediate but not the mature form (Koutnikova, H., Campuzano, V., and Koenig, M. (1998) Hum. Mol. Gen. 7, 1485-1489), suggesting that a different peptidase might be required for production of mature size frataxin. However, in the present study we show that MPP is solely responsible for maturation of yeast and human frataxin. MPP first cleaves the precursor to intermediate form and subsequently converts the intermediate to mature size protein. In this way, MPP could influence frataxin function and indirectly affect mitochondrial iron homeostasis.     

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.     

A functionally divergent hydrogenosomal peptidase with protomitochondrial ancestry

Matrix proteins of mitochondria, hydrogenosomes and mitosomes are typically targeted and translocated into their respective organelles using N-terminal presequences that are subsequently cleaved by a peptidase. Here we characterize a approximately 47 kDa metallopeptidase, from the hydrogenosome-bearing, unicellular eukaryote Trichomonas vaginalis, that contains the active site motif (HXXEHX(76)E) characteristic of the beta subunit of the mitochondrial processing peptidase (MPP) and localizes to hydrogenosomes. The purified recombinant protein, named hydrogenosomal processing peptidase (HPP), is capable of cleaving a hydrogenosomal presequence in vitro, in contrast to MPP which requires both an alpha and beta subunit for activity. T. vaginalis HPP forms an approximately 100 kDa homodimer in vitro and also exists in an approximately 100 kDa complex in vivo. Our phylogenetic analyses support a common origin for HPP and betaMPP and demonstrate that gene duplication gave rise to alphaMPP and betaMPP before the divergence of T. vaginalis and mitochondria-bearing lineages. These data, together with published analyses of MPPs and putative mitosomal processing peptidases, lead us to propose that the length of targeting presequences and the subunit composition of organellar processing peptidases evolved in concert. Specifically, longer mitochondrial presequences may have evolved to require an alpha/beta heterodimer for accurate cleavage, while shorter hydrogenosomal and mitosomal presequences did not.     

Autocatalytic Processing of m-AAA Protease Subunits in Mitochondria

m-AAA proteases are ATP-dependent proteolytic machines in the inner membrane of mitochondria which are crucial for the maintenance of mitochondrial activities. Conserved nuclear-encoded subunits, termed paraplegin, Afg3l1, and Afg3l2, form various isoenzymes differing in their subunit composition in mammalian mitochondria. Mutations in different m-AAA protease subunits are associated with distinct neuronal disorders in human. However, the biogenesis of m-AAA protease complexes or of individual subunits is only poorly understood. Here, we have examined the processing of nuclear-encoded m-AAA protease subunits upon import into mitochondria and demonstrate autocatalytic processing of Afg3l1 and Afg3l2. The mitochondrial processing peptidase MPP generates an intermediate form of Afg3l2 that is matured autocatalytically. Afg3l1 or Afg3l2 are also required for maturation of newly imported paraplegin subunits after their cleavage by MPP. Our results establish that mammalian m-AAA proteases can act as processing enzymes in vivo and reveal overlapping activities of Afg3l1 and Afg3l2. These findings might be of relevance for the pathogenesis of neurodegenerative disorders associated with mutations in different m-AAA protease subunits.     

Crystal structures of mitochondrial processing peptidase reveal the mode for specific cleavage of import signal sequences

BACKGROUND: Mitochondrial processing peptidase (MPP) is a metalloendopeptidase that cleaves the N-terminal signal sequences of nuclear-encoded proteins targeted for transport from the cytosol to the mitochondria. Mitochondrial signal sequences vary in length and sequence, but each is cleaved at a single specific site by MPP. The cleavage sites typically contain an arginine at position -2 (in the N-terminal portion) from the scissile peptide bond in addition to other distal basic residues, and an aromatic residue at position +1. Mitochondrial import machinery recognizes amphiphilic helical conformations in signal sequences. However, it is unclear how MPP specifically recognizes diverse presequence substrates. RESULTS: The crystal structures of recombinant yeast MPP and a cleavage-deficient mutant of MPP complexed with synthetic signal peptides have been determined. MPP is a heterodimer; its alpha and beta subunits are homologous to the core II and core I proteins, respectively, of the ubiquinol-cytochrome c oxidoreductase complex. Crystal structures of two different synthetic substrate peptides cocrystallized with the mutant MPP each show the peptide bound in an extended conformation at the active site. Recognition sites for the arginine at position -2 and the +1 aromatic residue are observed. CONCLUSIONS: MPP bound two mitochondrial import presequence peptides in extended conformations in a large polar cavity. The presequence conformations differ from the amphiphilic helical conformation recognized by mitochondrial import components. Our findings suggest that the presequences adopt context-dependent conformations through mitochondrial import and processing, helical for recognition by mitochondrial import machinery and extended for cleavage by the main processing component.     

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.     

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.