Processing of mitochondrial presequences

Mitochondrial proteins are synthesized as precursor proteins on either cytosolic or mitochondrial ribosomes. The synthesized precursors from both translation origins possess targeting signals that guide the protein to its final destination in one of the four subcompartments of the organelle. The majority of nuclear-encoded mitochondrial precursors and also mitochondrial-encoded preproteins have an N-terminal presequence that serves as a targeting sequence. Specific presequence peptidases that are found in the matrix, inner membrane and intermembrane space of mitochondria proteolytically remove the signal sequence upon import or sorting. Besides the classical presequence peptidases MPP, IMP and Oct1, several novel proteases have recently been described to possess precursor processing activity, and analysis of their functional relevance revealed a tight connection between precursor processing, mitochondrial dynamics and protein quality control. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.     

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.     

The presequence of fumarase is exposed to the cytosol during import into mitochondria

The majority of mitochondrial proteins can be imported into mitochondria following termination of their translation in the cytosol. Import of fumarase and several other proteins into mitochondria does not appear to occur post-translationally according to standard in vivo and in vitro assays. However, the nature of interaction between the translation and translocation apparatuses during import of these proteins is unknown. Therefore, a major question is whether the nascent chains of these proteins are exposed to the cytosol during import into mitochondria. We asked directly if the presequence of fumarase can be cleaved by externally added mitochondrial processing peptidase (MPP) during import, using an in vitro translation-translocation coupled reaction. The presequence of fumarase was cleaved by externally added MPP during import, indicating a lack of, or a loose physical connection between, the translation and translocation of this protein. Exchanging the authentic presequence of fumarase for that of the more efficient Su9-ATPase presequence reduced the exposure of fumarase precursors to externally added MPP en route to mitochondria. Therefore, exposure to cytosolic MPP is dependent on the presequence and not on the mature part of fumarase. On the other hand, following translation in the absence of mitochondria, the authentic fumarase presequence and that of Su9-ATPase become inaccessible to added MPP when attached to mature fumarase. Thus, folding of the mature portion of fumarase, which conceals the presequence, is the reason for its inability to be imported in classical post-translational assays. Another unique feature of fumarase is its distribution between the mitochondria and the cytosol. We show that in vivo the switch of the authentic presequence with that of Su9-ATPase caused more fumarase molecules to be localized to the mitochondria. A possible mechanism by which the cytosolic exposure, the targeting efficiency, and the subcellular distribution of fumarase are dictated by the presequence is discussed.     

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.     

A yeast mitochondrial presequence functions as a signal for targeting to plant mitochondria in vivo.

To date, the presequence of the mitochondrial beta-subunit of ATPase from tobacco is the only signal sequence that has been shown to target a foreign protein into plant mitochondria in vivo. Here we report that the presequence of a yeast mitochondrial protein directs bacterial beta-glucuronidase (GUS) specifically into the mitochondrial compartment of transgenic tobacco plants. Fusions between the presequence of the mitochondrial tryptophanyl-tRNA-synthetase gene from yeast and the GUS gene have been introduced into tobacco plants and yeast cells. In both systems, proteins containing the complete yeast mitochondrial presequence are efficiently imported in the mitochondria. Measurements of GUS activity in different subcellular fractions indicate that there is no substantial misrouting of the chimeric proteins in plant cells. In vitro synthesized GUS fusion proteins have a higher molecular weight than those found inside yeast and tobacco mitochondria, suggesting a processing of the precursors during import. Interestingly, fusion proteins translocated across the mitochondrial membranes of tobacco have the same size as those that are imported into yeast mitochondria. We conclude that the processing enzyme in plant mitochondria may recognize a proximate or even the same cleavage site within the mitochondrial tryptophanyl-tRNA-synthetase presequence as the matrix protease from yeast.     

A matrix-located processing peptidase of plant mitochondria

Nuclear-encoded mitochondrial precursor proteins are proteolytically processed inside the mitochondrion after import. The general mitochondrial processing activity in plant mitochondria has been shown to be integrated into the cytochrome bc₁ complex of the respiratory chain. Here we investigate the occurrence of an additional, matrix-located processing activity by incubation of the precursors of the soybean mitochondrial proteins, alternative oxidase, the F_Ad subunit of the ATP synthetase and the tobacco F₁ subunit of the ATP synthase, with the membrane and soluble components of mitochondria isolated from soybean cotyledons and spinach leaves. A matrix-located peptidase specifically processed the precursors to the predicted mature form in a reaction which was sensitive to orthophenanthroline, a characteristic inhibitor of mitochondrial processing peptidase (MPP). The specificity of the matrix peptidase was illustrated by the inhibition of processing of the alternative oxidase precursor in both soybean and spinach matrix extracts upon altering a single amino acid residue in the targeting presequence (-2 Arg to Gly). Additionally, there was no evidence for general proteolysis of precursor proteins incubated with the matrix. The purity of the matrix fractions was ascertained by spectrophotometric and immunological analyses. The results demonstrate that there is a specific processing activity in the matrix of soybean and spinach in addition to the previously well characterized membrane-bound MPP integrated into the cytochrome bc₁ complex of the respiratory chain.     

A Highlights from MBoC Selection: More than just a ticket canceller: the mitochondrial processing peptidase tailors complex precursor proteins at internal cleavage sites

Most mitochondrial proteins are synthesized as precursors that carry N-terminal presequences. After they are imported into mitochondria, these targeting signals are cleaved off by the mitochondrial processing peptidase (MPP). Using the mitochondrial tandem protein Arg5,6 as a model substrate, we demonstrate that MPP has an additional role in preprotein maturation, beyond the removal of presequences. Arg5,6 is synthesized as a polyprotein precursor that is imported into mitochondria and subsequently separated into two distinct enzymes. This internal processing is performed by MPP, which cleaves the Arg5,6 precursor at its N-terminus and at an internal site. The peculiar organization of Arg5,6 is conserved across fungi and reflects the polycistronic arginine operon in prokaryotes. MPP cleavage sites are also present in other mitochondrial fusion proteins from fungi, plants, and animals. Hence, besides its role as a “ticket canceller” for removal of presequences, MPP exhibits a second conserved activity as an internal processing peptidase for complex mitochondrial precursor proteins.     

Functional Analysis of the N-Terminal Prepeptides of Watermelon Mitochondrial and Glyoxysomal Malate Dehydrogenases*

Mitochondrial and glyoxysomal malate dehydrogenase (mMDH; gMDH; L-malate: NAD⁺ oxidoreductase; EC 1.1.1.37) of watermelon (Citrullus vulgaris) cotyledons are synthesized with N-terminal cleavable presequences which are shown to specify sorting of the two proteins. The two presequences differ in length (27 or 37 amino acids) and primary structure. Precursor proteins of the two isoenzymes with site-directed mutations in their presequences and hybrid precursor proteins with reciprocally exchanged presequences were analyzed for proper import using two approaches, namely in vitro using isolated watermelon organelles or in vivo after synthesis in the heterologous host, Hansenula polymorpha. The mitochondrial presequence is essential and sufficient to target the mature glyoxysomal isoenzyme into mitochondria (Gietl et al., 1994). As to the function of the mitochondrial presequence a substitution of ^?3R (considered important for one step precursor cleavage in yeast and mammals) with ^?3L permitted import into mitochondria but cleavage of the transit peptide and conversion into active mature enzyme was impeded. Substitution of ^?13R^?12S (in a sequence reminiscent of the octapeptide motif serving as a substrate for the mammalian and yeast intermediate peptidase) into ^?13L¹²F permitted mitochondrial import and processing like the wild type transit peptide. Purified rat mitochondrial processing protease, which can effect single step cleavage of mitochondrial protein precursors, cleaves in vitro translated watermelon mMDH precursor into its mature form. The glyoxysomal presequence is essential and sufficient to target the mature mitochondrial isoenzyme into peroxisomes of Hansenula polymorpha, but these peroxisomes lack a processing enzyme to cleave the presequence (Gietl et al., 1994). We here show that isolated watermelon organelles also import the hybrid proteins in vitro and process the glyoxysomal presequence. Site directed mutations within the conserved RI-X₅-HL-motif impede efficiency of import and cleavage by watermelon organelles.     

Mutant alcohol dehydrogenase (ADH III) presequences that affect both in vitro mitochondrial import and in vitro processing by the matrix protease.

Point mutations in the presequence of the mitochondrial alcohol dehydrogerase isoenzyme (ADH III) have been shown to affect either the import of the precursor protein into yeast mitochondria in vivo or its processing within the organelle. In the present work, the behavior of these mutants during in vitro import into isolated mitochondria was investigated. All point mutants tested were imported with a slower initial rate than that of the wild-type precursor. This defect was corrected when the precursors were treated with urea prior to import. Once imported, the extent of processing to the mature form of mutant precursors varied greatly and correlated well with the defects observed in vivo. This result was not affected by prior urea treatment. When matrix extracts enriched for the processing protease were used, this defect was shown to be due to failure of the protease to efficiently recognize or cleave the presequence, rather than to a lack of access to the precursor. The rate of import of two ADH III precursors bearing internal deletions in the leader sequence was similar to those of the point mutants, whereas a deletion leading to the removal of the 15 amino-terminal amino acids was poorly imported. The mature amino terminus of wild-type ADH III was determined to be Gln-25. Mutant m01 (Ser-26 to Phe), which reduced the efficiency of cleavage in vitro by 80%, was cleaved at the correct site.     

Isolated plant mitochondria import chloroplast precursor proteins in vitro with the same efficiency as chloroplasts.

Most chloroplast and mitochondrial proteins are synthesized with N-terminal presequences that direct their import into the appropriate organelle. In this report we have analyzed the specificity of standard in vitro assays for import into isolated pea chloroplasts and mitochondria. We find that chloroplast protein import is highly specific because mitochondrial proteins are not imported to any detectable levels. Surprisingly, however, pea mitochondria import a range of chloroplast protein precursors with the same efficiency as chloroplasts, including those of plastocyanin, the 33-kDa photosystem II protein, Hcf136, and coproporphyrinogen III oxidase. These import reactions are dependent on the Deltaphi across the inner mitochondrial membrane, and furthermore, marker enzyme assays and Western blotting studies exclude any import by contaminating chloroplasts in the preparation. The pea mitochondria specifically recognize information in the chloroplast-targeting presequences, because they also import a fusion comprising the presequence of coproporphyrinogen III oxidase linked to green fluorescent protein. However, the same construct is targeted exclusively into chloroplasts in vivo indicating that the in vitro mitochondrial import reactions are unphysiological, possibly because essential specificity factors are absent in these assays. Finally, we show that disruption of potential amphipathic helices in one presequence does not block import into pea mitochondria, indicating that other features are recognized.     

Investigations on the in vitro import ability of mitochondrial precursor proteins synthesized in wheat germ transcription-translation extract

Mitochondrial precursor proteins synthesized in rabbit reticulocyte lysate (RRL) are readily imported into mitochondria, whereas the same precursors synthesized in wheat germ extract (WGE) fail to be imported. We have investigated factors that render import incompetence from WGE. A precursor that does not require addition of extramitochondrial ATP for import, the F(A)d ATP synthase subunit, is imported from WGE. Import of chimeric constructs between precursors of the F(A)d protein and alternative oxidase (AOX) with switched presequences revealed that the mature domain of the F(A)d precursor defines the import competence in WGE as only the construct containing the presequence of AOX and mature portion of F(A)d (pAOX-mF(A)d) could be imported. Import competence of F(A)d and pAOX-mF(A)d correlated with solubility of these precursors in WGE, however, solubilization of import-incompetent precursors with urea did not restore import competence. Addition of RRL to WGE-synthesized precursors did not stimulate import but addition of WGE to the RRL-synthesized precursors or to the over-expressed mitochondrial precursor derived from the F1beta ATP synthase precursor inhibited import into mitochondria. The dual-targeted glutathione reductase precursor synthesized in WGE was imported into chloroplasts, but not into mitochondria. Antibodies against the 14-3-3 guidance complex characterized for chloroplast targeting were able to immunoprecipitate all of the precursors tested except the F(A)d ATP synthase precursor. Our results point to the conclusion that the import incompetence of WGE-synthesized mitochondrial precursors is not presequence dependent and is a result of interaction of WGE inhibitory factors with the mature portion of precursor proteins.     

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.     

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.     

Studies on the topology of the protein import channel in relation to the plant mitochondrial processing peptidase integrated into the cytochrome bc1 complex

The mitochondrial processing peptidase (MPP) specifically cleaves N-terminal targeting signals from hundreds of nuclear-encoded, matrix-targeted precursor proteins. In contrast to yeast and mammals, the plant MPP is an integral component of the respiratory cytochrome bc1 complex. The topology of the protein import channel in relation to MPP/bc1 in plants was studied using chimeric precursors containing truncated cytochrome b2 (cyt b2) proteins of 55-167 residues in length, fused to dihydrofolate reductase (DHFR). The DHFR domain could be tightly folded by methotrexate (MTX), generating translocation intermediates trapped in the import channel with only the cyt b2 pre-sequence/mature domain protruding into the matrix. Spinach and soybean mitochondria imported and processed unfolded precursors. MTX-folded intermediates were not processed in spinach but the longest (1-167) MTX-folded cyt b2-DHFR construct was processed in soybean, while yeast mitochondria successfully processed even shorter MTX-folded constructs. The MTX-folded precursors were cleaved with high efficiency by purified spinach MPP/bc1 complex. We interpret these results as indicating that the protein import channel is located distantly from the MPP/bc1 complex in plants, and that there is no link between protein translocation and protein processing.     

Active unfolding of precursor proteins during mitochondrial protein import.

Precursor proteins made in the cytoplasm must be in an unfolded conformation during import into mitochondria. Some precursor proteins have tightly folded domains but are imported faster than they unfold spontaneously, implying that mitochondria can unfold proteins. We measured the import rates of artificial precursors containing presequences of varying length fused to either mouse dihydrofolate reductase or bacterial barnase, and found that unfolding of a precursor at the mitochondrial surface is dramatically accelerated when its presequence is long enough to span both membranes and to interact with mhsp70 in the mitochondrial matrix. If the presequence is too short, import is slow but can be strongly accelerated by urea-induced unfolding, suggesting that import of these 'short' precursors is limited by spontaneous unfolding at the mitochondrial surface. With precursors that have sufficiently long presequences, unfolding by the inner membrane import machinery can be orders of magnitude faster than spontaneous unfolding, suggesting that mhsp70 can act as an ATP-driven force-generating motor during protein import.     

A mitochondrial import factor purified from rat liver cytosol is an ATP-dependent conformational modulator for precursor proteins.

Rat liver cytosol contained an activity that stimulated the import of wheat germ lysate-synthesized precursor proteins into mitochondria. The activity was purified 10,000-fold from the cytosol as a homogeneous heterodimeric protein. This protein (termed mitochondrial import stimulation factor or MSF) stimulated the binding and import of mitochondrial precursor proteins. MSF was also found to recognize the presequence portion of mitochondrial precursors and catalyze the depolymerization and unfolding of in vitro synthesized mitochondrial precursor proteins in an ATP-dependent manner; in this connection, MSF exhibited ATPase activity depending on the important-incompetent mitochondrial precursor protein. The mitochondrial binding and import-stimulating activities were strongly inhibited by the pretreatment of MSF with NEM, whereas the ATP-dependent depolymerization activity was insensitive to the NEM treatment, suggesting that the process subsequent to the unfolding was inhibited with the NEM treatment. We conclude that MSF is a multifunctional cytoplasmic chaperone specific for mitochondrial protein import.