The Cytochrome c Reductase Integrated Processing Peptidase from Potato Mitochondria Belongs to a New Class of Metalloendoproteases

The general mitochondrial processing peptidase that removes the N-terminal targeting signals from proteins imported into mitochondria forms part of a respiratory protein complex in potato (Solanum tuberosum L.). We have termed this complex the "cytochrome c reductase/processing peptidase complex" and show that it acts on a variety of precursor proteins from different intramitochondrial locations. In potato, biochemical methods fail to separate the ubiquinol cytochrome c oxidoreductase function from the function of the processing protease. On the other hand, inhibition of electron flow with antimycin A or myxothiazol does not affect processing activity. The integration into an oligomeric protein complex causes the unique properties of the processing enzyme. It is fully active at high pH and in the presence of high salt. It does not need externally added metal ions, but it is inhibited by EDTA and 1,10-phenanthroline. Other protease inhibitors have no effect on the processing activity. Taken together, the molecular genetic and physiological results indicate that the mitochondrial processing protease does not belong to the thermolysin superfamily of metalloproteinases but may be a member of a new class of metalloendoproteases.     

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.     

MAS1, a gene essential for yeast mitochondrial assembly, encodes a subunit of the mitochondrial processing protease.

We have previously described a yeast mutant (mas1) that accumulates mitochondrial precursor proteins at high temperature and is deficient in the activity of a matrix-localized protease which cleaves presequences from mitochondrial precursor proteins. We have now cloned and sequenced the wild-type MAS1 gene and found that it encodes a subunit of the mitochondrial processing protease, that it is essential for cell viability and that the protein product participates in its own cleavage during import into mitochondria. The MAS1 protein is thus the first genetically defined component of the mitochondrial protein import pathway.     

Affinity purification of cytochrome c reductase from potato mitochondria.

Ubiquinol-cytochrome-c oxidoreductase has been isolated from potato (Solanum tuberosum L.) mitochondria by cytochrome-c affinity chromatography and gel-filtration chromatography. The procedure, which up to now only proved applicable to Neurospora, yields a highly pure and active protein complex in monodisperse state. The molecular mass of the purified complex is about 650 kDa, indicating that potato cytochrome c reductase occurs as a dimer. Upon reconstitution into phospholipid membranes, the dimeric enzyme catalyzes electron transfer from a synthetic ubiquinol to equine cytochrome c with a turnover number of 50 s-1. The activity is inhibited by antimycin A and myxothiazol. A myxothiazol-insensitive and antimycin-sensitive transhydrogenation reaction, with a turnover number of 16 s-1, can be demonstrated as well. The protein complex consists of ten subunits, most of which have molecular masses similar to those of the nine-subunit fungal enzyme. Individual subunits were identified immunologically and spectral properties of b and c cytochromes were monitored. Interestingly, an additional 'core' polypeptide which is not present in other cytochrome bc1 complexes forms part of the enzyme from potato. Antibodies raised against individual polypeptides reveal that the core proteins are clearly immuno-distinguishable. The additional subunit may perform a specific function and contribute to the high molecular mass which exceeds those reported for other cytochrome-c-reductase dimers.     

Biogenesis of mitochondrial ubiquinol:cytochrome c reductase (cytochrome bc1 complex). Precursor proteins and their transfer into mitochondria

The precursor proteins to the subunits of ubiquinol:cytochrome c reductase (cytochrome bc1 complex) of Neurospora crassa were synthesized in a reticulocyte lysate. These precursors were immunoprecipitated with antibodies prepared against the individual subunits and compared to the mature subunits immunoprecipitated or isolated from mitochondria. Most subunits were synthesized as precursors with larger apparent molecular weights (subunits I, 51,500 versus 50,000; subunit II, 47,500 versus 45,000; subunit IV (cytochrome c1), 38,000 versus 31,000; subunit V (Fe-S protein), 28,000 versus 25,000; subunit VII, 12,000 versus 11,500; subunit VIII, 11,600 versus 11,200). Subunit VI (14,000) was synthesized with the same apparent molecular weight. The post-translational transfer of subunits I, IV, V, and VII was studied in an in vitro system employing reticulocyte lysate and isolated mitochondria. The transfer and proteolytic processing of these precursors was found to be dependent on the mitochondrial membrane potential. In the transfer of cytochrome c1, the proteolytic processing appears to take place in two separate steps via an intermediate both in vivo and in vitro. In vivo, the intermediate form accumulated when cells were kept at 8 degrees C and was chased into mature cytochrome c1 at 25 degrees C. Both processing steps were energy-dependent.     

Subunit VII, the ubiquinone-binding protein, of the cytochrome b-c1 complex of yeast mitochondria is involved in electron transport at center o and faces the matrix side of the membrane

The functional role and topographical orientation in the inner membrane of subunit VII, the ubiquinone-binding protein, of the cytochrome b-c1 complex of yeast mitochondria has been investigated. The apparent molecular weight of this subunit on sodium dodecyl sulfate-urea gels was calculated to be 15,500, while its amino acid composition was similar to that of the Q-binding proteins present in the cytochrome b-c1 complexes isolated from both beef heart and yeast mitochondria. The specific antibody obtained against subunit VII inhibited 30-47% of the ubiquinol-cytochrome c reductase activity in the isolated cytochrome b-c1 complex and in submitochondrial particles but had no effect on cytochrome c reductase activity in mitoplasts, mitochondria from which the outer membrane has been removed. Furthermore, the antibody against subunit VII strongly inhibited (74%) the reduction of cytochrome b by succinate in the presence of antimycin, an inhibitor of center i, but had no effect on cytochrome b reduction in the presence of myxothiazol, an inhibitor of center o. These results suggest that subunit VII, the Q-binding protein, is involved in electron transport at center o of the cytochrome b-c1 complex of the respiratory chain and that subunit VII is localized facing the matrix side of the inner mitochondrial membrane.     

The first twelve amino acids (less than half of the pre-sequence) of an imported mitochondrial protein can direct mouse cytosolic dihydrofolate reductase into the yeast mitochondrial matrix.

Yeast cytochrome c oxidase subunit IV (an imported mitochondrial protein) is made as a larger precursor with a transient pre-sequence of 25 amino acids. If this pre-sequence is fused to the amino terminus of mouse dihydrofolate reductase (a cytosolic protein) the resulting fusion protein is imported into the matrix space, and cleaved to a smaller size, by isolated yeast mitochondria. We have now fused progressively shorter amino-terminal segments of the subunit IV pre-sequence to dihydrofolate reductase and tested each fusion protein for import into the matrix space and cleavage by the matrix-located processing protease. The first 12 amino acids of the subunit IV pre-sequence were sufficient to direct dihydrofolate reductase into the mitochondrial matrix, both in vitro and in vivo. However, import of the corresponding fusion protein into the matrix was no longer accompanied by proteolytic processing. Fusion proteins containing fewer than nine amino-terminal residues from the subunit IV pre-piece were not imported into isolated mitochondria. The information for transporting attached mouse dihydrofolate reductase into mitochondria is thus contained within the first 12 amino acids of the subunit IV pre-sequence.     

Transport of proteins into yeast mitochondria

The amino-terminal sequences of several imported mitochondrial precursor proteins have been shown to contain all the information required for transport to and sorting within mitochondria. Proteins transported into the matrix contain a matrix-targeting sequence. Proteins destined for other submitochondrial compartments contain, in addition, an intramitochondrial sorting sequence. The sorting sequence in the cytochrome c1 presequence is a stop-transport sequence for the inner mitochondrial membrane. Proteins containing cleavable presequences can reach the intermembrane space by either of two pathways: (1) Part of the presequence is transported into the matrix; the attached protein, however, is transported across the outer but not the inner membrane (eg, the cytochrome c1 presequence). (2) The precursor is first transported into the matrix; part of the presequence is then removed, and the protein is reexported across the inner membrane (eg, the precursor of the iron-sulphur protein of the cytochrome bc1 complex). Matrix-targeting sequences lack primary amino acid sequence homology, but they share structural characteristics. Many DNA sequences in a genome can potentially encode a matrix-targeting sequence. These sequences become active if positioned upstream of a protein coding sequence. Artificial matrix-targeting sequences include synthetic presequences consisting of only a few different amino acids, a known amphiphilic helix found inside a cytosolic protein, and the presequence of an imported chloroplast protein. Transport of proteins across mitochrondrial membranes requires a membrane potential, ATP, and a 45-kd protein of the mitochondrial outer membrane. The ATP requirement for import is correlated with a stable structure in the imported precursor molecule. We suggest that transmembrane transport of a stably folded precursor requires an ATP-dependent unfolding of the precursor protein.     

Biosynthesis of the ubiquinol-cytochrome c reductase complex in yeast. Characterization of precursor forms of the 44-kDa, 40-kDa and 17-kDa subunits and identification of individual messenger RNAs for these and other imported subunits of the complex

The mitochondrial ubiquinol--cytochrome c reductase complex (complex III or cytochrome bc1 complex) is thought to consist of eight subunits, seven of which are specified by nuclear genes and synthesized in the cytoplasm. We have studied the synthesis of five of the nuclear-encoded subunits both in vivo and in vitro and show that of these the 44-kDa, 40-kDa and 17-kDa subunits are synthesized with cleavable extensions, while the 14-kDa and 11-kDa proteins are synthesized without detectable extra sequences. The sizes of the pre-sequences, as determined by the relative mobility of the precursor proteins in sodium dodecyl sulphate/polyacrylamide gels, range from 0.5-kDa for the 44-kDa and 40-kDa subunits to 9-kDa for the 17-kDa subunit. The existence in vivo of precursor forms to the 44-kDa, 40-kDa and 17-kDa subunits implies that import is at least partially a post-translational process. The precursor of the 44-kDa subunit can be processed post-translationally in vitro by isolated mitochondria. The messenger RNAs for subunits of the complex have been studied. Those coding for the 44-kDa, 40-kDa, 14-kDa and 11-kDa proteins and cytochrome c1 are of different sizes, indicating that each of these subunits is synthesized as a separate protein, rather than as part of a polyprotein precursor.     

Molecular features and mitochondrial import pathway of the 14-kilodalton subunit of cytochrome c reductase from potato.

The cytochrome c reductase complexes from fungi and mammals both contain a 14-kD protein (yeast, 14.4 kD; bovine, 13.4 kD) that does not directly participate in electron transfer but possibly is indirectly involved in the function of the complex and has a role in assembly of the multimeric enzyme. A subunit of comparable size was identified for the bc1 complex of higher plants. The 14-kD protein from potato (Solanum tuberosum) was specifically separated from the isolated protein complex in the presence of 6 M urea and is, therefore, assumed to be a peripheral component. Direct sequence analysis of the proteins from potato and wheat (Triticum aestivum) and isolation of corresponding cDNA clones for the subunit from potato revealed clear similarity to the equivalent proteins from yeast and bovine. The wheat 14-kD protein seems to occur in two isoforms. The 14-kD protein from plants is very hydrophilic, has a characteristic charge distribution, and contains no potential membrane-spanning helices. In vitro import of the radiolabeled 14-kD protein from potato into isolated mitochondria depends on the membrane potential across the inner mitochondrial membrane. The protein seems to lack a cleavable mitochondrial presequence, because it is not processed upon translocation. Possible intramolecular regions involved in targeting of the 14-kD protein to plant mitochondria are discussed.     

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 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.     

Molecular identification of the ten subunits of cytochrome-c reductase from potato mitochondria

Cytochrome-c reductase (EC 1.10.2.2.) from Solanum tuberosum L. comprises ten subunits with apparent molecular sizes of 55, 53, 51, 35, 33, 25, 14, 12, 11 and 10 kDa on 14% SDS-PAGE. The identity of the subunits was analysed by direct amino-acid sequencing via cyclic Edman degradation. A large-scale purification procedure for the enzyme complex based on affinity chromatography and gelfiltraton is described. All subunits were enzymatically fragmented and the generated peptides were separated by reverse-phase HPLC. Complete or partial sequence determination of 33 peptides comprising a total of nearly 500 amino acids showed, that cytochrome-c reductase from potato contains three respiratory proteins (cytochrome b, cytochrome c ₁ and the Rieske iron-sulfur protein), four small proteins with molecular sizes below 15 kDa (so-called Q-binding, hinge, cytochrome-c ₁-linked and core-linked proteins) and three proteins in the 50-kDa range which show similarity to members of the core/PEP/MPP protein family (core/processing enhancing protein/mitochondrial processing peptidase). In fact these subunits show highest sequence identity either to MPP or PEP, which is in line with earlier findings, that isolated cytochrome-c reductase from potato exhibits processing activity towards mitochondrial precursor proteins.Abbreviations MPP mitochondrial processing peptidase - PEP processing enhancing protein This research was supported by the Deutsche Forschungsgemeinschaft.     

Isolation and characterization of complex I, rotenone-sensitive NADH: ubiquinone oxidoreductase, from the procyclic forms of Trypanosoma brucei.

Additional characterization of complex I, rotenone-sensitive NADH:ubiquinone oxidoreductase, in the mitochondria of Trypanosoma brucei brucei has been obtained. Both proline:cytochrome c reductase and NADH:ubiquinone oxidoreductase of procyclic T. brucei were inhibited by the specific inhibitors of complex I rotenone, piericidin A, and capsaicin. These inhibitors had no effect on succinate: cytochrome c reductase activity. Antimycin A, a specific inhibitor of the cytochrome bc1 complex (ubiquinol:cytochrome c oxidoreductase), blocked almost completely cytochrome c reductase activity with either proline or succinate as electron donor, but had no inhibitory effect on NADH:ubiquinone oxidoreductase activity. The rotenone-sensitive NADH:ubiquinone oxidoreductase of procyclic T. brucei was partially purified by sucrose density centrifugation of mitochondria solubilized with dodecyl-beta-D-maltoside, with an approximately eightfold increase in specific activity compared to that of the mitochondrial membranes. Four polypeptides of the partially purified enzyme were identified as the homologous subunits of complex I (51 kDa, PSST, TYKY, and ND4) by immunoblotting with antibodies raised against subunits of Paracoccus denitrificans and against synthetic peptides predicted from putative complex I subunit genes encoded by mitochondrial and nuclear T. brucei DNA. Blue Native polyacrylamide gel electrophoresis of T. brucei mitochondrial membrane proteins followed by immunoblotting revealed the presence of a putative complex I with a molecular mass of 600 kDa, which contains a minimum of 11 polypeptides determined by second-dimensional Tricine-SDS/PAGE including the 51 kDa, PSST and TYKY subunits.     

Mitochondrial protein import

Most mitochondrial proteins are synthesized as precursor proteins on cytosolic polysomes and are subsequently imported into mitochondria. Many precursors carry amino-terminal presequences which contain information for their targeting to mitochondria. In several cases, targeting and sorting information is also contained in non-amino-terminal portions of the precursor protein. Nucleoside triphosphates are required to keep precursors in an import-competent (unfolded) conformation. The precursors bind to specific receptor proteins on the mitochondrial surface and interact with a general insertion protein (GIP) in the outer membrane. The initial interaction of the precursor with the inner membrane requires the mitochondrial membrane potential (delta psi) and occurs at contact sites between outer and inner membranes. Completion of translocation into the inner membrane or matrix is independent of delta psi. The presequences are cleaved off by the processing peptidase in the mitochondrial matrix. In several cases, a second proteolytic processing event is performed in either the matrix or in the intermembrane space. Other modifications can occur such as the addition of prosthetic groups (e.g., heme or Fe/S clusters). Some precursors of proteins of the intermembrane space or the outer surface of the inner membrane are retranslocated from the matrix space across the inner membrane to their functional destination ('conservative sorting'). Finally, many proteins are assembled in multi-subunit complexes. Exceptions to this general import pathway are known. Precursors of outer membrane proteins are transported directly into the outer membrane in a receptor-dependent manner. The precursor of cytochrome c is directly translocated across the outer membrane and thereby reaches the intermembrane space. In addition to the general sequence of events which occurs during mitochondrial protein import, current research focuses on the molecules themselves that are involved in these processes.     

An unusual mitochondrial import pathway for the precursor to yeast cytochrome c oxidase subunit Va

We have studied the import of the precursor to yeast cytochrome c oxidase subunit Va, a protein of the mitochondrial inner membrane. Like the majority of mitochondrial precursor proteins studied thus far, import of presubunit Va was dependent upon both a membrane potential (delta psi) and the hydrolysis of ATP. However, the levels of ATP necessary for the import of presubunit Va were significantly lower than those required for the import of a different mitochondrial precursor protein, the beta subunit of the F1-ATPase. The rate of import of presubunit Va was found to be unaffected by temperature over the range 0 to 30 degrees C, and was not facilitated by prior denaturation of the protein. These results, in conjunction with those of an earlier study demonstrating that presubunit Va could be efficiently targeted to mitochondria with minimal presequences, suggest that the subunit Va precursor normally exists in a loosely folded conformation. Presubunit Va could also be imported into mitochondria that had been pretreated with high concentrations of trypsin or proteinase K (1 mg/ml and 200 micrograms/ml, respectively). Furthermore, the rate of import into trypsin-treated mitochondria, at both 0 and 30 degrees C, was identical to that observed with the untreated organelles. Thus, import of presubunit Va is not dependent upon the function of a protease-sensitive surface receptor. When taken together, the results of this study suggest that presubunit Va follows an unusual import pathway. While this pathway uses several well-established translocation steps, in its entirety it is distinct from either the receptor-independent pathway used by apocytochrome c, or the more general pathway used by a majority of mitochondrial precursor proteins.