Targeting of pre-ornithine transcarbamylase to mitochondria: definition of critical regions and residues in the leader peptide

The cytoplasmically synthesized precursor of the mitochondrial matrix enzyme, ornithine transcarbamylase (OTC), is directed to mitochondria by its amino-terminal leader peptide. To define the critical residues and/or regions in the OTC leader peptide, we have synthesized OTC precursors with alterations in the leader portion. Analysis of deletions reveals that the middle portion of the 32 residue leader peptide is absolutely required for both mitochondrial uptake and proteolytic processing, whereas NH2-terminal and penultimate COOH-terminal portions are not. Analysis of precursors with single substitutions revealed complete loss of function when arginine 23 was substituted with glycine. Additional substitutions suggested that the critical role of this arginine residue may be mediated by participation in a local secondary structure, very likely an alpha-helix, which is proposed to be an essential element in the midportion of the leader peptide.     

The ornithine transcarbamylase leader peptide directs mitochondrial import through both its midportion structure and net positive charge

The cytoplasmically synthesized precursor of the mitochondrial matrix enzyme, ornithine transcarbamylase (OTC), is targeted to mitochondria by its NH2-terminal leader peptide. We previously established through mutational analysis that the midportion of the OTC leader peptide is functionally required. In this article, we report that study of additional OTC precursors, altered in either a site-directed or random manner, reveals that (a) the midportion, but not the NH2-terminal half, is sufficient by itself to direct import, (b) the functional structure in the midportion is unlikely to be an amphiphilic alpha-helix, (c) the four arginines in the leader peptide contribute collectively to import function by conferring net positive charge, and (d) surprisingly, proteolytic processing of the leader peptide does not require the presence of a specific primary structure at the site of cleavage, in order to produce the mature OTC subunit.     

Mechanisms of mitochondrial protein import.

1. Most proteins of cell organelles are synthesized as precursor proteins on cytosolic polysomes and are directed by signal sequences into the correct compartments. 2. In this review, the characteristics of mitochondrial protein uptake will be described, including the specific recognition, membrane translocation, proteolytic processing and folding of nuclear-encoded precursor proteins. 3. Recent studies indicate that a proteinaceous machinery located in the mitochondrial membranes and matrix performs these key steps of protein import.     

Import of rat ornithine transcarbamylase precursor into mitochondria: two-step processing of the leader peptide

The mitochondrial matrix enzyme ornithine transcarbamylase (OTC) is synthesized on cytoplasmic polyribosomes as a precursor (pOTC) with an NH2-terminal extension of 32 amino acids. We report here that rat pOTC synthesized in vitro is internalized and cleaved by isolated rat liver mitochondria in two, temporally separate steps. In the first step, which is dependent upon an intact mitochondrial membrane potential, pOTC is translocated into mitochondria and cleaved by a matrix protease to a product designated iOTC, intermediate in size between pOTC and mature OTC. This product is in a trypsin-protected mitochondrial location. The same intermediate-sized OTC is produced in vivo in frog oocytes injected with in vitro-synthesized pOTC. The proteolytic processing of pOTC to iOTC involves the removal of 24 amino acids from the NH2 terminus of the precursor and utilizes a cleavage site two residues away from a critical arginine residue at position 23. In a second cleavage step, also catalyzed by a matrix protease, iOTC is converted to mature OTC by removal of the remaining eight residues of leader sequence. To define the critical regions in the OTC leader peptide required for these events, we have synthesized OTC precursors with alterations in the leader. Substitution of either an acidic (aspartate) or a "helix-breaking" (glycine) amino acid residue for arginine 23 of the leader inhibits formation of both iOTC and OTC, without affecting translocation. These mutant precursors are cleaved at an otherwise cryptic cleavage site between residues 16 and 17 of the leader. Interestingly, this cleavage occurs at a site two residues away from an arginine at position 15. The data indicate that conversion of pOTC to mature OTC proceeds via the formation of a third discrete species: an intermediate-sized OTC. The data suggest further that, in the rat pOTC leader, the essential elements required for translocation differ from those necessary for correct cleavage to either iOTC or mature OTC.     

Molecular organization (topography) of cytochrome P-450(11)beta in mitochondrial membrane and phospholipid vesicles as studied by trypsinolysis

Cytochrome P-450(11)beta from adrenal cortex is an intrinsic membrane protein embedded in the inner mitochondrial membrane. Topography of the protein inside a phospholipid bilayer was examined using controlled proteolysis of purified cytochrome P-450(11)beta following its integration into artificial liposomes. Inclusion of the protein into phospholipid vesicles led to a marked stabilization of the cytochrome activity. Trypsin treatment of the liposome-integrated cytochrome resulted in the rapid disappearance of the native protein moiety (47 kDa), while a major 34 kDa peptide component was formed. This peptide core retained the heme moiety and part of the cytochrome steroid-11 beta hydroxylase activity. Very similar observations were obtained when inside-out vesicles prepared from isolated adrenocortical mitoplasts were examined with the same approach. It is thus suggested that adrenocortical cytochrome P-450(11)beta is embedded in the inner mitochondrial membrane as well as in artificial liposomes by a major hydrophobic domain associated with the heme moiety while a limited domain remains accessible on the matrix side of the membrane surface. The previous described phosphorylation of the cytochrome P-450(11)beta on a serine residue, by the cAMP-dependent protein kinase is suggested to occur in the protein domain oriented toward the membrane surface, the phosphorylation site being lost under mild proteolytic digestion of the membrane-integrated protein.     

Tight folding of a passenger protein can interfere with the targeting function of a mitochondrial presequence.

The first seven residues of the yeast cytochrome oxidase subunit IV presequence are insufficient to target attached mouse dihydrofolate reductase into isolated yeast mitochondria. However, the targeting function of this truncated presequence can be restored by presenting the fusion protein to isolated mitochondria either as nascent, unfolded chains, or as full-length chains whose dihydrofolate reductase moiety had been destabilized either by urea treatment or by point mutations. The targeting efficiency of a mitochondrial presequence can thus be strongly influenced by the conformation of the attached 'passenger protein'. These results also underscore the difficulty of defining a 'minimal' mitochondrial targeting signal.     

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.     

Molecular organization (topography) of cytochrome P-45011β in mitochondrial membrane and phospholipid vesicles as studied by trypsinolysis

Cytochrome P-450_11β from adrenal cortex is an intrinsic membrane protein embedded in the inner mitochondrial membrane. Topography of the protein inside a phospholipid bilayer was examined using controlled proteolysis of purified cytochrome P-450_11β following its integration into artificial liposomes. Inclusion of the protein into phospholipid vesicles led to a marked stabilization of the cytochrome activity. Trypsin treatment of the liposome-integrated cytochrome resulted in the rapid disappearance of the native protein moiety (47 kDa), while a major 34 kDa peptide component was formed. This peptide core retained the heme moiety and part of the cytochrome steroid-11β hydroxylase activity. Very similar observations were obtained when inside-out vesicles prepared from isolated adrenocortical mitoplasts were examined with the same approach. It is thus suggested that adrenocortical cytochrome P-450_11β is embedded in the inner mitochondrial membrane as well as in artificial liposomes by a major hydrophobic domain associated with the heme moiety while a limited domain remains accessible on the matrix side of the membrane surface. The previous described phosphorylation of the cytochrome P-450_11β on a serine residue, by the cAMP-dependent protein kinase is suggested to occur in the protein domain oriented toward the membrane surface, the phosphorylation site being lost under mild proteolytic digestion of the membrane-integrated protein.     

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.     

Amphiphilicity is essential for mitochondrial presequence function.

We have shown earlier that a mitochondrial presequence peptide can form an amphiphilic helix. However, the importance of amphiphilicity for mitochondrial presequence function became doubtful when an artificial presequence, designed to be non-amphiphilic, proved to be active as a mitochondrial import signal. We now show experimentally that this 'non-amphiphilic' presequence peptide is, in fact, highly amphiphilic as measured by its ability to insert into phospholipid monolayers and to disrupt phospholipid vesicles. This result, and similar tests on three additional artificial presequences (two functionally active and one inactive), revealed that all active presequences were amphiphilic whereas the inactive presequence was non-amphiphilic. One of the active presequence peptides was non-helical in solution and in the presence of detergent micelles. We conclude that amphiphilicity is necessary for mitochondrial presequence function whereas a helical structure may not be essential.     

Signal peptidases and signal peptide hydrolases

Signal peptidases, the endoproteases that remove the amino-terminal signal sequence from many secretory proteins, have been isolated from various sources. Seven signal peptidases have been purified, two fromE. coli, two from mammalian sources, and three from mitochondrial matrix. The mitochondrial enzymes are soluble and function as a heterogeneous dimer. The mammalian enzymes are isolated as a complex and share a common glycosylated subunit. The bacterial enzymes are isolated as monomers and show no sequence homology with each other or the mammalian enzymes. The membrane-bound enzymes seem to require a substrate containing a consensus sequence following the –3, –1 rule of von Heijne at the cleavage site; however, processing of the substrate is strongly influenced by the hydrophobic region of the signal peptide. The enzymes appear to recognize an unknown three-dimensional motif rather than a specific amino acid sequence around the cleavage site. The matrix mitochondrial enzymes are metallo-endopeptidases; however, the other signal peptidases may belong to a unique class of proteases as they are resistant to chelators and most protease inhibitors. There are no data concerning the substrate binding site of these enzymes. In vivo, the signal peptide is rapidly degraded. Three different enzymes inEscherichia coli that can degrade a signal peptidein vitro have been identified. The intact signal peptide is not accumulated in mutants lacking these enzymes, which suggests that these peptidases individually are not responsible for the degredation of an intact signal peptidein vivo. It is speculated that signal peptidases and signal peptide hydrolases are integral components of the secretory pathway and that inhibition of the terminal steps can block translocation.     

A leader peptide is sufficient to direct mitochondrial import of a chimeric protein.

Most mitochondrial proteins are encoded in the nucleus and synthesized in the cytoplasm as larger precursors containing NH2-terminal 'leader' peptides. To test whether a leader peptide is sufficient to direct mitochondrial import, we fused the cloned nucleotide sequence encoding the leader peptide of the mitochondrial matrix enzyme ornithine transcarbamylase (OTC) with the sequence encoding the cytosolic enzyme dihydrofolate reductase (DHFR). The fused sequence, joined with SV40 regulatory elements, was introduced along with a selectable marker into a mutant CHO cell line devoid of endogenous DHFR. In stable transformants, the predicted 26-K chimeric precursor protein and two additional proteins, 22 K and 20 K, were detected by immunoprecipitation with anti-DHFR antiserum. In the presence of rhodamine 6G, an inhibitor of mitochondrial import, only the chimeric precursor was detected. Immunofluorescent staining of stably transformed cells with anti-DHFR antiserum produced a pattern characteristic of mitochondrial localization of immunoreactive material. When the chimeric precursor was synthesized in a cell-free system and incubated post-translationally with isolated rat liver mitochondria, it was imported and converted to a major product of 20 K that associated with mitochondria and was resistant to proteolytic digestion by externally added trypsin. Thus, both in intact cells and in vitro, a leader sequence is sufficient to direct the post-translational import of a chimeric precursor protein by mitochondria.     

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.     

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.     

Replication of nuclear and mitochondrial DNA in X-ray-damaged cells: evidence for a nuclear-specific mechanism that down-regulates replication.

The mechanism by which X rays inhibit DNA replication has been investigated in three distinct populations of DNA molecules in human cells: (a) large chromosomal DNA, (b) a population of 50-100 10.3-kb nuclear episomal plasmids per cell, and (c) a population of about 500 16-kb cytoplasmic mitochondrial DNA molecules per cell. DNA replication was inhibited by X rays in nuclear chromosomal and plasmid DNA, but not in mitochondrial DNA. The mechanism by which ionizing radiation inhibits DNA replication must therefore be nuclear-specific and is unlikely to involve diffusible low-molecular-weight substances. Since mitochondrial DNA exists in the cell as independent 16-kb circular molecules and responds to radiation as would be expected for small targets, the implication for nuclear plasmids is that their replication is regulated by a large target. A current model for DNA replication involves the movement of DNA through replication centers made up of polymerases, helicases, and associated replication enzymes that are attached to a matrix. The difference in the response to X rays between mitochondrial DNA and nuclear plasmid DNA can be explained if nuclear plasmids are tightly associated with chromosomal DNA and attached to the matrix, and are coordinately replicated.     

Mitochondrial shaping cuts

A broad range of cellular processes are regulated by proteolytic events. Proteolysis has now also been established to control mitochondrial morphology which results from the balanced action of fusion and fission. Two out of three known core components of the mitochondrial fusion machinery are under proteolytic control. The GTPase Fzo1 in the outer membrane of mitochondria is degraded along two independent proteolytic pathways. One controls mitochondrial fusion in vegetatively growing cells, the other one acts upon mating factor-induced cell cycle arrest. Fusion also depends on proteolytic processing of the GTPase Mgm1 by the rhomboid protease Pcp1 in the inner membrane of mitochondria. Functional links of AAA proteases or other proteolytic components to mitochondrial dynamics are just emerging. This review summarises the current understanding of regulatory roles of proteolytic processes for mitochondrial plasticity.     

Mitochondrial shaping cuts

A broad range of cellular processes are regulated by proteolytic events. Proteolysis has now also been established to control mitochondrial morphology which results from the balanced action of fusion and fission. Two out of three known core components of the mitochondrial fusion machinery are under proteolytic control. The GTPase Fzo1 in the outer membrane of mitochondria is degraded along two independent proteolytic pathways. One controls mitochondrial fusion in vegetatively growing cells, the other one acts upon mating factor-induced cell cycle arrest. Fusion also depends on proteolytic processing of the GTPase Mgm1 by the rhomboid protease Pcp1 in the inner membrane of mitochondria. Functional links of AAA proteases or other proteolytic components to mitochondrial dynamics are just emerging. This review summarises the current understanding of regulatory roles of proteolytic processes for mitochondrial plasticity.     

Efficient priming and expansion of antigen-specific CD8+ T cells by a novel cell-based artificial APC

The ex vivo priming and expansion of human CTL by APC, such as autologous monocyte-derived dendritic cells (DC), has the potential for use in immunotherapy for infectious diseases and cancer. To overcome the difficulty of obtaining sufficient number of autologous DC from patients, we have developed cell-based artificial APC (aAPC), designated Med-APC. These aAPC rapidly activate and expand the corresponding Ag-specific CD8+ T cells when pulsed with CTL epitope peptide(s) as efficiently as mature DC (mDC). We have also shown that Med-APC possess an innate cellular machinery that is sufficient to support the processing of complete Ag into immunodominant peptides, which considerably extends the usefulness of this technology. In addition, we have developed a novel expression vector system that expresses ubiquitinated Ag, resulting in an enhanced APC function of this system. Genetically encoded Ag can be easily introduced into Med-APC by transfection with this vector. Med-APC transfected with ubiquitinated Ag can efficiently expand the corresponding Ag-specific CTL without exogenous peptides. Therefore, Med-APC may have important therapeutic implications for adoptive immunotherapy and can be used for the detection of Ag-specific CTL for immunomonitoring.     

Generation of rho0 cells utilizing a mitochondrially targeted restriction endonuclease and comparative analyses

Eukaryotic cells devoid of mitochondrial DNA (rho0 cells) were originally generated under artificial growth conditions utilizing ethidium bromide. The chemical is known to intercalate preferentially with the mitochondrial double-stranded DNA thereby interfering with enzymes of the replication machinery. Rho0 cell lines are highly valuable tools to study human mitochondrial disorders because they can be utilized in cytoplasmic transfer experiments. However, mutagenic effects of ethidium bromide onto the nuclear DNA cannot be excluded. To foreclose this mutagenic character during the development of rho0 cell lines, we developed an extremely mild, reliable and timesaving method to generate rho0 cell lines within 3-5 days based on an enzymatic approach. Utilizing the genes for the restriction endonuclease EcoRI and the fluorescent protein EGFP that were fused to a mitochondrial targeting sequence, we developed a CMV-driven expression vector that allowed the temporal expression of the resulting fusion enzyme in eukaryotic cells. Applied on the human cell line 143B.TK- the active protein localized to mitochondria and induced the complete destruction of endogenous mtDNA. Mouse and rat rho0 cell lines were also successfully created with this approach. Furthermore, the newly established 143B.TK- rho0 cell line was characterized in great detail thereby releasing interesting insights into the morphology and ultra structure of human rho0 mitochondria.     

Regulation of mitochondrial morphology through proteolytic cleavage of OPA1

The dynamin-like GTPase OPA1, a causal gene product of human dominant optic atrophy, functions in mitochondrial fusion and inner membrane remodeling. It has several splice variants and even a single variant is found as several processed forms, although their functional significance is unknown. In yeast, mitochondrial rhomboid protease regulates mitochondrial function and morphology through proteolytic cleavage of Mgm1, the yeast homolog of OPA1. We demonstrate that OPA1 variants are synthesized with a bipartite-type mitochondrial targeting sequence. During import, the matrix-targeting signal is removed and processed forms (L-isoforms) are anchored to the inner membrane in type I topology. L-isoforms undergo further processing in the matrix to produce S-isoforms. Knockdown of OPA1 induced mitochondrial fragmentation, whose network morphology was recovered by expression of L-isoform but not S-isoform, indicating that only L-isoform is fusion-competent. Dissipation of membrane potential, expression of m-AAA protease paraplegin, or induction of apoptosis stimulated this processing along with the mitochondrial fragmentation. Thus, mammalian mitochondrial function and morphology is regulated through processing of OPA1 in a DeltaPsi-dependent manner.