Reconstitution of mitochondrial protein transport with purified ornithine carbamoyltransferase precursor expressed in Escherichia coli

Ornithine carbamoyltransferase (OTC; subunit, 36,000 Da) is initially synthesized as a precursor (pOTC) with a transient NH2-terminal presequence of 32 amino acid residues and imported posttranslationally into the mitochondrial matrix. The rat pOTC was synthesized in Escherichia coli using an expression vector containing a thermoinducible lambda pL promoter. The recombinant pOTC represented 5-10% of the total bacterial protein and was present in the precipitate of the disrupted bacteria. The precipitate was washed and pOTC was extracted with 8 M urea or 0.1% cetyltrimethylammonium bromide. The extracted pOTC was essentially homogeneous, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified pOTC was cleaved to the intermediate-sized product of 37,000 Da by a processing protease partially purified from the matrix fraction of rat liver mitochondria. The purified recombinant pOTC, but not the mature form of OTC synthesized in E. coli and purified, competed with the in vitro-synthesized, radiolabeled pOTC for uptake and processing by the isolated rat liver mitochondria. The radiolabeled and purified recombinant pOTC could be imported into the isolated mitochondria and processed to the mature form in an energy- and rabbit reticulocyte lysate-dependent manner. When the purified pOTC was subjected to sucrose gradient centrifugation, it sedimented as a large aggregate of greater than 60 S in the absence of reticulocyte lysate, whereas it sedimented as a complex of about 5 S in the presence of the lysate. These observations together with our previous results indicate that a protein factor(s) present in the lysate interacts with pOTC and holds it in an import-competent form.     

Cleavage of precursors by the mitochondrial processing peptidase requires a compatible mature protein or an intermediate octapeptide

Many precursors of mitochondrial proteins are processed in two successive steps by independent matrix peptidases (MPP and MIP), whereas others are cleaved in a single step by MPP alone. To explain this dichotomy, we have constructed deletions of all or part of the octapeptide characteristic of a twice cleaved precursor (human ornithine transcarbamylase [pOTC]), have exchanged leader peptide sequences between once-cleaved (human methylmalonyl-CoA mutase [pMUT]; yeast F1ATPase beta-subunit [pF1 beta]) and twice-cleaved (pOTC; rat malate dehydrogenase (pMDH); Neurospora ubiquinol-cytochrome c reductase iron-sulfur subunit [pFe/S]) precursors, and have incubated these proteins with purified MPP and MIP. When the octapeptide of pOTC was deleted, or when the entire leader peptide of a once-cleaved precursor (pMUT or pF1 beta) was joined to the mature amino terminus of a twice-cleaved precursor (pOTC or pFe/S), no cleavage was produced by either protease. Cleavage of these constructs by MPP was restored by re-inserting as few as two amino-terminal residues of the octapeptide or of the mature amino terminus of a once-cleaved precursor. We conclude that the mature amino terminus of a twice-cleaved precursor is structurally incompatible with cleavage by MPP; such proteins have evolved octapeptides cleaved by MIP to overcome this incompatibility.     

Import of the malate dehydrogenase precursor by mitochondria. Cleavage within leader peptide by matrix protease leads to formation of intermediate-sized form

The mitochondrial matrix enzyme malate dehydrogenase (MDH) is synthesized on cytoplasmic polysomes as a larger precursor (pMDH) with an NH2-terminal leader peptide of 24 amino acids. Import of in vitro synthesized MDH into mitochondria results in formation of the mature-sized subunit. We report here that the conversion of pMDH to mMDH occurs via two distinct cleavage events within the leader peptide. First, pMDH is cleaved to an intermediate form (iMDH) of MDH. Conversion of the precursor to the intermediate form is catalyzed by a protease localized to the mitochondrial matrix. The cleavage of pMDH to iMDH involves the removal of 15 amino acids from the NH2 terminus of the pMDH leader peptide. The iMDH is subsequently cleaved, also by a matrix protease, to mature MDH in a reaction which is O-phenanthroline-sensitive. Cleavage to iMDH and to mature MDH occurs prior to completion of translocation of the MDH polypeptide chain into the mitochondrial matrix.     

Presequence does not prevent folding of a purified mitochondrial precursor protein and is essential for association with a reticulocyte cytosolic factor(s)

Ornithine carbamoyltransferase (OTC; subunit, 36,000 Da) [EC 2.1.3.3] is initially synthesized as a precursor (pOTC) with a transient NH2-terminal presequence of 32 amino acid residues, then is imported posttranslationally nto the mitochondrial matrix. We expressed rat pOTC in Escherichia coli, purified it in a denatured form, and showed that could be transported into isolated mitochondria in the presence of rabbit reticulocyte lysate [Murakami et al. (1988) J. Biol. Chem. 263, 18437-18442]. In order to compare the properties of the precursor and mature form of OTC, the rat mature OTC was synthesized in E. coli and purified. The recombinant OTC represented about 5% of the total bacterial protein and was present in both the supernatant and precipitate of the disrupted bacteria. The OTC, extracted from the precipitate with 8 M urea or 6 M guanidine.HCl, was essentially homogeneous, as judged by SDS-PAGE. When guanidine.HCl-denatured mature OTC was diluted and incubated at 0 degrees C for 40-60 h, it was reactivated to a specific activity of 170 mumol/min/mg protein at 37 degrees C (18% of that of the purified mature enzyme). Guanidine.HCl-denatured pOTC was activated to a specific activity of 125 mumol/min/mg protein under similar conditions. The native and reactivated OTC sedimented with an s20.w value of 6.2S, whereas the activated pOTC sedimented with an s20.w of 5.2S. The activated pOTC was more unstable than the reactivated OTC at 50 degrees C. These observations indicate that the presequence does not prevent pOTC from folding into an enzymatically active trimeric form, although the pOTC trimer appears to be less compact than the mature trimer.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-level expression of a mitochondrial enzyme, ornithine transcarbamylase from rat liver, in a baculovirus expression system

The mitochondrial enzyme, ornithine transcarbamylase (OTC) from rat liver was expressed in Spodoptera frugiperda (Sf) insect cells using a baculovirus vector. When insect cells were infected with recombinant Autographica californica nuclear polyhedrosis virus (AcNPV) containing a cDNA encoding the precursor form of OTC (pOTC) inserted into the polyhedrin gene, they expressed catalytically active enzyme at levels of approximately 2.5 micrograms/10(6) cells. About 25% of the active enzyme was a novel, partially processed product of pOTC containing four extra amino acids at the amino terminus of OTC. The most abundant protein found in mitochondria from infected insect cells was the normal processing intermediate iOTC, which contains 8 extra amino acids at the amino terminus of OTC. Whereas this species, present at 20 micrograms/10(6) cells, was not active and did not bind the transition-state analog inhibitor of OTC, delta-PALO, the novel processing product did bind and was affinity-purified, along with mature OTC, on a PALO-affinity column. The OTC expressed in insect cells was located in the same compartment of the mitochondrion as in rat liver. The incomplete processing occurred in vitro in both noninfected and infected insect cells. The high level of expression of iOTC using the baculoviral expression system provides a means of overproducing an obligatory intermediate in the mitochondrial import process.     

The mature size of rat 4-aminobutyrate aminotransferase is different in liver and brain.

The amino acid sequence predicted from a rat liver cDNA library indicated that the precursor of beta-AlaAT I (4-aminobutyrate aminotransferase, beta-alanine-oxoglutarate aminotransferase) consists of a mature enzyme of 466 amino acid residues and a 34-amino acid terminal segment, with amino acids attributed to the leader peptide. However, the mass of beta-AlaAT I from rat brain was larger than that from rat liver and kidney, as assessed by Western-blot analysis, mass spectroscopy and N-terminal sequencing. The mature form of beta-AlaAT I from the brain had an ISQAAAK- peptide on the N-terminus of the liver mature beta-AlaAT I. Brain beta-AlaAT I was cleaved to liver beta-AlaAT I when incubated with fresh mitochondrial extract from rat liver. These results imply that mature rat liver beta-AlaAT I is proteolytically cleaved in two steps. The first cleavage of the motif XRX( downward arrow)XS is performed by a mitochondrial processing peptidase, yielding an intermediate-sized protein which is the mature brain beta-AlaAT I. The second cleavage, which generates the mature liver beta-AlaAT I, is also carried out by a mitochondrial endopeptidase. The second peptidase is active in liver but lacking in brain.     

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.     

Presequence binding factor-dependent and -independent import of proteins into mitochondria.

A cytosolic protein factor(s) is involved in the import of precursor proteins into mitochondria. PBF (presequence binding factor) is a protein factor which binds to the precursor form (pOTC) of rat ornithine carbamoyltransferase (OTC) but not to the mature OTC, and is required for the mitochondrial import of pOTC. The precursors for aspartate aminotransferase and malate dehydrogenase as well as pOTC synthesized in a reticulocyte lysate were efficiently imported into the mitochondria. However, the precursors synthesized in the lysate depleted for PBF by treatment with pOTC-Sepharose were not imported. Readdition of the purified PBF to the depleted lysate fully restored the import. pOTC synthesized in the untreated lysate sedimented as a complex with a broad peak of around 9 S, whereas pOTC synthesized in the PBF-depleted lysate sedimented at an expected position of monomer (2.5 S). When the purified PBF was readded to the depleted lysate, pOTC sedimented as a complex of about 7 S. In contrast to most mitochondrial proteins, rat 3-oxoacyl-CoA thiolase is synthesized with no cleavable presequence and an NH2-terminal portion of the mature protein functions as a mitochondrial import signal. The thiolase synthesized in the PBF-depleted lysate could be efficiently imported into the mitochondria, and readdition of PBF had little effect on the import. The thiolase synthesized in the untreated, the PBF-depleted, or the PBF-readded lysate sedimented at an expected position of monomer (2.5 S). These observations provide support for the existence of PBF-dependent and -independent pathways of mitochondrial protein import.     

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.     

Expression of amplified DNA sequences for ornithine transcarbamylase in HeLa cells: arginine residues may be required for mitochondrial import of enzyme precursor

Expression of ornithine transcarbamylase (OTC), a nuclear-coded mitochondrial enzyme, was programmed in HeLa cells by the use of a strategy of gene co-amplification. HeLa cells, ordinarily devoid of OTC activity, were transfected with a plasmid containing viral regulatory elements joined with two cDNA sequences, one encoding the human OTC precursor and a second encoding a mutant mouse dihydrofolate reductase. After transfection and selection in increasing concentrations of methotrexate, several hundred copies per cell of the sequence encoding OTC were detected by blot analysis. Immunoprecipitation of extracts of radiolabeled cells with anti-OTC antiserum revealed newly synthesized mature OTC subunits. Furthermore, OTC enzymatic activity in cell extracts was comparable to that of control human liver, and mitochondrial localization of OTC was demonstrated by immunofluorescence. When we incubated transfected HeLa cells with dinitrophenol, a known inhibitor of mitochondrial import, the only form of newly synthesized OTC detected was the precursor. We estimated the rate of import of precursor by performing an inhibitor-free chase; precursor was converted to mature subunit with a half-life of less than two minutes. When a HeLa transformant was incubated with the arginine analogue canavanine, the major form of newly synthesized OTC detected was a species migrating slightly more slowly than the normal precursor; little mature-sized subunit was recovered. This indicates that substitution of the analogue for arginine in the OTC precursor interferes with mitochondrial import and processing. Thus, arginine residues in the OTC precursor--most likely the four residues contained in its NH2-terminal leader sequence--probably play an important role in mitochondrial import and/or processing.     

Mitochondrial import of rat pre-ornithine transcarbamylase: accurate processing of the precursor form is not required for uptake into mitochondria, nor assembly into catalytically active enzyme

Mitochondrial uptake of the cytoplasmically synthesized precursor of the mammalian enzyme ornithine transcarbamylase is mediated by an N-terminal leader sequence of 32 amino acids. In the mitochondrial matrix, the precursor form is processed to the mature subunit by proteolytic removal of this pre-sequence and in the enzyme from rat liver it has been suggested that this occurs in a two-step process which involves an intermediate cleavage at residue 24. We show that deletion of residues 20-26 spanning this intermediate cleavage site prevents correct processing to the mature subunit but it does not prevent mitochondrial targeting and internalization or assembly of the incorrectly processed product into a catalytically active enzyme. The incorrectly processed enzyme, which is larger than the normal mature enzyme, is nevertheless more susceptible to proteolytic degradation in permanently transfected human cells than the correctly processed enzyme.     

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 import and processing of mutant human ornithine transcarbamylase precursors in cultured cells.

We have investigated mitochondrial import and processing of the precursor for human ornithine transcarbamylase (OTC; carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) in HeLa cells stably transformed with cDNA sequences encoding OTC precursors carrying mutations in their leader peptides. The mutant precursors studied included two with amino acid substitutions in the 32-amino-acid leader peptide (glycine for arginine at position 23, designated gly23; glycines for arginines at positions 15, 23, and 26, designated gly15,23,26) and two with deletions (deletion of residues 8 to 22, designated d8-22; deletion of residues 17 to 32, designated N16). Specific immunoprecipitation with anti-OTC antiserum of extracts of L-[35S]methionine-labeled cells expressing these mutations yielded only precursor species; neither mature nor intermediate-size OTC subunits were observed. Fractionation of radiolabeled cells, however, revealed important differences among the various mutants: the gly23 precursor was associated with mitochondria and was not detected in the cytosol; the d8-22 and N16 precursors were found with both the mitochondrial fraction and the cytosol; only the gly15,23,26 precursor was detected exclusively in the cytosol. A large fraction of each of the mitochondrially associated OTC species was in a trypsin-protected compartment. In particular, the gly23 precursor behaved in trypsin protection and mitochondrial fractionation studies in a manner consistent with its translocation into the mitochondrial matrix. On the other hand, the lack of binding of the gly23 protein to a delta-N-phosphonoacetyl-L-ornithine affinity column, which specifically recognizes active OTC enzyme, indicated that, despite its intramitochondrial location, the mutant protein did not assemble into the normal, active trimer. Further, the gly23 mutant precursor was unstable within the mitochondria and was degraded with a t1/2 of less further than 4 h. Thus, we have shown that, in intact HeLa cells, cleavage of the OTC leader peptide is not required for translocation into mitochondria, but is required for assembly into active enzyme.     

Purified presequence binding factor (PBF) forms an import-competent complex with a purified mitochondrial precursor protein.

In vitro mitochondrial import of the purified precursor form (pOTC) of rat ornithine carbamoyltransferase (OTC) is stimulated by a cytosolic factor(s) contained in rabbit reticulocyte lysate. A protein factor that binds to pOTC but not to mature OTC and was named presequence binding factor or PBF, was purified 91,000-fold from the lysate by affinity chromatography using pOTC-bound Sepharose, DEAE-5PW HPLC and sucrose gradient centrifugation. The purified PBF migrated as a single polypeptide of 50,000 daltons on SDS-PAGE. On sucrose gradients, urea-denatured pOTC sedimented to the bottom, whereas PBF sedimented with an S20,w value of 5.5S. When pOTC and PBF were centrifuged together, both polypeptides sedimented as a complex of 7.1S. Formation of the pOTC-PBF complex was inhibited by micromolar concentrations of the synthetic presequence of pOTC and those of other mitochondrial precursor proteins. The purified PBF markedly stimulated the import of purified or in vitro synthesized pOTC into the mitochondria. PBF-stimulated pOTC import was further enhanced by a 70 kd heat shock protein (hsp 70) purified from yeast; the hsp70 alone had little effect. Thus, PBF binds to the presequence portion of the precursors and may hold them in a transport-competent form in cooperation with hsp70.     

Aberrant mitochondrial processing of chimaeric import precursors containing subunits 8 and 9 of yeast mitochondrial ATP synthase

A set of chimaeric precursors which contain the same leader sequences but different passenger proteins has been analyzed for the site of protease cleavage following import into yeast mitochondria. Each precursor comprises the leader of Neurospora crassa subunit 9 of mitochondrial ATP synthase fused to subunit 8 or 9 of the corresponding yeast enzyme. Precursors containing the first five residues of mature N. crassa subunit 9 interposed between the leader and the yeast passenger protein were cleaved at the natural site of the N. crassa subunit 9 precursor. Direct fusions without interposed sequences were cleaved at novel sites. Cleavage occurred between the 3rd and 4th residues of yeast subunit 8, but for yeast subunit 9, cleavage occurred within the leader, 8 residues upstream of the passenger protein.     

Processing of artificial peptide-DNA-conjugates by the mitochondrial intermediate peptidase (MIP).

Import of DNA from the cytoplasm into the mitochondrial matrix is an obligatory step for an in organello site-directed mutagenesis or gene therapy approach on mitochondrial DNA diseases. In this context, we have developed an artificial DNA translocation vector that is composed of the mitochondrial signal peptide of the ornithine transcarbamylase (OTC) and a DNA moiety. While this vector is capable of directing attached passenger molecules to the mitochondrial matrix, the recognition of this artificial molecule by the endogenous mitochondrial signal peptide processing machinery as well as the cleavage of the peptide plays a pivotal role in the release of the attached DNA. To study the proteolytic processing of the artificial vector, various signal peptide-DNA-conjugates were treated with purified mitochondrial intermediate peptidase. When the leader peptide is directly linked to the DNA moiety without an intervening spacer, MIP processing is prevented. Cleavage of the peptide can be restored, however, when the first ten amino acid residues of the mature part of OTC are appended at the carboxy-terminal end of the signal peptide. Our results show that artificial peptide-DNA-conjugates are recognized by the mitochondrial proteolytic machinery, and therefore an interference of the peptide with the DNA function can be excluded.     

A noncleavable signal for mitochondrial import of 3-oxoacyl-CoA thiolase

Rat 3-oxoacyl-CoA thiolase, an enzyme of the fatty acid beta-oxidation cycle, is located in the mitochondrial matrix. Unlike most mitochondrial matrix proteins, the thiolase is synthesized with no transient presequence and possesses information for mitochondrial targeting and import in the mature protein of 397 amino acid residues. cDNA sequences encoding various portions of the thiolase were fused in frame to the cDNA encoding the mature portion of rat ornithine transcarbamylase (lacking its own presequence). The fusion genes were transfected into COS cells, and subcellular localization of the fusion proteins was analyzed by cell fractionation with digitonin. When the mature portion of ornithine transcarbamylase was expressed, it was recovered in the soluble fraction. On the other hand, the fusion proteins containing the NH2-terminal 392, 161, or 61 amino acid residues of the thiolase were recovered in the particulate fraction, whereas the fusion protein containing the COOH-terminal 331 residues (residues 62-392) was recovered in the soluble fraction. Enzyme immunocytochemical and immunoelectron microscopic analyses using an anti-ornithine transcarbamylase antibody showed mitochondrial localization of the fusion proteins containing the NH2-terminal portions of the thiolase. These results indicate that the NH2-terminal 61 amino acids of rat 3-oxoacyl-CoA thiolase function as a noncleavable signal for mitochondrial targeting and import of this enzyme protein. Pulse-chase experiments showed that the ornithine transcarbamylase precursor and the thiolase traveled from the cytosol to the mitochondria with half-lives of less than 5 min, whereas the three fusion proteins traveled with half-lives of 10-15 min. Interestingly, in the cells expressing the fusion proteins, the mitochondria showed abnormal shapes and were filled with immunogold-positive crystalloid structures.     

Sequence of the precursor to rat ornithine aminotransferase deduced from a cDNA clone

The nucleotide sequence of ornithine aminotransferase mRNA from rat liver, including the entire coding and 3' untranslated regions, was determined from two overlapping cDNA clones. The mRNA encodes a precursor polypeptide of 439 amino acid residues with a molecular weight of 48,332. The deduced amino acid composition of the proposed mature enzyme sequence (residues 35 through 439) was in good agreement with that reported for the purified protein. The amino-terminal segment of the precursor corresponding to residues 1 through 34 has an overall positive charge, containing 6 basic residues and only a single acidic residue, and is postulated to be the mitochondrial leader sequence. The first 22 amino acid residues of the proposed leader sequences share 54% homology with the leader peptide of rat ornithine transcarbamylase precursor and more limited homology to the leader peptides of other nuclear-encoded mitochondrial matrix proteins. Homology was also observed between residues 286 through 362 ornithine aminotransferase precursor and a region containing the pyridoxyl phosphate binding domain of mitochondrial aspartate aminotransferase.