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

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

Two forms of DNA-dependent RNA polymerase alpha subunit in streptomycetes.

We demonstrated two different DNA-dependent RNA polymerase (RNAP) alpha subunits in spores of Streptomyces granaticolor with apparent molecular masses of 40 and 43 kDa. The 43-kDa subunit was also found in vegetative cells. These two proteins are highly similar to each other as well as to other bacterial RNAP alpha subunits. The 40-kDa subunit is shortened from its C-terminus, in the portion of the protein, required for binding of DNA and transcription regulators. The gene for RNAP alpha from S. granaticolor was cloned and sequenced and the corresponding protein was overproduced in Escherichia coli. In vitro experiments using purified RNAP alpha showed that the cell free extract from spores of S. granaticolor exhibits proteolytic activity responsible for the alpha subunit shortening, whereas that from vegetative cells does not. This modification of alpha subunit might point to a novel mechanism of transcriptional control in streptomycetes.

Complementation between mitochondrial processing peptidase (MPP) subunits from different species.

Mitochondrial processing peptidase (MPP), a dimer of nonidentical subunits, is the primary peptidase responsible for the removal of leader peptides from nuclearly encoded mitochondrial proteins. Alignments of the alpha and beta subunits of MPP (alpha- and beta-MPP) from different species show strong protein sequence similarity in certain regions, including a highly negatively charged region as well as a domain containing a putative metal ion binding site. In this report, we describe experiments in which we combine the subunits of MPP from yeast, rat, and Neurospora crassa, both in vivo and in vitro and mesure the resultant processing activity. For in vivo complementation, we used the temperature sensitive mif1 and mif2 yeast mutants, which lack MPP activity at the nonpermissive temperature (37 degrees C). We found that the defective alpha-MPP of mif2 cannot be substituted for by the alpha-MPP from rat or Neurospora. On the other hand, the beta-MPP from rat and Neurospora can fully substitute for the defective beta-MPP in the mif1 mutant. These results were confirmed in in vitro experiments in which individually expressed subunits were combined. Only combinations of the alpha-MPP from yeast with the beta-MPP from rat or Neurospora produced active MPP.

PCR method for generating multiple mutations at adjacent sites.

Procedures to introduce point mutations, restriction sites and insert or delete DNA fragments are very important tools to study protein function. We describe here two-step PCR-based method for generating single or multiple mutations, insertions and deletions in a small region of the sequence. In the first step, a unique restriction site is introduced near the part of DNA sequence to be changed, without changing the amino acid sequence. For this step, one of the methods already described can be used. In the second step, mutations are introduced using mutagenic primers containing the unique restriction site from the first step at the 5' end, paired with a universal primer crossing another unique restriction site present originally in the sequence. The method is very simple, economic and rapid. In comparison with the traditional in vitro mutagenesis methods, one can generate large numbers of mutated plasmids in hours.

Eukaryotic precursor proteins are processed by Escherichia coli outer membrane protein OmpP.

A new specific endopeptidase that cleaves eukaryotic precursor proteins has been found in Escherichia coli K but not in E. coli B strains. After purification, protein sequencing and Western blotting, the endopeptidase was shown to be identical with E. coli outer membrane protein OmpP [Kaufmann, A., Stierhof, Y.-D. & Henning, U. (1994) J. Bacteriol. 176, 359-367]. Further characterization of enzymatic properties of the new peptidase was performed. Comparison of the cleavage specificities of the newly found endopeptidase and that of rat mitochondrial processing peptidase (MPP) showed that patterns of proteolytic cleavage on the investigated precursor proteins by both enzymes are similar. By using three mitochondrial precursor proteins, the specificity assigned to OmpP previously, a cleavage position between two basic amino-acid residues, was extended to a three amino-acid recognition sequence. Positions +1 to +3 of this extended recognition site consist of an amino-acid residue with a small aliphatic side chain such as alanine or serine, a large hydrophobic residue such as leucine or valine followed by an arginine residue. Additionally, structural motifs of the substrate seem to be required for OmpP cleavage.

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

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

A cysteine endopeptidase with a C-terminal KDEL motif isolated from castor bean endosperm is a marker enzyme for the ricinosome, a putative lytic compartment.

A papain-type cysteine endopeptidase with a molecular mass of 35 kDa for the mature enzyme, was purified from germinating castor bean (Ricinus communis L.) endosperm by virtue of its capacity to process the glyoxysomal malate dehydrogenase precursor protein to the mature subunit in vitro (C. Gietl et al., 1997, Plant Physiol 113: 863-871). The cDNA clones from endosperm of germinating seedlings and from developing seeds were isolated and sequence analysis revealed that a very similar or identical peptidase is synthesised in both tissues. Sequencing established a presequence for co-translational targeting into the endoplasmic reticulum, an N-terminal propeptide and a C-terminal KDEL motif for the castor bean cysteine endopeptidase precursor. The 45-kDa pro-enzyme stably present in isolated organelles was enzymatically active. Immunocytochemistry with antibodies raised against the purified cysteine endopeptidase revealed highly specific labelling of ricinosomes, organelles which co-purify with glyoxysomes from germinating Ricinus endosperm. The cysteine endopeptidase from castor bean endosperm, which represents a senescing tissue, is homologous to cysteine endopeptidases from other senescing tissues such as the cotyledons of germinating mung bean (Vigna mungo) and vetch (Vicia sativa), the seed pods of maturing French bean (Phaseolus vulgaris) and the flowers of daylily (Hemerocallis sp.).

A cysteine endopeptidase isolated from castor bean endosperm microbodies processes the glyoxysomal malate dehydrogenase precursor protein.

A plant cysteine endopeptidase with a molecular mass of 35 kD was purified from microbodies of germinating castor bean (Ricinus communis) endosperm by virtue of its capacity to specifically process the glyoxysomal malate dehydrogenase precursor protein to the mature subunit in vitro. Processing of the glyoxysomal malate dehydrogenase precursor occurs sequentially in three steps, the first intermediate resulting from cleavage after arginine-13 within the presequence and the second from cleavage after arginine-33. The endopeptidase is unable to remove the presequences of prethiolases from rape (Brassica napus) glyoxysomes and rat peroxisomes at the expected cleavage site. Protein sequence analysis of N-terminal and internal peptides revealed high identity to the mature papain-type cysteine endopeptidases from cotyledons of germinating mung bean (Vigna mungo) and French bean (Phaseolus vulgaris) seeds. These endopeptidases are synthesized with an extended pre-/prosequence at the N terminus and have been considered to be processed in the endoplasmic reticulum and targeted to protein-storing vacuoles.

Mutational analysis of both subunits from rat mitochondrial processing peptidase.

Rat liver mitochondrial processing peptidase (MPP) is the primary peptidase that cleaves leader peptides from nuclearly encoded mitochondrial proteins following their transport from the cytosol to the mitochondrial matrix. This enzyme consists of two nonidentical subunits that have overall similarity to each other and share certain amino acid motifs. These include the putative metal-ion binding HFLEH motif in the beta-subunit and the HFLEK motif of the alpha-subunit, as well as a possibly helical amino acid stretch bearing a high concentration of negatively charged residues about 70 amino acids downstream of these motifs in both subunits. In order to achieve a better understanding of the role of certain amino acids in rat MPP, we performed site-directed mutagenesis on both of its subunits. Our results show that whereas both histidines and the glutamate of the HFLEH motif in the beta-subunit are crucial for MPP function, this holds true only for the glutamate in the related HFLEK motif in the alpha-subunit. In addition, functionally important negatively charged residues in the region 70 amino acids downstream occur only in the beta-subunit and not in the alpha-subunit. This indicates a functional asymmetry between the subunits, with the beta-subunit containing a majority of residues participating in the active center.

Studies on protein processing for membrane-bound spinach leaf mitochondrial processing peptidase integrated into the cytochrome bc1 complex and the soluble rat liver matrix mitochondrial processing peptidase.

The plant mitochondrial processing peptidase (MPP) that catalyses the cleavage of the presequences from precursor proteins during or after protein import is a membrane-bound enzyme that constitutes an integral part of the bc1 complex of the respiratory chain. In contrast, MPP from mammals is soluble in the matrix space and does not form part of the respiratory chain. In the present study, we have compared the substrate specificity of the isolated spinach leaf bc1/MPP with rat liver MPP using synthetic signal peptides and different mitochondrial precursor proteins. Inhibition studies of processing with synthetic peptides showed a similar inhibition pattern for plant and rat MPP activity. A peptide derived from the presequence of rat liver mitochondrial aldehyde dehydrogenase (ALDH) was a potent inhibitor of the spinach and rat MPP. Two nonprocessed signal peptides, rhodanese and linker-deleted ALDH (a form of ALDH that lacks the RGP linker connecting two helices in the presequence) had lower inhibitory effects towards each protease. The signal peptide from thiolase, another nonprocessed protein, had little inhibitory effect on MPP. Peptides derived from presequence of the plant Nicotiana plumbaginifolia F1 beta also showed a similar inhibitory pattern with rat MPP as with spinach MPP processing. In-vitro synthesised precursors of plant N. plumbaginifolia F1 beta and rat liver ALDH were cleaved to mature form by both spinach and rat MPP. However, the efficiency of processing was higher with the homologous precursor. Linker-deleted ALDH, rhodanese, and thiolase were not processed by the mammalian or plant MPP. However, both forms of MPP cleaved a mutated form of rhodanese that possesses a typical MPP cleavage motif, RXY S. Addition of the same cleavage motif to thiolase did not result in processing by either MPP. These results show that similar higher-order structural elements upstream from the cleavage site are important for processing by both the membrane-bound plant and the soluble mammalian MPP.

Folding, flavinylation, and mitochondrial import of 6-hydroxy-D-nicotine oxidase fused to the presequence of rat dimethylglycine dehydrogenase.

We analyzed the folding, covalent flavinylation, and mitochondrial import of the rabbit reticulocyte lysate-translated bacterial 6-hydroxy-D-nicotine oxidase (6-HDNO) fused to the mitochondrial targeting sequence of rat liver dimethylglycine dehydrogenase. Translation of 6-HDNO in FAD-supplemented reticulocyte lysate resulted in a protein that contained covalently incorporated FAD, exhibited enzyme activity, and was trypsin-resistant, a characteristic of the tight conformation of the holoenzyme. The attached mitochondrial presequence did not prevent folding, binding of FAD, or enzyme activity of the 6-HDNO moiety of the fusion protein (pre-6-HDNO). Pre-6-HDNO was imported into rat liver mitochondria and processed by the mitochondrial processing peptidase. Incubation of the trypsin-resistant pre-holo-6-HDNO protein with deenergized rat liver mitochondria demonstrated that upon contact with mitochondria, the protein was unfolded and became trypsin sensitive. Mitochondrial import assays showed that the unfolded pre-holo-6-HDNO with covalently attached FAD was imported into rat liver mitochondria. Inside the mitochondrion the holo-6-HDNO was refolded into the trypsin-resistant conformation. However, when pre-apo-6-HDNO was imported only part of the protein became trypsin resistant (approximately 20%). Addition of FAD and the allosteric effector glycerol 3-phosphate to apo-6-HDNO containing mitochondrial matrix was required to transform the protein into the trypsin-resistant conformation characteristic of holo-6-HDNO.

In vitro characterization of the mitochondrial processing and the potential function of the 68-kDa subunit of renal glutaminase.

Rat renal mitochondrial glutaminase (GA) is initially synthesized in primary cultures of proximal tubule cells as a 74-kDa precursor and is processed via a 72-kDa intermediate to generate a heterotetrameric enzyme which contains three 66-kDa subunits and one 68-kDa subunit (Perera, S. Y., Chen, T. C., and Curthoys, N. P. (1990) J. Biol. Chem. 265, 17764-17770). The two mature subunits may be derived by either of two possible mechanisms: 1) alternative proteolytic processing or 2) initial synthesis of the 66-kDa subunit followed by its covalent modification to generate the 68-kDa subunit. An in vitro system was utilized to further characterize this unique processing pathway and to investigate the potential function of the 68-kDa subunit. In vitro transcription and translation of the GA cDNA yields a single 74-kDa precursor. Upon incubation with isolated rat liver mitochondria, the precursor is translocated into the mitochondria and processed via a 72-kDa intermediate to yield a 3:1 ratio of the 66- and 68-kDa subunits, respectively. The kinetics of the in vitro processing reaction also closely approximate the kinetics observed in cultured cells. Mitochondrial processing is blocked by o-phenanthroline, an inhibitor of the matrix processing peptidase (MPP). The 72-amino acid presequence of the 66-kDa subunit contains a large proportion of basic amino acids. Two-dimensional gel electrophoresis of mature GA established that the 68-kDa subunit is slightly more basic than the 66-kDa subunit. In addition, incubation of the 74-kDa precursor with purified MPP yields equimolar amounts of the two mature peptides. A cDNA construct, p delta GA, was created which lacks the nucleotides that encode the amino acid residues 32 through 72 of GA. When transcribed and translated in vitro, p delta GA yields a 70-kDa precursor. This precursor is processed by mitochondria to a single mature subunit with a M of 66 kDa. This observation suggests that the 68-kDa subunit is not produced by covalent modification of the 66-kDa subunit and further supports the conclusion that the two mature subunits of GA are produced by alternative processing reactions which can be catalyzed by MPP. However, the yield of products obtained in intact mitochondria may be determined by some unidentified accessory factor. Submitochondrial fractionation of imported GA and delta GA precursors suggest that the 68-kDa subunit may function to retain the mature GA within the mitochondrial matrix.

Role of the N-terminal 118 amino acids in the processing of the rat renal mitochondrial glutaminase precursor.

Rat renal mitochondrial glutaminase (GA) is synthesized as a 74-kDa cytosolic precursor that is translocated into mitochondria and processed via a 72-kDa intermediate to yield a 3:1 ratio of mature 66- and 68-kDa subunits, respectively. The 66-kDa subunit is derived by removal of a 72-amino-acid presequence. The structural determinants necessary for translocation and proteolytic processing were further delineated by characterizing the processing of different chimeric constructs formed by fusing various segments of the N-terminal sequence of the GA precursor to chloramphenicol acetyl transferase (CAT). GA1-118 CAT is translocated and processed in isolated rat liver mitochondria or cleaved by purified mitochondrial processing peptidase (MPP) to yield an intermediate peptide and two mature subunits that are analogous to the products of processing of the GA precursor. The two reactions also occur with kinetics which are similar to those observed for processing of the GA precursor. Thus, all of the information required for the translocation and synthesis of the mature subunits of GA reside in the N-terminal 118 amino acids of the GA precursor. In contrast, GA1-72 CAT, a construct that contains the GA presequence fused to CAT, is apparently translocated and processed less efficiently. It yields only two peptides that are analogous to the intermediate and 68 kDa forms of GA. In addition, GA1-31 CAT associates with mitochondria but is not proteolytically processed and GA1-31,73-118 CAT is slowly translocated and processed to a single peptide that is analogous to the 66 kDa form of GA. The latter results suggest that the MPP cleavage reactions which yield the GA intermediate and the 66-kDa subunit depend primarily on information that is present C-terminal to the respective sites of cleavage.

Rat liver mitochondrial processing peptidase. Both alpha- and beta-subunits are required for activity.

Most nuclearly encoded mitochondrial proteins are synthesized with an amino-terminal leader peptide that is cleaved by the mitochondrial processing peptidase (MPP). Purified rat liver MPP, like the Neurospora and yeast enzymes, consists of two nonidentical subunits, alpha (55 kDa) and beta (50 kDa). To confirm the functional authenticity of the recently cloned and sequenced cDNAs for the alpha- and beta-MPP subunits from rat liver and to study each subunit's participation in MPP activity, we have subcloned and expressed separately in Escherichia coli the mature sequence of each subunit as a fusion protein with the maltose-binding protein. After induction, about 80% of each expressed fusion protein was insoluble in aggregates or inclusion bodies, and 20% remained soluble in the supernatant. The fusion proteins in the soluble fraction were purified by affinity chromatography and treated with factor Xa, and the MPP subunits were purified to homogeneity. When mixed together, these subunits showed no activity, suggesting that they might be misfolded. Therefore, a reconstitution protocol was developed which consisted of denaturation in urea, dithiothreitol, and 2-mercaptoethanol, followed by renaturation by dilution and dialysis under reducing conditions. With this procedure, active MPP was recovered from the mixed subunits, and it could be demonstrated that both alpha- and beta-MPP subunits were necessary for activity. Reconstituted recombinant MPP resembled the native rat liver enzyme as judged by its molecular weight, its inhibition by EDTA, and its ability to process a variety of mitochondrial precursor proteins appropriately to either an intermediate or a mature form.

The beta subunit of the mitochondrial processing peptidase from rat liver: cloning and sequencing of a cDNA and comparison with a proposed family of metallopeptidases.

Most nuclearly encoded mitochondrial proteins are synthesized with amino-terminal leader peptides that are removed by the mitochondrial processing peptidase (MPP) after translocation. Earlier we reported cloning and sequencing of a cDNA for the larger subunit (MPP alpha subunit) of this enzyme from rat liver mitochondria. We have now completed the cloning and sequencing of a cDNA encoding the smaller subunit of the enzyme (MPP beta subunit) from the same source. The cDNA consists of 1570 bp: 17 bp of 5'-untranslated sequence, 1467 bp of coding sequence, and 86 bp of 3'-untranslated sequence. The predicted protein consists of 489 amino acid residues, including a 45-amino acid leader peptide at the amino terminus and a 444-amino acid mature protein. The amino acid sequences of four tryptic peptides derived from purified MPP beta subunit precisely match those predicted by the cDNA sequence, as does the predicted mature amino terminus. The amino-terminal sequence is typical of a mitochondrial leader peptide, with eight positively charged arginine residues and a single negatively charged aspartate residue. When the amino acid sequence of rat MPP beta subunit is compared with sequences in the protein data bases, significant homology is found with the protease-enhancing protein of Neurospora crassa, the smaller subunit of MPP from Saccharomyces cerevisiae, and the core I protein of bovine ubiquinol:cytochrome c reductase. Lower homology is found with other members of a recently proposed class of endoproteases, which includes human insulinase and protease III from Escherichia coli.

GroEL, GroES, and ATP-dependent folding and spontaneous assembly of ornithine transcarbamylase.

When purified rat liver ornithine transcarbamylase (OTC), a trimer of 36 kDa subunits, was denatured in 6 M guanidine hydrochloride and then diluted 50-100-fold, no activity was recovered, and the OTC subunits aggregated. In contrast, when the chaperonin groEL was included in the dilution buffer, OTC did not aggregate but instead comigrated in a sucrose density gradient with the groEL oligomer, indicating that a complex had been formed. Upon addition of the cochaperonin groES and ATP to the isolated OTC-groEL complex, OTC monomers were folded, released, and assembled into active trimer. Neither groES nor ATP alone was sufficient to release active OTC from groEL. The extent of recovery of activity was proportional to the concentration of the complex, reaching approximately 80-90% at monomer concentrations above 0.6 microM. At low complex concentrations, kinetic studies revealed an initial lag in the reconstitution reaction, suggesting that assembly is the rate-limiting step under these conditions. We could trap folded, released, inactive OTC monomers at early times that assembled into active trimers with longer incubation. A nonhydrolyzable ATP analog could release bound OTC from groEL in the presence of groES, but the OTC monomers were not competent for assembly. These data show that recovery of OTC activity in vitro can be efficiently directed by the bacterial chaperonins in the presence of ATP and suggest that the mechanism of reconstitution involves ATP and groES-dependent folding and release of OTC monomers from groEL, followed by spontaneous assembly of trimers.

Sequence analysis of rat mitochondrial intermediate peptidase: similarity to zinc metallopeptidases and to a putative yeast homologue.

Proteolytic removal of amino-terminal octapeptides from mitochondrial intermediate proteins is a required step for a subgroup of nuclear-encoded mitochondrial precursors and is specifically catalyzed by mitochondrial intermediate peptidase (MIP). We recently reported the purification of MIP from rat liver and showed that the enzyme is a monomer of 75 kDa. We now report the sequence of a full-length rat MIP cDNA. This cDNA codes for a protein of 710 amino acids, including an amino-terminal mitochondrial leader peptide of 33 residues. The region surrounding the mature MIP amino terminus shows a cleavage site typically recognized by the general mitochondrial processing peptidase (MPP). In vitro synthesized MIP precursor is cleaved to mature MIP by purified MPP, and thus MIP is not required for its own proteolytic maturation. Comparison of the deduced MIP sequence with other sequences in the GenBank data base reveals two important similarities. The first is to a sequence encoding a putative MIP homologue in the recently reported sequence of yeast chromosome III. The putative yeast protein is predicted to be 712 amino acids long and includes a putative 23-residue mitochondrial leader peptide also with a MPP processing site. It shows 47% similarity and 24% identity to rat MIP. The second similarity is to members of a subfamily of metallopeptidases that includes rat metalloendopeptidase EC 3.4.24.15 and two bacterial proteases, oligopeptidase A and dipeptidyl carboxypeptidase. A region of greater than 50% similarity over 400 residues between MIP and these proteins is centered around the sequence motif HEXXH, typical of zinc metallopeptidases.

Rat liver mitochondrial intermediate peptidase (MIP): purification and initial characterization.

A number of nuclearly encoded mitochondrial protein precursors that are transported into the matrix and inner membrane are cleaved in two sequential steps by two distinct matrix peptidases, mitochondrial processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP). We have isolated and purified MIP from rat liver mitochondrial matrix. The enzyme, purified 2250-fold, is a monomer of 75 kDa and cleaves all tested mitochondrial intermediate proteins to their mature forms. About 20% of the final MIP preparation consists of equimolar amounts of two peptides of 47 kDa and 28 kDa, which are apparently the products of a single cleavage of the 75 kDa protein. These peptides are not separable from the 75 kDa protein, nor from each other, under any conditions used in the purification. The peptidase has a broad pH optimum between pH 6.6 and 8.9 and is inactivated by N-ethylmaleimide (NEM) and other sulfhydryl group reagents. The processing activity is divalent cation-dependent; it is stimulated by manganese, magnesium or calcium ions and reversibly inhibited by EDTA. Zinc, cobalt and iron strongly inhibit MIP activity. This pattern of cation dependence and inhibition is not clearly consistent with that of any known family of proteases.