©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Conversion of a Nonprocessed Mitochondrial Precursor Protein into One That Is Processed by the Mitochondrial Processing Peptidase (*)

(Received for publication, March 27, 1995; and in revised form, August 29, 1995)

Mary Waltner Henry Weiner (§)

From the Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Mitochondrial processing peptidase (MPP) cleaves the signal sequence from a variety of mitochondrial precursor proteins. A subset of mitochondrial proteins, including rhodanese and 3-oxoacyl-CoA thiolase, are imported into the matrix space, yet are not processed. Rhodanese signal peptide and translated protein were recognized by MPP, as both were inhibitors of processing. The signal peptide of precursor aldehyde dehydrogenase consists of a helix-linker-helix motif but when the RGP linker is removed, processing no longer occurs (Thornton, K., Wang, Y., Weiner, H., and Gorenstein, D. G.(1993) J. Biol. Chem. 268, 19906-19914). Disruption of the helical signal sequence of rhodanese by the addition of the RGP linker did not allow cleavage to occur. However, addition of a putative cleavage site allowed the protein to be processed. The same cleavage site was added to 3-oxoacyl-CoA thiolase, but this protein was still not processed. Thiolase and linker-deleted aldehyde dehydrogenase signal peptides were poor inhibitors of MPP. It can be concluded that both a processing site and the structure surrounding this site are important for MPP recognition.


INTRODUCTION

The majority of mitochondrial proteins are synthesized on cytosolic polyribosomes as precursor proteins with a signal sequence that targets these proteins to the mitochondria. Import into the matrix is usually accompanied by the removal of the signal sequence from the precursor protein in one or two steps (Attardi and Schatz, 1988; Hartl et al., 1989). A general processing peptidase called mitochondrial processing peptidase (MPP) (^1)catalyzes the removal of most signal sequences (Ou et al., 1989). For precursors that are cleaved in two steps, the initial part of the signal sequence is removed by MPP, leaving an octapeptide that is cleaved from the precursor by the mitochondrial intermediate peptidase (Kalousek et al., 1992).

MPP, which has been purified from Neurospora crassa (Hawlitschek et al., 1988), yeast (Yang et al., 1988), and rat liver (Ou et al., 1989), is a metalloprotease consisting of two nonidentical subunits termed alpha-MPP and beta-MPP (Kleiber et al., 1990; Paces et al., 1993). This processing enzyme belongs to a new class of endoproteases since it does not appear to fit into any of the known classes of proteases. Recently, attention has been given to the processing activity in plants. In contrast to mammals and fungi, MPP processing activity is integrated into the cytochrome c reductase complex of the respiratory chain. This suggests that, in plants, this complex functions both in respiration and protein processing (Glaser et al., 1994).

Although the processing enzyme does not recognize a specific amino acid sequence, a positional bias of amino acids has been identified. Most notably, arginine tends to exist at position -2, -3, -10, or -11 relative to the cleavage site (Gavel and von Heijne, 1990). Based on this observation, four cleavage motifs were suggested and are listed in Table 1. A further examination of a set of mitochondrial signal sequences revealed a preference for a large non-hydrophobic residue at position -2; a slight preference for hydrophobic residues at positions +1, -1, and -5; and an additional slight preference for large residues at position -5 (Schneider et al., 1995). Despite the identification of different cleavage motifs and positional preferences for various residues, there are signal sequence cleavage sites that do not have these characteristics. Thus, since there is no obvious cleavage motif for all processed proteins, it is believed that MPP also recognizes some unidentified higher-order structure.



Until recently, the only structural information about a mitochondrial signal peptide was its theoretical ability to form an amphipathic alpha-helical structure. With the use of two-dimensional NMR, a more detailed structural evaluation of several signal peptides has been undertaken. The structure of four signal peptides from proteins in which the signal sequence is processed has been determined. NMR data indicates that the rat mitochondrial aldehyde dehydrogenase (ALDH) signal peptide forms a helix-linker-helix (Karslake et al., 1990); the signal peptide of yeast cytochrome oxidase subunit IV (COX IV) forms a helix followed by a random coil (Endo et al., 1989); F(1)-ATPase beta-subunit signal peptide forms a helix followed by a less stable helix (Bruch and Hoyt, 1992), and the signal peptide from malate dehydrogenase forms a flexible helix (MacLachlan et al., 1994). It appears that each of these signal sequences has a helix with some degree of flexibility before the MPP cleavage site.

A group of mitochondrial matrix proteins that do not possess a cleavable signal sequence exists. Each of these proteins has a signal necessary for import into the mitochondria, yet lack an element(s) essential for processing. Such proteins include rhodanese (Miller et al., 1991), 3-oxoacyl-CoA thiolase (thiolase) (Arakawa et al., 1987), isopropyl malate synthase (Beltzer et al., 1988), chaperonin 10 (Ryan et al., 1994), and the beta-subunit of the human electron-transfer flavoprotein (Finocchiaro et al., 1993). Recently, the structure of four of the nonprocessed signal peptides has been determined by NMR. It appears that rhodanese and thiolase signal peptides are similar in that each forms a long, continuous helix through the initial 14 or 21 residues, respectively (Hammen et al., 1994). Additionally, if the linker from the ALDH signal peptide is deleted, this protein is imported into mitochondria, yet is no longer processed. The structure of this linker-deleted signal peptide has also been solved and has been shown to form a long, continuous helix (Thornton et al., 1993). The signal peptide from the mitochondrial chaperonin 10 is different because its signal peptide forms a helix-turn-helix (Jarvis et al., 1995). Before the structure of the chaperonin 10 signal peptide was solved, it was suggested that MPP recognizes a flexible signal peptide since the nonprocessed signal peptides from rhodanese, thiolase, and linker-deleted ALDH form a continuous helix (Hammen et al., 1994).

Although a flexible signal peptide may be an important recognition factor for MPP, we show here that the lack of processing of certain proteins may be more complex and other factors may be necessary for recognition by MPP. In this paper, we demonstrate that certain nonprocessed proteins lack a processing site, while other nonprocessed proteins weakly interact with MPP.


EXPERIMENTAL PROCEDURES

Deletion of Residues 23-55 within Rhodanese

cDNA for rhodanese, a gift from Dr. Paul Dooley (Southwest Foundation for Biomedical Research), was amplified using polymerase chain reaction (PCR), with the addition of a 5` NdeI site and the sequence encoding the initial Met and Val. A BamHI site was added after the stop codon at the 3` end of this gene. After NdeI and BamHI digestion, rhodanese was cloned into the plasmid pT7-7. Residues 23-55 were deleted using PCR. Briefly, cDNA encoding residues 56 through the stop codon of rhodanese was amplified with flanking BamHI sites. The cDNA encoding the initial 22 residues of rhodanese and pT7-7 was also amplified with flanking BamHI sites. The two different PCR products were subsequently digested with BamHI and ligated. The orientation of the DNA insert was confirmed with PstI digestion and sequencing.

Addition of an MPP Site to Rhodanese and Thiolase

cDNA for thiolase, a gift from Dr. Masataka Mori (Kumamoto University School of Medicine), was initially cloned into the plasmid pT7-7 between the NdeI and SalI sites. Subsequently, cDNA for both rhodanese and thiolase was cloned into pBluescript SK+/-, with rhodanese being flanked by BamHI sites and thiolase between the XbaI and HindIII sites. Addition of an MPP site was carried out using oligonucleotide-directed mutagenesis (Bio-Rad kit). The oligonucleotide used to create this site in rhodanese was 5`-AGGCTTGGCCCCACGCTGTAGGACCGAATGGATTCCGCCA-3` and the oligonucleotide used to create this site in thiolase was 5`GGCAGTGAAGTCCTTGAGAAGACCGGAATAAGCTCTAAAGGGTGTTCGCTTCGCAGCAAC-3` (underlined bases indicate the site of mutation). Residues RGP were added to rhodanese and rhodanese with an MPP site using PCR. The oligonucleotide used to add these three residues was 5`-TTTCATATGGTGCATCAGGTGCTCTACCGAGCGCTGGTCTCCAGAGGTCCAACCAAGTGGCTGGCG-3` with the codons corresponding to these amino acids underlined. All mutations were confirmed using sequencing. Mutant cDNA was subsequently cloned into plasmid pT7-7 between the NdeI and BamHI sites for in vitro transcription.

Transcription and Translation

In vitro transcription was performed using Ampliscribe T7 transcription kit (Epicentre Technologies). mRNAs were translated in rabbit reticulocyte lysate with [S]methionine (Amersham) as a labeled amino acid (Pelham and Jackson, 1976).

In Vitro Import of Precursor Proteins into Isolated Mitochondria

Rat liver mitochondrial isolation and in vitro import were performed as described previously (Pak and Weiner, 1990; Wang et al., 1989). Briefly, 14 µl of translated reticulocyte lysate was incubated with 25 µl of isolated mitochondria (7 mg of protein/ml) in import buffer (Pak and Weiner, 1990) with a final volume of 100 µl at 30 °C for 30 min. Half the reaction mixture was treated with 4 µl of proteinase K (2 mg/ml) (Sigma) at 0 °C for 15 min to digest protein which was not imported. Mitochondria were reisolated using centrifugation and treated with phenylmethylsulfonyl fluoride (2 µl of a 200 mM solution) to stop the proteinase K reaction. Additionally, samples of reisolated mitochondria were disrupted with 1% Triton X-100 and imported protein was digested with proteinase K. Subsequently, samples were subjected to SDS-polyacrylamide gel electrophoresis and autoradiography. Import was quantitated using densitometry (LKB Ultrascan XL Laser densitometer interfaced with an IBM personal computer).

In Vitro Processing Peptidase Activity

A solution containing both MPP (1 mg/ml) and mitochondrial intermediate peptidase (0.6 mg/ml) was a gift from Dr. Frantisek Kalousek (Yale University School of Medicine). A standard assay consisted of 2 µl of reticulocyte lysate, 1 µl of the MPP/mitochondrial intermediate peptidase solution, 10 mM Hepes-KOH, 1 mM dithiothreitol, and 0.1 mM MnCl(2) (with a final volume of 10 µl) and was incubated for various times at 27 °C. The reactions were terminated by the addition of an equal volume of SDS treatment buffer. For the inhibition of processing, various amounts of different peptides dissolved in processing peptidase assay buffer (above) or different volumes of rabbit reticulocyte lysate were added to the processing assay. Incubations were performed for 1 h under standard assay conditions. Samples were subjected to SDS-PAGE and autoradiography. Protein amounts were quantitated using densitometry or scintillation counting.

Synthesis of Peptides

All signal peptides were synthesized by the Macromolecular Structure Laboratory at Purdue University and were purified by a semipreparative C18 reverse-phase high performance liquid chromatography column (20 cm times 1 cm diameter). Peptides were eluted by an acetonitrile gradient. The purity of the peptides was verified by high performance liquid chromatography and mass spectroscopy. Glucagon was purchased from Sigma and purity was assessed using mass spectroscopy.

Trypsin Digestion of Translated Proteins

The rate of folding of the nonprocessed proteins was examined as described previously (Mattingly et al., 1993). Briefly, the translation of protein was allowed to proceed at 30 °C for either 30 or 60 min. The reaction was terminated by placing the translation product on ice and by adding cycloheximide to a final concentration of 50 µM. Subsequently, a 2.5-µl aliquot of translation product was diluted into 22.5 µl of trypsin digestion buffer (20 mM HEPPS, 150 mM NaCl, 0.1 mM EDTA, pH 8.3). The remaining translation reaction was incubated at 15 °C for an additional 1 or 3 h to allow protein folding to occur. From the diluted translation product, 2.5 µl was removed and added to 22.5 µl of SDS-PAGE treatment buffer. Trypsin digestion was begun by the addition of 1 µl of 0.3 mg/ml L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin to the remaining diluted translation product and the incubation proceeded for 1, 5, 10, and 20-min time periods at 0 °C. When each time period had elapsed, 2.5 µl was removed and diluted into SDS-PAGE treatment buffer. The samples were analyzed on a 12.5% polyacrylamide gel and proteins were visualized by autoradiography.

Miscellaneous

pBluescript SK+/- was purchased from Stratagene. The reagents used for PCR were from Perkin-Elmer. Restriction enzymes and T4 DNA Ligase were obtained from New England BioLabs. DNA sequencing was performed using Sequenase 2.0 from U. S. Biochemical Corp. (Sanger et al., 1977). SDS-PAGE was run according to Laemmli(1970). Chou-Fasman analysis and helical wheel plots were performed using the Genetics Computer Group (GCG) Package, Version 7.2. Hydropathy plots were performed using Gene Runner 3.0.


RESULTS

The Initial 22 Residues of Rhodanese Are Sufficient for Mitochondrial Import

Rhodanese is a mitochondrial matrix protein that possesses a noncleaved signal sequence (Miller et al., 1991). The crystal structure of rhodanese showed that, although the initial N-terminal residues cannot be located with certainty, residues 11-22 can form an alpha-helix (Ploegman et al., 1978). Two-dimensional NMR results revealed that residues 4-21 can form an alpha-helix in a hydrophobic environment (Hammen et al., 1994). Since an N-terminal helix may be necessary for import (Roise and Schatz, 1988; Wang and Weiner, 1993), the initial 22 residues of rhodanese were expected to be sufficient for targeting to the mitochondria. To confirm this hypothesis, a construct with a deletion from residue 23 to 55 within rhodanese (referred to as rhodanese Delta23-55) was created. This form of rhodanese was imported, demonstrating that the initial 22 residues are sufficient for mitochondrial targeting (Fig. 1). These results were confirmed by fusing the same 22 residues to dihydrofolate reductase and observing import (data not shown).


Figure 1: The N-terminal residues of rhodanese are sufficient for import. Residues 23-55 were deleted from rhodanese with the resultant protein remaining competent for import. In vitro import was performed as described under ``Experimental Procedures.'' Lanes 1-5 represent rhodanese. Lanes 6-10 represent rhodanese Delta23-55. For each assay, translated protein (lanes 1 and 6) was incubated with rat liver mitochondria for 30 min at 30 °C. One reaction mixture was subsequently treated with proteinase K to remove nonimported protein (lanes 4 and 9), while another reaction mixture was left untreated (lanes 3 and 8). The protected protein in lanes 4 and 9 represents imported protein. To determine susceptibility to protease digestion, rabbit reticulocyte lysate was treated with proteinase K (lanes 2 and 7). Additionally, mitochondria were disrupted with 1% Triton X-100 and subsequently treated with proteinase K to confirm the presence of protein within the mitochondria (lanes 5 and 10).



After incubation of mitochondria with rhodanese or rhodanese Delta23-55, proteinase K was added to destroy protein not imported into the mitochondria. Presumably, the protein remaining after proteinase K digestion resides within the organelle. For proteins processed after import, one can observe a smaller, cleaved protein on an SDS-polyacrylamide gel. Since rhodanese, a known matrix-space enzyme, is not processed after import, it was difficult to know if rhodanese was imported into the mitochondria or remained outside the mitochondria and was resistant to proteinase K. Newly translated rhodanese was digested by proteinase K, as shown in Fig. 1, lane 2. To confirm the presence of rhodanese within the organelle after import, mitochondria were disrupted with Triton X-100. Following disruption, protein released from the mitochondrial interior was digested with proteinase K. Approximately 10% of native rhodanese remained protease-resistant after mitochondrial disruption (Fig. 1, lane 5). However, this nondigested protein represented a small fraction of the total imported protein. As expected, the molecular weight of the protein imported into the mitochondria did not change, indicating that both rhodanese and rhodanese Delta23-55 were not processed.

Rhodanese with a Disrupted N-terminal Helix Is Still Not Processed

It had been suggested that signal sequences with more than three turns of a helix may not be recognized by MPP (Hammen et al., 1994). It seemed possible then that disruption of the signal sequence of a nonprocessed protein, such as rhodanese, might allow processing by MPP. Therefore, the N-terminal helix of native rhodanese was disrupted by placing the three-amino acid linker of pALDH (RGP) within it (referred to as rhodanese-RGP) (Fig. 2). Since the initial 22 residues of rhodanese form a helix, RGP was added after residue 11 to disrupt this structure. Although the structure of the rhodanese-RGP signal peptide was not determined by NMR, it is a reasonable assumption that the presence of glycine and proline, both helix-breaking residues, disrupt the continuous helix of the rhodanese signal peptide. Chou-Fasman analysis showed that these residues could disrupt secondary structure (data not shown). If MPP recognized a disrupted helix, it would be expected that rhodanese-RGP would be processed. However, this was not the case. Rhodanese-RGP was not processed after import into mitochondria. The addition of the linker had no adverse effect on import, as the amount of rhodanese-RGP imported was comparable to native rhodanese. Approximately 30% of both native rhodanese and rhodanese-RGP added to the mitochondria was imported (as determined by densitometry).


Figure 2: Sequence and structure of pALDH, rhodanese, and thiolase signal peptides. A, structure of native and linker-deleted pALDH signal peptides. The helical segments were determined by NMR analysis in a hydrophobic environment and are underlined (Karslake et al., 1990; Hammen et al., 1994). B, structure of native rhodanese and rhodanese-RGP signal peptides. Underlined regions indicate helical regions of the native rhodanese signal peptide as determined using NMR (Hammen et al., 1994). Broken lines under the rhodanese-RGP signal peptide indicate presumed disruption of the helix in this signal peptide. C, the amino acid sequence of rhodanese/R-3 and rhodanese-RGP/R-3 with an engineered R-3 (RXYS) recognition site (Gavel and von Heijne, 1990). The processing site is underlined. D, the amino acid sequence of thiolase with an engineered R-3 processing site. The native thiolase sequence, GAYG, was changed to RAYS.



To confirm this lack of processing, rhodanese and rhodanese-RGP, as well as pALDH and linker-deleted ALDH, were incubated with purified MPP. In accordance with the results after import, rhodanese, rhodanese-RGP, and linker-deleted ALDH were not processed by the purified enzyme, while pALDH was processed. Hence, it appears that MPP must recognize more than a disrupted N-terminal signal sequence for processing.

Rhodanese, A Nonprocessed Protein, Is Capable of Interacting with MPP

Previously, it had been shown that synthetic signal peptides from cleavable precursor proteins act as competitive inhibitors of processing catalyzed by MPP (Yang et al., 1991; Arretz et al., 1994). To determine if nonprocessed signal peptides interact with MPP, the rhodanese peptide (corresponding to the N-terminal 23 residues) was used as an inhibitor of pCOX IV processing by MPP. The processing of pCOX IV has been characterized previously and has been shown to occur in two steps, with the initial cleavage event being catalyzed by MPP. A remaining octapeptide is cleaved by mitochondrial intermediate peptidase (Maarse et al., 1984; Hendrick et al., 1989). Both the rhodanese peptide and the pALDH peptide (corresponding to the N-terminal 22 residues) acted equally well as inhibitors of pCOX IV processing (Fig. 3). Half-maximal inhibition of processing occurred with approximately 60 µM of either added peptide. Glucagon (a 29-mer), as a control, had little effect on processing, even at concentrations above 1 mM.


Figure 3: Rhodanese and pALDH signal peptides inhibit pCOX IV processing. Various concentrations of pALDH (bullet) and rhodanese (circle) signal peptides were used to inhibit the processing of pCOX IV, which was translated in reticulocyte lysate and incubated with purified MPP for 1 h, as described under ``Experimental Procedures.'' The percent processing was the amount of product produced (mature protein), divided by the total amount of protein in the reaction (mature + precursor (M + P)). Processing in the absence of peptide was arbitrarily set at 100%.



Although the rhodanese signal peptide inhibited MPP, it remained unknown whether full-length rhodanese interacted with MPP. To address this question, fully translated rhodanese (from rabbit reticulocyte lysate) was used as a potential inhibitor of pCOX IV processing. Rhodanese, translated for 1 h in reticulocyte lysate, was capable of inhibiting pCOX IV processing, with half-maximal inhibition estimated to be approximately 10 pM. Rabbit reticulocyte lysate alone had no effect on processing, indicating that the inhibition of processing was due to rhodanese and not some other component present in the lysate. Additionally, bovine serum albumin at 10 µM, as a control for nonspecific protein interactions, had no effect on processing. Furthermore, purified commercial rhodanese, folded into its native conformation, had no effect on processing at a concentration of 10 µM. Therefore, it appears that rhodanese translated in rabbit reticulocyte lysate could interact with MPP, even though it lacked an element essential for processing.

A Specific Processing Site Is Necessary for Recognition by MPP

Since newly translated rhodanese inhibits MPP, it seemed plausible that this protein may simply lack a specific cleavage site recognized by MPP. To test this hypothesis, an R-3 site (Table 1) (Gavel and von Heijne, 1990) was added after residue 22 of rhodanese and after residue 25 of rhodanese-RGP (referred to as rhodanese/R-3 and rhodanese-RGP/R-3, respectively) (Fig. 2C). Placement of the R-3 site was chosen because the targeting signal for mitochondria import lies within the first 22 N-terminal residues, as shown in Fig. 1. Both forms of rhodanese with an R-3 site were cleaved after import into mitochondria (Fig. 4A). Disruption of a continuous helix by insertion of RGP had little effect on processing, as rhodanese/R-3 and rhodanese-RGP/R-3 were processed to the same extent. Both of these proteins were also processed when incubated with purified MPP (Fig. 4B). Since femtomoles of product were produced in these reactions, there was not a sufficient amount of protein for sequencing. However, the size of the faster migrating protein corresponded to the loss of approximately 20-25 residues, consistent with cleavage after residue 22. It is interesting to note that approximately 50-60% of imported rhodanese is processed, as compared to approximately 80-100% for imported pALDH or pCOX IV (data not shown). Although rhodanese/R-3 was not processed to the same extent as the native precursors, it is possible that a different processing site or slight variations in the amino acid sequence of the rhodanese signal peptide would optimize processing.


Figure 4: Cleavage of rhodanese possessing an added MPP recognition site. In vitro import and processing assays were performed as described under ``Experimental Procedures.'' A, import of rhodanese/R-3 and rhodanese-RGP/R-3. Lanes 1-3 represent rhodanese/R-3. Lanes 4-6 represent rhodanese-RGP/R-3. Lanes 1 and 4, rhodanese/R-3 and rhodanese-RGP/R-3 translated in reticulocyte lysate, prior to import. Lanes 2 and 5, import in the absence of proteinase K treatment. Lanes 3 and 6, addition of proteinase K. B, processing after incubation with purified MPP. Lanes 1 and 2 represent rhodanese/R-3. Lanes 3 and 4 represent rhodanese-RGP/R-3. Lanes 1 and 3, translated rhodanese/R-3 and rhodanese-RGP/R-3. Lanes 2 and 4, incubation of translated protein with MPP for 1 h.



Addition of an MPP Recognition Site to Thiolase

To determine if thiolase also was not processed because it lacked an MPP recognition site, an R-3 site was added to this protein (Fig. 2D). Since it was reported that the initial 16 residues of thiolase act as a signal for targeting to the mitochondria (Arakawa et al., 1990), the R-3 site was added at residue 20 (referred to as thiolase/R-3) to ensure mitochondrial import function. As in the case of rhodanese, both native thiolase and thiolase/R-3 were imported to a comparable extent. However, thiolase, unlike rhodanese, was still not processed when it possessed an R-3 site. The same results were obtained when this thiolase construct was incubated with purified MPP.

Thiolase/R-3 behaves like the nonprocessed protein, linker-deleted ALDH. Both proteins have a signal peptide that forms a helix which is followed by a processing site, yet neither is processed. To determine if either native thiolase or linker-deleted ALDH was recognized by MPP, the corresponding signal peptides were used as potential inhibitors of pCOX IV processing. Although both inhibited, inhibition was half-maximal at approximately 130 µM for thiolase peptide and approximately 210 µM for linker-deleted ALDH peptide. These concentrations were two to three times higher than the concentration necessary for rhodanese and pALDH peptides to inhibit processing. Additionally, both proteins were translated in reticulocyte lysate for use as potential inhibitors of pCOX IV processing. Unlike rhodanese, these proteins were poor inhibitors of MPP processing. Thus, not all nonprocessed proteins are capable of inhibiting MPP, even if they possess an R-3 site.

Protein Folding Rates in Rabbit Reticulocyte Lysate

The possibility existed that the proteins not processed in this study may fold too rapidly to allow presentation of a putative cleavage site to MPP. However, it has been shown that other precursors, such as the rat mitochondrial aspartate aminotransferase (pmAspAT), fold slowly in reticulocyte lysate. It was shown that pmAspAT translated in reticulocyte lysate at 30 °C was initially very susceptible to digestion by trypsin, but with time, this protein underwent a slow conversion to a trypsin-resistant, presumably folded, state (Mattingly et al., 1993). A similar result was obtained for the nonprocessed proteins used in this study. Each of these proteins was translated for either 30 or 60 min at 30 °C. The proteins were then subjected to a 1-min trypsin digestion and the protein remaining after this digestion was quantitated (Fig. 5). If the proteins were digested for longer time periods, most of the protein was digested to smaller fragments. Only thiolase formed a smaller, approximately 20-kDa, fragment that remained resistant to trypsin. Interestingly, thiolase/R-3 did not form this trypsin-resistant fragment, indicating that thiolase/R-3 may misfold or not fold at all.


Figure 5: Trypsin-resistance of nonprocessed proteins. Each of the nonprocessed proteins, rhodanese (), thiolase (&cjs2100;), thiolase/R-3 (&cjs2110;), and linker-deleted ALDH (&cjs2113;), was translated for 30 or 60 min at 30 °C. After the 60-min translation, an aliquot was removed and incubated at 15 °C for an additional 180 min, or a total of 240 min after the start of translation. Protein remaining after a 1-min trypsin digestion was quantitated and compared at the indicated time points. Experiments were repeated in duplicate or triplicate and the results were averaged.



The pmAspAT had been shown to become trypsin-resistant after an extended incubation at 15 °C. Therefore, after translating the nonprocessed proteins for 60 min at 30 °C, these proteins were incubated for an additional 180 min (a total of 240 min after the start of translation) at 15 °C so protein folding may occur. Each nonprocessed protein became more resistant to trypsin with time (Fig. 5), indicating that proteins were slowly folding or possibly aggregating.

Experiments with MPP reported above had been performed on proteins translated for 60 min. The nonprocessed proteins, although, were the most sensitive to trypsin after a 30-min translation. It is presumed that most of the protein remained unfolded at this time. Therefore, as a further verification that folding was not a factor for the lack of processing by MPP, processing assays were performed on these proteins which had been translated for 30 min. Processing by MPP still did not occur. Thus, it appears that these nonprocessed proteins, even in an unfolded conformation, are lacking an element essential for cleavage by MPP.


DISCUSSION

Many endopeptidases are known to exist. Some, such as trypsin or chymotrypsin, cleave nonspecifically, while others, such as thrombin, recognize a specific cleavage site. MPP is unique among endopeptidases in that it acts on hundreds of different signal sequences, yet removes the signal sequence in a single and specific cleavage reaction. MPP falls into a new class of metalloendoproteases since it lacks the zinc-binding site -His-Glu-Xaa-Xaa-His- found in the majority of metalloendoproteases, such as thermolysin (Rawlings and Barrett, 1991). The specificity and mechanism of MPP remain enigmatic in that MPP cleavage sites differ among precursor proteins and not all precursors have one of the cleavage motifs listed in Table 1. Since there is a significant amount of variability in the signal sequence among different precursors, it appears that higher structural features may also be important for MPP recognition and cleavage.

It is unknown in what state of folding a protein is processed after import into the mitochondria. It is believed that when proteins first reach the matrix space, they are maintained in a loosely folded conformation by heat shock protein 70 (hsp 70) until they are able to interact with mitochondrial heat shock protein 60 (hsp 60). Subsequent to this interaction, the protein is folded into its native conformation (Stuart et al., 1994). Does MPP recognize the loosely folded protein associated with a heat shock protein, or does MPP cleave a folded protein? Rhodanese, translated in rabbit reticulocyte lysate (which contains various cytosolic factors and heat shock proteins that maintain proteins in a loosely folded state), inhibits MPP processing, suggesting that MPP may recognize an unfolded or partially folded protein. Purified rhodanese, commercially available, is not an inhibitor of processing by MPP, even at concentrations a thousand times greater than rhodanese translated in rabbit reticulocyte lysate, showing that folded proteins may not interact with MPP.

One may speculate that the rate of folding or extent of folding of an imported protein may influence its ability to be processed. A protein which immediately folds after import may not have time to interact with MPP. Perhaps certain nonprocessed proteins are folded more quickly than are processed proteins. It may be argued that the proteins not processed in this study may fold too rapidly to allow presentation of a putative cleavage site to MPP. However, we do not feel that this is the case for two reasons. First, each of the altered proteins used in this study remained import-competent. It has been proposed that proteins are imported in a loosely-folded conformation and are only imported if they do not have an opportunity to refold or aggregate (Glick and Schatz, 1991). This suggests that each of our nonprocessed proteins were also maintained in a loosely-folded conformation. Second, we showed that the nonprocessed proteins used in this study were initially very susceptible to digestion by trypsin, but with time, underwent a slow conversion to a trypsin-resistant state. These presumably unfolded proteins were not recognized by MPP, suggesting that these nonprocessed proteins lack a feature necessary for cleavage.

Generally, signal peptides are believed to form amphiphilic alpha-helices, at least in a hydrophobic milieu. The amino acid composition of mitochondrial signal peptides is characterized by the presence of numerous basic (especially arginine) and hydrophobic residues. Acidic residues are rarely found (Roise and Schatz 1988; von Heijne et al., 1989). Although the three-dimensional structure of the alpha- and beta-subunits of MPP has not been solved, secondary structural predictions indicate that both subunits possess an amphiphilic, highly negative-charged helix (Paces et al., 1993). Perhaps this region of MPP interacts with the positive-charged signal sequence. Previous studies have indicated the importance of basic residues, not only near the site of cleavage, but also in N-terminal regions of the signal peptide (Ou et al., 1994). For instance, it has been shown that synthetic peptides with a greater number of arginine residues are better inhibitors of MPP than less highly charged peptides. Additionally, the precursor protein, pre-adrenodoxin, which possesses a 58-amino acid signal sequence, was examined to show the importance of arginine residues for processing. Deletion of the positive-charged middle portion of this signal sequence prevented processing, as did mutation of the arginine residues between the 25th and 37th residue (Ou et al., 1994).

Although positive-charged residues in the signal sequence are important, it is not yet well understood what structural requirements are essential for MPP recognition and processing. By making a nonprocessed protein processable, and, conversely, by making a processed protein nonprocessable, we may be in a better position to elucidate characteristics necessary for the cleavage of a signal sequence from a precursor protein. ALDH was made nonprocessable by removal of the RGP linker (Thornton et al., 1993). The addition of the linker to rhodanese did not make it processable. Rhodanese, was made processable by the addition of an R-3 cleavage site (rhodanese/R-3). However, the presence of a cleavage site is not sufficient for the processing of all proteins. Both linker-deleted ALDH and thiolase/R-3 have a helix and cleavage site, yet, unlike rhodanese/R-3, these proteins were not processed. Additionally, studies using synthetic peptides as inhibitors of MPP indicate that, unlike pALDH and rhodanese peptides, linker-deleted ALDH and thiolase peptides were poor inhibitors of MPP.

The thiolase signal peptide has a low hydrophobic moment and does not appear to be as amphiphilic as the rhodanese or pALDH signal peptide. A helical wheel representation of the thiolase signal peptide (the N-terminal 14 amino acids) shown in Fig. 6indicates that, instead of being an amphiphilic helix, hydrophobic residues are found around the entire wheel. The linker-deleted ALDH peptide, although amphiphilic and having a relatively high hydrophobic moment, displays one face of a helix with six hydrophobic residues, which differs from native ALDH (Fig. 6). Perhaps MPP does not recognize a hydrophobic signal peptide. Hydropathy plot analysis indicates that three nonprocessed proteins, thiolase, isopropyl malate synthase, and the beta-subunit of the electron-transfer flavoprotein, all have a greater degree of hydrophobicity at their N-terminal relative to pALDH and rhodanese (data not shown).


Figure 6: Helical wheel diagram. The helical wheel diagrams are indicated for residues 1-14 thiolase, 1-22 rhodanese, 1-16 linker-deleted ALDH, and 1-19 native pALDH signal peptides. These helical wheels correspond to helical regions determined by two-dimensional NMR. The native ALDH signal peptide consists of a helix-linker-helix. Since there are 3.6 residues per turn of a helix, the 3-amino acid linker (residues RGP) may maintain the N- and C-terminal helices in phase with each other. Therefore, the linker was included in the helical wheel representation of native ALDH. Hydrophobic residues are boxed.



Other possibilities may be envisioned for the lack of processing of certain proteins. Rigidity of the signal sequence or the presence of negative charges within the signal sequence may affect processing by MPP. A greater number of negative charges in the signal sequence may prevent an interaction with the negative-charged regions within MPP. Three nonprocessed proteins with negative charges in the signal sequence are isopropyl malate synthase (Beltzer et al., 1988), chaperonin 10 (Ryan et al., 1994), and the beta-subunit of the electron-transfer flavoprotein (Finocchiaro et al., 1993). Each of these proteins possess two negative charges within the first 20 N-terminal residues. However, of the nonprocessed proteins used in this study, only rhodanese has one negative charge.

Many mitochondrial signal sequences contain one or more glycine or proline residues which may confer flexibility. Perhaps the positive-charged signal sequence interacts with MPP and then must be properly positioned to allow cleavage. Without this flexibility, the cleavage site may not be accessible to catalytic residues within MPP. Structural analysis by two-dimensional NMR has demonstrated that signal peptides from cleaved precursors have a certain degree of flexibility, while signal peptides from noncleaved proteins tend to form longer, more stable helices in a hydrophobic environment (Thornton et al., 1993; Hammen et al., 1994). We previously proposed that this feature of nonprocessed proteins may be one reason for the lack of processing. However, the addition of a linker to rhodanese (rhodanese-RGP) did not allow processing, indicating that lack of flexibility may not be a major reason for native rhodanese being noncleavable.

We show here that the structure of the signal peptide and a processing site are factors essential for cleavage by MPP. However, since the known structures of signal sequences were determined in a micellar environment, which is known to induce helicity, it is difficult to make definite conclusions about structural features of the signal peptide which are necessary for MPP recognition. A signal sequence may display different structural features in different environments, such as the environment in a proteinaceous import receptor or in the matrix. Additionally, an interaction with MPP may induce a conformation in the signal sequence that is compatible with cleavage. Although the nonprocessed proteins used in this study did not appear to fold too rapidly to allow presentation of a processing site, further investigation is necessary to pinpoint structural features necessary for cleavage. As more is learned about the structure of MPP and the relationship between this structure and its interaction with a signal sequence, a greater understanding of signal peptide recognition will be obtained.


FOOTNOTES

*
This work was supported in part by Grant AA05812 from the National Institute on Alcohol Abuse and Alcoholism. This is journal paper 14798 from the Purdue University Agricultural Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Senior Scientist Award AA00028 from the National Institute on Alcohol Abuse and Alcoholism. To whom correspondence should be addressed: Dept. of Biochemistry, Purdue University, West Lafayette, IN 47907-1153. Tel.: 317-494-1650; Fax: 317-494-7897.

(^1)
The abbreviations used are: MPP, mitochondrial processing peptidase; pALDH, precursor aldehyde dehydrogenase; pCOX IV, precursor cytochrome oxidase subunit IV; pmAspAT, precursor mitochondrial aspartate aminotransferase; rhodanese-RGP/R-3, rhodanese with the RGP linker and an R-3 site; thiolase/R-3, thiolase with an R-3 site; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; HEPPS, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Prof. Paul Dooley for rhodanese cDNA, Dr. Masataka Mori for thiolase cDNA, and Prof. Frantisek Kalousek for his generous gift of purified MPP and mitochondrial intermediate peptidase.


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