From the Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City, Missouri 64110-2499
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ABSTRACT |
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Of the two homologous isozymes of aspartate aminotransferase that are also nearly identical in their folded structures, only the mitochondrial form (mAAT) is synthesized as a precursor (pmAAT). After its in vitro synthesis in rabbit reticulocyte lysate, it can also be efficiently imported into isolated rat liver mitochondria, where it is processed to its native form by removal of the N-terminal presequence. The homologous cytosolic isoenzyme (cAAT) is not imported into mitochondria, even after fusion of the mitochondrial presequence from pmAAT to its N-terminal end. Substitution of the 30-residue N-terminal peptide of the mature portion of pmAAT with the corresponding sequence from the homologous, import-incompetent cytosolic isozyme (pcmAAT) does not prevent import but reduces substantially its processing in the matrix. A detectable amount of the pcmAAT chimera is found associated with the inner mitochondrial membrane. Single and double substitution mutants of Trp-5 and Trp-6 at the N-terminal end of the mature protein are imported into mitochondria with efficiency similar to that of wild type. However, replacement of Trp-5 with proline, or of both tryptophans with either alanine (W5A/W6A mutant) or valine and alanine (W5V/W6A mutant), allows import but interferes with the correct processing of the imported protein despite the presence of an intact cleavage site for the processing peptidase. Similar cleavage results were obtained using newly synthesized proteins and mitochondrial matrix extracts. These results indicate that translocation and processing for a precursor are independent events and that sequences C-terminal to the cleavage site are indeed important for the correct maturation of pmAAT in the matrix, probably because of their contribution to the conformation and flexibility of the peptide region surrounding the cleavage site required for efficient processing. The same region from the mature component of the protein may play a role in the commitment of the passenger protein to complete its translocation into the matrix.
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INTRODUCTION |
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Over 90% of mitochondrial proteins are encoded by the nuclear
DNA, synthesized in the cytoplasm as precursors with an N-terminal targeting sequence, and imported into the organelle. For proteins destined to the mitochondrial matrix such as mitochondrial aspartate aminotransferase (mAAT),1 the
translocation process involves the initial binding of the precursor
form of the protein to the surface of mitochondria, its transport
across both mitochondrial membranes mediated by the translocation
apparatus, and the final processing and folding of the protein in the
matrix (1, 2). In addition to energy in the form of ATP hydrolysis in
the matrix and a membrane potential across the inner membrane ()
(1, 2), a partially unfolded, loose conformation of the imported
protein appears to be also necessary for efficient translocation (3,
4). The ATP requirement seems to be due primarily to the
ATP-dependent action of mitochondrial hsp70 (mt-hsp70) (5).
It has been proposed that hsp70 binds the N terminus of the incoming
polypeptide as it emerges into the matrix (6, 7). This binding would
secure the irreversibility of the process by acting as a molecular
ratchet in collaboration with components of the protein import
machinery in the inner membrane (8, 9) and additional mitochondrial
chaperones (10). Other molecular chaperones in the matrix, including
the cpn60/cpn10 chaperonins (5, 11) and mitochondrial cyclophilins (12, 13), mediate the final folding of at least some proteins into their
native, active conformations.
At some point during the translocation process, the presequence of most
matrix precursors is proteolytically removed to render the mature,
naturally found protein. Many mitochondrial precursors are processed in
a single step catalyzed by the mitochondrial processing peptidase
(14-16), whereas others require an additional cleavage by
mitochondrial intermediate peptidase (17). Several bivalent cations
stimulate the activity of the mitochondrial processing peptidase (15).
Inhibition of processing activity has been observed in the presence of
several metal chelators such as EDTA and O-phenanthroline (15, 18, 19). Processing peptidases are highly specific for the
precursor forms of mitochondrial proteins (14), the presequences of
which are very heterogeneous in sequence although they share some
general properties such as the presence of hydroxylated and positively
charged amino acids as well as the ability to form amphiphilic
-helices in hydrophobic environments (20). While these properties
are known to be important for the targeting function of the
presequences (21), the structural features responsible for the specific
cleavage of the presequence within mitochondria at the exact junction
with the N terminus of the mature protein are much less clear. Several
cleavage motifs have been suggested, based on mutagenesis analyses and
comparison of large numbers of sequences from extension peptides (22,
23).
The contribution of sequences C-terminal to the cleavage site
(i.e. N-terminal end of mature protein) to the import and
correct processing of mitochondrial precursors is still under debate. Whereas manipulations of sequences 5 residues downstream of the normal
processing site of mitochondrial pre-ornithine carbamyltransferase were
reported to affect neither import nor correct processing (24), deletion
of mature sequences 17 residues down from the maturation site was found
to mainly prevent processing of the F1-ATPase -subunit
within mitochondria (25). More recent studies using either synthetic
peptides (26) or chimeric proteins (27, 28) indicate that the amino
acid in position +1 C-terminal to the processing site may have a role
in determining the fidelity of processing. The lack of apparent
consensus among the sequences at the processing sites of mitochondrial
precursors has led to the proposal that the conformation of the
presequence peptide and the chain flexibility in the region surrounding
the cleavage site may be important for the recognition and maturation
of precursors in the matrix (29-32). In addition, it is not known
whether the overall conformational state of the imported protein or its
interaction with molecular chaperones in the matrix might also
contribute to the specificity of the cleavage.
Aspartate aminotransferase exists in animal cells in two homologous
(over 50% sequence similarity) molecular forms, one located exclusively in the cytosol (cAAT) and the other in the mitochondrial matrix (mAAT). Both are encoded in the nuclear genome and synthesized in the cytosol, yet only the mitochondrial form is competent for translocation into mitochondria. mAAT is synthesized as a precursor with a cleavable extension at its N-terminal end (pmAAT). This N-terminal segment also contains one of the regions of greater dissimilarities between the two AAT isozymes. The presequence of pmAAT
from rat liver is 29 residues long and contains two arginine residues,
one of them located in position 2 from the maturation site (33).
pmAAT therefore belongs to the R-2 class of mitochondrial precursors
containing the RX1
X2S
cleavage motif (22, 32), where X1 is Ala and
X2 is Ser for pmAAT. As we show here, processing of imported pmAAT appears to occur in a single step and is dependent on
a metalloprotease activity. In this work, we focus on the analysis of
the effect of alterations introduced at the N-terminal end of the
mature portion of AAT on the processing of the imported protein as well
as on the full translocation of the protein to the matrix
compartment.
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EXPERIMENTAL PROCEDURES |
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Construction of Chimeric Proteins and Site-specific Mutagenesis-- The cDNA for rat liver pmAAT previously cloned in Bluescript KS (pBSKS-4) (4) was used as the template for all site-directed mutations. The coding region of pmAAT was excised from pBSKS-4 by EcoRI/BamHI digestion and subcloned into the pALTER vector (Promega). The DNA was mutagenized according to the protocol provided by the vendor. In native pmAAT, the tryptophan residues in positions 5 and 6 are coded by TGG. Mutations at either or both of these positions were achieved by using oligonucleotide primers (ranging from 27 to 36 nucleotides in size) in which the Trp codons in the center of the oligonucleotide were changed to the appropriate codon: GCC GCA for W5A/W6A; GTG GCC for W5V/W6A; TTT TTT for W5F/W6F; TTT GCC for W5F/W6A; TTT (A/G)TC for W5F/W6I and W5F/W6V; A/GTC TTT for W5I/W6F and W5V/W6F; GCC TTT for W5A/W6F; (G/A)(C/G)C for W5A, W5T, and W5S; (G/C)(C/G)C for W5A, W5P, and W5R; G(C/T)C for W6A and W6V, and GTG for W5V. Mutated DNAs were used to transform Escherichia coli JM109, and the derived mutations were selected by polymerase chain reaction dideoxy sequencing using the Fentomol sequencing kit from Promega.
The cDNA encoding the pcmAAT chimera, pBSKS-8, was assembled from two previously isolated plasmids, pBSKS-4 encoding pmAAT (4) and pBSKS-6 encoding pcAAT (34) using recombinant polymerase chain reaction as described elsewhere (35).In Vitro Transcription and Translation-- mRNAs were prepared by in vitro transcription of BamHI-linearized templates using the mCAP kit from Stratagene and either T3 RNA polymerase for the pBluescript KS plasmids or T7 RNA polymerase for the pALTER plasmids. The mRNAs obtained were translated in rabbit reticulocyte lysate (RRL) (Promega) for 20 min at 30 °C using 25 µg/ml of mRNA and [35S]methionine as the radiolabeled amino acid as described previously (4, 34). Translation reactions were stopped by chilling on ice and adding cycloheximide to a final concentration of 50 µM.
In Vitro Import of Precursor Proteins into Isolated Mitochondria-- Mitochondria were isolated from male Wistar rat liver by a simple differential centrifugation (36) in a slightly modified buffer (MESH buffer: 220 mM mannitol, 0.1 mM EDTA, 70 mM sucrose, 20 mM HEPPS, pH 7.4). The import reaction was performed essentially as described previously (4, 37). Briefly, 20 µl of 35S-labeled precursor protein freshly translated in RRL were incubated with 20 µl of isolated mitochondria (5 mg/ml) in MESH buffer for 30 min at 30 °C. After chilling on ice, mitochondria were reisolated by centrifugation at 16,000 × g for 4 min at 4 °C. The supernatant was removed and mixed with an equal volume of 2 × SDS-PAGE sample buffer. This fraction represents the protein that does not bind to mitochondria. The pelleted mitochondria were then washed twice by resuspending in 200 µl of MESH, recovered by centrifugation, and finally resuspended in an appropriate volume of MESH (65 µl or higher). This mitochondrial fraction was either mixed directly with 2 × SDS-PAGE sample buffer (total protein bound to mitochondria), digested with 20 µg/ml L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin for 30 min on ice (protein that has been imported), or digested under the same conditions after disrupting mitochondria with 0.1% Triton X-100 (imported protein that is properly folded). Subsequently, samples were analyzed by SDS-PAGE and, at least, an overnight exposure to a Molecular Dynamics PhosphorImagerTM screen. The intensity of the radiolabeled mature size in the pelleted mitochondria relative to that of the precursor plus mature bands observed in the complete import reaction was used to calculate the percentage of precursor protein imported.
Processing of the imported protein was inhibited by incubating mitochondria with 2 mM O-phenanthroline for 10 min at 4 °C before starting import of the pmAAT translation product as described above. Due to the requirement of extramitochondrial Mg-ATP for pmAAT import and therefore the interference of metal chelators with translocation, the import reaction was supplemented with 5 mM magnesium acetate and 3 mM ATP. Under these conditions, about 30% of the protease-resistant mAAT associated with mitochondria remained as unprocessed precursor (data not shown).Subfractionation of Mitochondria-- After reisolation by centrifugation, mitochondrial pellets were treated with 100 µg/ml TPCK- trypsin for 30 min on ice to remove the protein associated with mitochondria but not imported. Trypsin digestion was stopped by adding 240 µg/ml soybean trypsin inhibitor. When indicated, a mixture of protease inhibitors (leupeptin (5 µg/ml), pepstatin, aprotinin, antipain, elastinal (1.5 µg/ml), and phenylmethylsulfonyl fluoride (PMSF) (2 mM)) was added to mitochondria before disruption of the outer membrane by osmotic swelling. Mitochondria were subjected to osmotic swelling by dilution with 10 volumes of ice-cold hypotonic buffer (10 mM potassium phosphate, pH 7.5). After incubation on ice for 30 min, mitoplasts were reisolated by centrifugation (16,000 × g for 4 min at 4 °C) and resuspended in the same hypotonic buffer. When indicated, the protease inhibitors (leupeptin (5 µg/ml), and aprotinin and pepstatin (1.5 µg/ml)) were added to the mitoplast preparation. To determine the amount of protein that has been completely translocated into the matrix, mitoplasts were digested for 15 or 30 min on ice with different concentrations of proteinase K (5, 10, and 20 µg/ml), followed by inhibition of proteinase K with 4 mM PMSF. The integrity of the mitoplasts was examined by monitoring the leakage of the matrix enzyme malic dehydrogenase into the solution using a standard activity assay for this enzyme (38).
To fractionate mitoplasts into their soluble (matrix) and membrane components, samples were frozen in liquid nitrogen and thawed in a sonicating water bath (room temperature, 60 s). The freezing-thawing/sonication cycle was repeated three times, and the extract was centrifuged in a Beckman Airfuge for 10 min at 100,000 × g to separate the soluble matrix (supernatant) from the membrane fraction (pellet).In Vitro Processing Peptidase Activity-- Mitochondrial matrix extracts were prepared from rat liver mitochondria (5 mg/ml) by osmotic swelling in hypotonic buffer, followed by disruption of the recovered mitoplasts by cycles of freezing-thawing and sonication as described above in the presence of protease inhibitors leupeptin and pepstatin (2 µg/ml) and PMSF (0.1 mM). Serine protease inhibitors and microbial protease inhibitors do not affect the processing peptidase activity. Processing reactions contained (in a final volume of 10 µl): 2 µl of RRL translation reaction containing 35S-labeled pmAAT, 2 µl of mitochondrial matrix extract, 1 µl of 5 mM MnCl2, 43 units/ml of apyrase, and 5 µl of 10 mM phosphate buffer, pH 7.5. Processing reactions were incubated at 27 °C for 5, 15, or 30 min. Reactions were terminated by the addition of an equal volume of 2 × SDS-PAGE loading buffer, and samples were analyzed by SDS-PAGE and autoradiography. The amount of processing was estimated from the intensity of the radioactive electrophoretic bands measured with a PhosphorImager and is expressed as the fraction of precursor that has been converted to mature (processed protein) relative to the total amount of protein (precursor + mature) present.
Miscellaneous Procedures-- SDS-PAGE was performed with the Bio-Rad Mini-PROTEAN II apparatus using the discontinuous buffer system (39) in 12% polyacrylamide separating gels approximately 5 cm in length. After fixing and drying the gels, the intensity of the radiolabeled bands was determined using a Molecular Dynamics PhosphorImager as described previously (35, 37). All oligonucleotides were synthesized at the Molecular Core facility in the School of Biological Sciences of the University of Missouri Kansas City and purified on NENSORB PREP columns (Du Pont). Restriction enzymes and T4 ligase were obtained from Life Technologies, Inc., New England Biolabs, and Promega. Proteinase K was from IBI.
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RESULTS |
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Sequence comparisons between the N-terminal end of mature mAAT isolated from rat liver (40) and the complete cDNA of rat precursor pmAAT (33) indicate that processing of pmAAT in vivo occurs by cleavage between Ala-29 and Ser-30 of the precursor polypeptide (Fig. 1). The sequence of the cleavage site and its immediate flanking region is identical in pmAATs from other animal sources (41, 42) but differs from those found in other mitochondrial matrix proteins (23). To analyze the contribution of sequences downstream from the cleavage site to both efficient import and correct processing of rat liver pmAAT, we prepared various fusion protein constructs (Fig. 1) and substitution mutants (Table I). One of the fusion constructs, pcAAT, encodes the presequence of pmAAT fused in frame to the entire cDNA of the homologous cytosolic AAT isozyme, which differs substantially from mAAT in its N-terminal segment (34). The other, pcmAAT, codes both the mitochondrial presequence and 33 amino acids from the N terminus of the cytosolic cAAT fused to the cDNA for residues 34-401 of the mitochondrial mAAT (35). In addition, a set of single and double substitution mutants was prepared targeting the initial part of the N-terminal segment of the mature form of the protein, which is known to be critical for proper protein folding and stability of AAT. Thus, either or both of the tryptophan residues found in positions 5 and 6 of the mAAT N-terminal peptide (third and fourth residue downstream from the cleavage site) were replaced with a variety of hydrophobic, hydroxylated, or even charged residues (Table I). Nevertheless, these mutants retained the wild type Ala-Ser cleavage site.
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Import and Processing of Wild Type pmAAT and pcmAAT into Isolated
Mitochondria--
SDS-PAGE electrophoretic patterns of import
reactions of pmAAT and pcmAAT are shown in Fig.
2. As reported previously (43), freshly
synthesized pmAAT was efficiently imported into isolated rat liver
mitochondria and processed to a mature-size form. The small amount of
precursor-size protein recovered with mitochondria (Fig. 2, lane
3) appears to be adsorbed unfolded material as it was degraded by
trypsin, whereas most of the processed mature protein remained intact
(Fig. 2, lane 4). The intensity of the radiolabeled mature
size protein in the pelleted mitochondria treated with trypsin relative
to that of the precursor plus mature bands observed in the complete
import reaction was used to calculate the percentage of precursor
protein imported (ranging from 60% to 70%). Treatment with trypsin in
the presence of 0.1% Triton X-100, which disrupts the mitochondrial
membranes and thereby allows access of the protease to the internalized
protein, showed that about 60-65% of the imported protein was
properly folded (Fig. 2, lane 5). This test for folding
relies on the well known extreme resistance displayed by folded
(native) mAAT toward proteolysis (44). The precursor form pmAAT is also
able to fold in to a conformation extremely similar to the known
structure of the mature protein (4, 43); only the presequence peptide
is susceptible to hydrolysis by low concentrations of trypsin (4),
which cleaves after Arg at positions 22 and
2 in the presequence
peptide (45). On the other hand, a hybrid protein (pcAAT) consisting of
the pmAAT presequence fused to the N terminus of the cytosolic isozyme, which can fold rapidly even in RRL (34), not only was not imported into
isolated mitochondria, it did not even bind to the organelle (data not
shown).
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Translocation of pmAAT Trp-5 and Trp-6 Mutants into Isolated
Mitochondria--
The results presented above indicate that
alterations introduced C-terminal to the peptide bond cleaved by the
mitochondrial processing peptidase in pmAAT, i.e. in the
N-terminal region of the mature protein, might be important for precise
processing. However, the sequence alterations introduced in pcmAAT
extend from residue +33 to the amino acid in the +1 position (Met for Ser, Fig. 1). Although methionine residues have been found at both the
1 and + 1 positions of maturation sites of mitochondrial precursors
(23), it is not clear which of the modifications in the sequence
downstream from the processing site in pcmAAT is responsible for the
inhibition of processing. To begin to address this question more
specifically, we prepared a family of mutants containing the wild type
cleavage site sequence, but with single or double substitutions in
positions 5 and 6 of the N-terminal peptide of mature mAAT that
correspond to residues +3 and +4 C-terminal from the processing site in
pmAAT (in the numbering of AAT, the N terminus of mature mAAT is
numbered as residue number 3 to maximize sequence alignments with the
cytosolic isozyme of various species, which contains two additional
residues at the N-terminal end; Ref. 46). These two tryptophan residues
are particularly intriguing because they are conserved in all
mitochondrial mAATs for which sequences are available, whereas the
equivalent positions in the cytosolic forms contain either
phenylalanine or other hydrophobic residue but not tryptophan (33, 46,
47). They are also known to contribute to the stability of the dimeric
structure of the native protein.
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Location of Imported Proteins within Mitochondria-- The results presented in Figs. 2 and 3 indicate that a substantial fraction of protease-resistant pcmAAT and W5P-pmAAT accumulates within mitochondria as unprocessed species after import in vitro. Between 60% and 70% of the protease-resistant material remained associated with mitoplasts prepared by disruption of the outer membrane by swelling mitochondria under hypotonic conditions (Fig. 4, B and C). This treatment leaves the inner membrane intact (38) but releases the contents of the intermembrane space. The distribution between precursor and processed material remained unchanged after preparation of mitoplasts. Similar treatment of mitochondria that had imported either wild type (Fig. 4A) or a pmAAT mutant that is processed normally (Fig. 4D) shows that also about 60% of the translocated proteins is recovered with the mitoplast fraction.
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In Vitro Processing of pmAAT Variants by Mitochondrial Matrix
Extracts--
Processing of wild type and mutant pmAAT was also
assessed by monitoring the conversion of precursor to mature form upon
incubation of aliquots of in vitro translation reactions
with a freshly prepared matrix extract as a source of processing
peptidase (18, 32). Incubation of wild type pmAAT freshly translated in
RRL with a matrix extract for 15 min at 27 °C results in about 25%
conversion of precursor to mature size translation product (Fig.
5, lane 2). This extent of
conversion is comparable to the yield of processing observed using
other translation products and purified mitochondrial processing
peptidase (18, 30, 32). We should point out that the freshly
synthesized pmAAT translation product we use as substrate for
processing is completely digested by a small concentration of trypsin
to fragments too small to be detected in the SDS-PAGE gel (data not
shown). Therefore, it is unlikely that the observed hydrolytic
conversion of a fraction of precursor into mature species results from
the action of unspecific matrix endoproteases. Hydrolysis of an
incompletely folded translation product by those proteases would be
expected to be at least as extensive as that observed with trypsin.
Nonetheless, the processing reaction was supplemented with various
protease inhibitors (leupeptin, pepstatin, and PMSF) to minimize the
hydrolysis by matrix proteases. In addition, the processing of pmAAT
was completely inhibited in the presence of EDTA and
O-phenanthroline (Fig. 5, lane 3), which
indicates that the reaction is mediated by a metalloprotease present in
the matrix crude extract, most likely a mitochondrial processing
peptidase whose activity is influenced my metal chelators (15, 18, 19). Interestingly, no processing was observed (Fig. 5, lane 14)
when the translation product was preincubated to allow its folding into
a trypsin-resistant conformation (4). Apparently, the processing site
is not accessible to the processing peptidase once the translation
product has acquired a native-like conformation. Furthermore, this lack
of processing is not due to the overall inaccessibility of the
presequence since trypsin rapidly removes the presequence from the
folded translation product by cleaving after Arg at position 2 just
one peptide bond upstream from the processing site (4).
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DISCUSSION |
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One of the final steps in the translocation of most mitochondrial precursors into the matrix is the proteolytic removal of the N-terminal presequence peptide that has initially guided the protein to the organelle. Since no intermediate size species are detected during import of wild type rat liver pmAAT into isolated mitochondria (Fig. 2), this precursor is probably processed in a single cleavage step, most likely mediated by the chelator-sensitive general mitochondrial processing peptidase.
One of the unusual features of the action of processing peptidases appears to be their ability to cleave with great fidelity at the presequence-mature junctions of many different precursor proteins. The almost complete lack of consensus among the sequences at these cleavage sites (22, 23) has led to the proposal that higher levels of structure might be responsible for the specificity of the proteolytic cleavage (24, 30). However, the exact nature of the above mentioned structural requirements is still obscure. This question has been addressed primarily by analyzing the structure of small synthetic peptides comprising the presequence itself or the presequence plus a few residues of the N-terminal mature sequence (31, 50) in membrane-mimetic environments. However, the conformational properties of the cleavage region or its accessibility to the protease may be determined not just by the sequences surrounding the nicked peptide but by the overall conformation of the protein substrate at the time of processing, which in turn might be influenced by its interaction with mitochondrial chaperones. Indeed, our results indicate that the folding state of the precursor substrate affects dramatically the efficiency of the reaction. Analysis of the processing of pmAAT translated in vitro in RRL by a matrix extract clearly shows that the folded, trypsin-resistant polypeptide is no longer processed to the mature form. By contrast, a significant fraction of the newly synthesized unfolded pmAAT can be processed to a mature-size form by this matrix extract (Fig. 5). These results suggest that processing of pmAAT in intact mitochondria probably precedes at least the latest stages of folding of the imported protein. Similar behavior has also been reported for mitochondrial rhodanese (32).
Thus, matrix processing peptidases resemble other endoproteases in
their general requirements for cleavage of peptide bonds in
non-denatured proteins. Proteolysis of native structures is limited not
just by the primary specificity of the endoprotease or the
accessibility of the peptide bond but also by the flexibility of the
region proximate to the cleavage site and its ability to adopt a
cleavable conformation at the protease active site (51). Hence, local
unfolding of the potential cleavage region can be the key factor in
determining limited proteolysis. Analysis of the structure of the
N-terminal region of mAAT in the folded structure provides a plausible
explanation for the inability of matrix extracts to process the folded
precursor. In the native folded dimer, the N-terminal arm is part of
the subunit interface with residues 3-14 of the mature sequence
forming an extended strand that interacts with the other subunit (52).
Residues Trp-5 and Trp-6 bind in hydrophobic pockets on the surface of
the other subunit, whereas residues Ser-3 and Ser-4 are more exposed to
the solvent. Even though the processing site Ala-(1)-Ser-3 might be
still relatively accessible on the surface of the protein, restrictions
in the flexibility or conformational adaptability of the polypeptide chain downstream from the cleavage site as a result of quaternary interactions might interfere with the processing peptidase. It is
interesting to note that the peptide bond after Arg at position
2
(one peptide unit upstream from the pmAAT maturation site) is readily
cleaved by small concentrations of another protease, trypsin (4),
confirming that in the folded protein the N-terminal end is exposed on
the surface of the folded precursor.
In addition to a partially unfolded conformation of the precursor protein, a number of structural features of the region surrounding the cleavage site have been identified as essential for efficient processing. Within the presequence peptide itself, the presence of positively charged residues both near the cleavage site and at the N-terminal region of the presequence seems to be important for processing (29, 53). A certain degree of flexibility of the presequence peptide also appears to be necessary (31, 32). In this report, we show that the region C-terminal to the processing site is also important for recognition and processing by the liver matrix peptidase. Several alterations affecting the amino acid sequence of the N terminus of the mature subunit of pmAAT interfere with processing. First, the chimeric precursor pcmAAT remains essentially unprocessed after import into mitochondria or incubation with a matrix extract. In this precursor, the residues at positions +1 to +4 relative to the cleavage site and beyond have been replaced by the corresponding sequence form the import-incompetent cytosolic isozyme. Despite the fact that methionine residues are found at +1 positions of maturation sites in other precursors (23), in the context of the pmAAT processing region this substitution per se might be responsible for the inhibition of processing. In fact, it has been reported that amino acid substitutions at position +1 affect processing but not import of a chimeric protein containing the presequence of cytochrome b2 fused to dihydrofolate reductase (28).
Second, our results on the processing of a set of pmAAT mutants with
substitutions exclusively in the +3 and +4 positions indicate that
efficient processing is also directly or indirectly dependent on amino
acid determinants present at these locations. From the summary
presented in Table I, we can see that the processing peptidase can
tolerate almost any residue in either of these positions as long as the
other residue remains aromatic (either wild type tryptophan or
phenylalanine). The main exception to this trend is the incorporation
of Pro in the +3 position (the mutant containing proline in the +4
position is not available). The processing of this mutant (W5P-pmAAT)
by intact mitochondria is very inefficient, although better than for
pcmAAT. It is interesting to note that the pcmAAT chimera contains two
consecutive proline residues at the +3 and +4 positions. A possible
interpretation of these findings is that the substrate recognition site
of the peptidase extends to at least the +3 position in the substrate
and the enzyme is unable to accommodate a Pro residue in this location,
perhaps because of the configuration of the Ser-Pro peptide bond at
position +2/+3. Many proteolytic enzymes, including trypsin, show a
strong preference for cleaving peptide substrates when the cleaved bond or other bonds close to the cleavage site are in the trans
configuration (54). A similar restriction may apply to the matrix
processing peptidase substrates if the X-Pro bond in the
pcmAAT and W5P-pmAAT precursors exists in the cis form or as
a mixture of the cis and trans forms at
equilibrium. Alternatively, the presence of Pro in this position may
have a long range effect on the conformation of the cleavage region
that may affect its specific recognition by the peptidase or the proper
orientation of the Ala-Ser scissile bond toward the catalytic residues
within the peptidase's active site. Interestingly, even trypsin has
difficulties cleaving after Arg at position 2 in the folded
W5P-pmAAT.2
The reasons for the incorrect processing of the mutants containing
non-aromatic residues at the +3 and +4 positions are unclear. Only two
such mutants are available (W5A/W6A and W5V/W6A), but in both cases the
precursors are converted not to the mature species but to
intermediate-size species by intact mitochondria, indicating that
cleavage at the normal processing site is prevented. Comparison of the
electrophoretic mobility of these intermediate species to that of a
shortened precursor produced by trypsin hydrolysis of purified folded
pmAAT after Arg at 22 (45) indicates that a cleavage in a region
near this Arg at
22 is occurring instead. This alternative processing
most likely results from the action of the processing peptidase and not
other matrix proteases since the presequence of the unprocessed pcmAAT
and W5P-pmAAT species that accumulate in the matrix remains intact, and
a similar, chelator-sensitive conversion is observed with matrix
extracts. It is possible that incorporation in these positions of a
residue with a relatively high helical tendency such as Ala might alter
the conformation of the processing site. Secondary structure
predictions indicate the potential for formation of
-helices at the
N-terminal, middle, and C-terminal portions of the presequence
separated by flexible linkers (residues
19 to
22 and
12 to
10)
(45). The introduction of alanine residues in the region immediately
following the junction with the mature sequence might favor the
extension of the helical structure through the maturation site. This
conformational alteration of the initial residues of the mature protein
might preclude recognition by the processing peptidase as proposed for
the linker-deleted presequence of another mammalian protein, the
aldehyde dehydrogenase precursor (31). This may also lead to the
positioning of the wrong peptide region in the active site of the
peptidase, which could explain the incorrect processing observed.
Analysis of the sequence of the region where the alternative cleavage
appears to occur reveals the presence of a sequence motif
22RVLS
19 that resembles the normal
maturation site in pmAAT (
2RASS+2) in that it
contains the essential Arg in position
2.
Our results raise the issue of how a region of the presequence about 20 residues upstream from the cleavage site can end up being positioned in
the catalytic site of the peptidase. A possible explanation is that
this region of the presequence represents a normal substrate
recognition motif during processing of the wild type protein by the
processing peptidase and therefore has a specific binding site in the
peptidase. Alterations in the conformation of the processing site may
lead to changes in the positioning of the N-terminal region of the
presequence in the catalytic site that result in the cleavage of the
wrong peptide bond. This interpretation is supported by previous
studies indicating the importance of basic residues, particularly
arginine, at positions distant from the cleavage site (position 22 in
pmAAT) for recognition by the matrix processing peptidase (29, 30).
Based on these findings, it was proposed that both the proximal and
distal arginine residues found in presequences interact with the
peptidase active site (29). The presence of a flexible linker region
containing glycine and/or proline residues in the middle of the
presequence (positions
11 and
10 in pmAAT) would facilitate the
positioning of these two distant regions of the presequence in the
active site. Indeed, removal of the RPG linker found in aldehyde
dehydrogenase has been shown to render the protein unprocessable (31),
suggesting that presequence flexibility might be necessary, although
probably not sufficient, for correct processing.
Several of the pmAAT mutants that show altered processing appear to have difficulties folding properly after import into the matrix (Table I). Therefore, it may be argued that misfolding or aggregation of the partially folded substrate is the reason for the anomalous processing observed. However, no such correlation exists between the folding and the processing properties of other mutants. Whereas the W5F/W6A mutant, which remains partially unfolded after translocation, is processed correctly and as efficiently as the wild type, only a fraction of imported W5P-pmAAT, which folds properly, is converted to mature.
Even though all of the pmAAT forms analyzed in this work are translocated to a protease-inaccessible location inside mitochondria with yields similar to the wild type, a fraction (about 50%) of internalized pcmAAT is recovered in the inner membrane fraction. Apparently, alteration of the N-terminal section of pmAAT interferes with some of the last stages of its translocation into the matrix. According to current models for protein import into mitochondria, irreversible translocation across the inner membrane requires the ATP-dependent interaction of precursors with mt-hsp70 (13, 55, 56), which recognizes both the presequence peptide and incoming mature segments of precursor proteins (7). The presence of the N-terminal sequence from cAAT at the N terminus of pcmAAT might hinder the initial interaction of mt-hsp70 with emerging segments of the mature protein immediately following the presequence. As a consequence, some of the protein may slide back into the membrane. This interpretation is consistent with our previous findings, indicating that the N-terminal segment of the mature protein sequence represents a recognition site in pmAAT for cytosolic hsp70 (35, 57) and that cAAT does not bind to cytosolic hsp70 (35, 58). Thus, the N-terminal region of mature mAAT contains information for both correct processing and full translocation of the mAAT precursor into the matrix. It is possible that both processes involve the action of molecular chaperones in the matrix. We propose that intermolecular interactions with molecular chaperones involved in the translocation and folding of the imported protein may contribute to the correct conformation and orientation of the processing region to allow recognition and correct processing by the processing peptidase. According to this hypothesis, mutations C-terminal from the maturation site may affect its interaction with other matrix proteins and thereby its accessibility and general suitability as substrate for the processing peptidase.
In summary, this study provides valuable insight into several aspects
of the translocation and maturation of precursors in mitochondria.
First, the hybrid precursor pcAAT is not translocated into mitochondria
in vitro, which indicates that the presequence peptide is
necessary but not sufficient for import. Additional information must
reside within the sequence of the passenger protein. Second,
translocation and processing are independent events. However, certain
alterations of the N-terminal region of the mature protein affect the
efficiency of both. Third, the requirements for substrate recognition
and specific hydrolysis by processing peptidases might not be that
different from those of other endoproteases for hydrolysis of peptide
bonds in native proteins. In addition to information contained in the
presequence peptide, efficient and correct processing are dependent on
amino acid determinants located C-terminal to the cleavage site. The
permissive primary specificity characteristic of the mitochondrial
processing peptidases probably arises from a catalytic site that can
accommodate a great variety of different amino acid residues at the 1
and +1 positions. However, to achieve the correct orientation of the
active bond toward the catalytic groups, the enzymes may display
"conformational specificity" for regions both upstream and
downstream from the cleavage site. Any amino acid substitution
perturbing this as yet unknown structure would affect the processing
reaction. The desired conformation can be directed by the amino acid
sequence of the region or imposed by intermolecular interactions with
components present in the mitochondrial matrix, particularly molecular
chaperones involved in the translocation and folding of many imported
proteins.
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ACKNOWLEDGEMENT |
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We thank Dr. Joseph Mattingly for preparing the pcmAAT chimera used in this study.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL-38412 and GM-38341.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Instituto de Bioquímica, Facultad de
Ciencias, Universidad Austral de Chile, Valdivia, Chile.
§ To whom correspondence should be addressed. Tel.: 816-235-5246; Fax: 816-235-5158.
1
The abbreviations used are: mAAT, mitochondrial
aspartate aminotransferase; cAAT, cytosolic aspartate
aminotransferase; pmAAT, precursor to aspartate aminotransferase;
pcAAT, precursor chimera to cytosolic aspartate aminotransferase;
pcmAAT, chimeric precursor; , membrane potential; mt-hsp70,
mitochondrial hsp70; PAGE, polyacrylamide gel electrophoresis; PMSF,
phenylmethylsulfonyl fluoride; RRL, rabbit reticulocyte lysate; TPCK,
L-1-tosylamido-2-phenylethyl chloromethylketone; HEPPS,
4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid.
2 A. Yañez, A. Iriarte, and M. Martinez-Carrion, manuscript in preparation.
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REFERENCES |
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