From the Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095-1569
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ABSTRACT |
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In Neurospora crassa, the
mitochondrial arginine biosynthetic enzymes,
N-acetylglutamate kinase (AGK) and
N-acetyl--glutamyl-phosphate reductase (AGPR), are
generated by processing of a 96-kDa cytosolic polyprotein precursor
(pAGK-AGPR). The proximal kinase and distal reductase domains are
separated by a short connector region. Substitutions of arginines at
positions
2 and
3 upstream of the N terminus of the AGPR domain or
replacement of threonine at position +3 in the mature AGPR domain
revealed a second processing site at position
20. Substitution of
arginine at position
22, in combination with changes at
2 and
3,
prevented cleavage of the precursor and identified two proteolytic
cleavage sites, Arg-Gly
Tyr-Leu-Thr at the N terminus of the AGPR
domain and Arg-Gly-Tyr
Ser-Thr located 20 residues upstream.
Inhibitors of metal-dependent peptidases blocked
proteolytic cleavage at both sites. Amino acid residues required for
proteolytic cleavage in the connector were identified, and processing
was abolished by mutations changing these residues. The unprocessed
AGK-AGPR fusion had both catalytic activities, including feedback
inhibition of AGK, and complemented AGK
AGPR
mutants. These results indicate that cleavage of pAGK-AGPR is not
required for functioning of these enzymes in the mitochondria.
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INTRODUCTION |
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In eukaryotic organisms, arginine biosynthesis is
compartmentalized. In Neurospora crassa, the first six steps
occur in the mitochondria and the last two steps in the cytosol. Flux
through the arginine biosynthetic pathway is regulated primarily by
feedback inhibition of the enzymes that catalyze the first and second
reactions. The second and third steps of the pathway are catalyzed by
N-acetylglutamate kinase
(AGK)1 (EC 2.7.2.8) and
N-acetyl--glutamyl-phosphate reductase (AGPR) (EC
1.2.1.38) (1, 2). These enzymes are produced from a polyprotein
precursor (pAGK-AGPR), which is targeted to the mitochondria and
processed into mature AGK and AGPR (3, 4). The polyprotein consists of
a mitochondrial targeting sequence followed by two protein domains, AGK
and AGPR, separated by a connector region (Fig. 1A).
Most mitochondrial proteins are synthesized in the cytosol and targeted
to the organelle by leader sequences at the N terminus of their
precursors. Mitochondrial leader sequences are recognized by specific
receptors on the mitochondrial outer membrane and translocated from the
receptors to downstream components of the import machinery (5-8).
Removal of N-terminal targeting sequences in the matrix is performed by
a mitochondrial processing peptidase (MPP), composed of two similar
subunits, -MPP and
-MPP;
-MPP is soluble in the matrix, and
-MPP is associated with the mitochondrial inner membrane (9-12). As
import and processing take place, proteins are folded into functional
enzymes or assembled into functional multienzyme complexes. We
previously showed that two proteins were obtained that comigrated with
mature AGK and AGPR upon incubation of in vitro synthesized
wild-type pAGK-AGPR with purified MPP (4). However, identification of
the the precise cleavage site(s) in the connector region of the
precursor remained to be determined.
Targeting sequences have the capability to form an amphipathic
-helix (13); however, defined sequences or structural motifs involved in proteolytic processing are not well understood (14-17). The connector of pAGK-AGPR contains an internal processing sequence, which has some of the characteristics of a mitochondrial targeting sequence, although it is not predicted to form an amphipathic
-helix
(4). Arginine residues at positions
2 or
3 and positions
10 or
11 relative to the first amino acid in the mature protein are often
found in targeting sequences of mitochondrial precursor proteins and
appear to form part of the not well defined motifs found at cleavage
sites (15, 16). Some similarities between the connector region of
pAGK-AGPR and mitochondrial targeting sequences are apparent (Fig.
1B).
Several questions are addressed in this study. What are the sequences or structural motifs that specify the cleavage at the connector region of pAGK-AGPR? How many cleavage events are necessary to process the polyprotein precursor into two proteins? Is processing of the precursor into two independent proteins required for function in the mitochondria? Processing of pAGK-AGPR and its biological function were analyzed in vitro and in vivo. The roles of several amino acid residues as signals for processing were examined by introducing point mutations in the connector region of the precursor. Two sites for proteolytic processing were identified, and processing into two mature proteins was prevented by mutagenesis of these sites. Proteolytic cleavage of pAGK-AGPR in the connector region did not appear to be required for the activity of either enzyme or for feedback inhibition of AGK by arginine. Processing at the connector region of pAGK-AGPR is discussed in the context of putative advantages that targeting of fusion proteins may have versus the targeting of independent proteins.
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MATERIALS AND METHODS |
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Strains and Growth Conditions--
Escherichia coli
strains DH5 and JM101 were used to propagate plasmid DNA. Strain
GM48 was used to prepare nonmethylated DNA for digestion of
methylation-sensitive restriction sites. E. coli RZ1032 and
helper phage VCSM13 were used for the generation of single-stranded
plasmid DNA for site-directed mutagenesis. Bacterial cultures were
grown in LB medium or terrific broth (18) as specified.
Neurospora wild-type strain LA1 (74A) and arg-6 strain LA358 (allele CD118) were obtained from R. H. Davis (19). Neurospora cultures were grown in Vogel's minimal medium N
(20) or in minimal medium supplemented with 0.2 mg/ml arginine.
Construction of Plasmids--
Plasmid constructs were derived
from pUC19 (New England Biolabs) or pBluescript KSII(+/)
(Stratagene). Constructs pRW5 and pJK2 have been previously described
(4); pRW5 contains a copy of the wild-type arg-6 gene, which
encodes pAGK-AGPR in an 8.0-kilobase pair SphI fragment
cloned into pUC19; and pJK2 contains a full-length wild-type
arg-6 cDNA in a 3.0-kilobase pair
EcoRI-HindIII fragment cloned into pBluescript
KSII(
). Plasmid pRW7 is a derivative of pRW5 that contains the
hph gene on a 1.69-kb SalI-BamHI
fragment. Plasmid pgH2 contains the arg-6 gene from pRW7 on
a 4.1-kilobase pair HindIII fragment cloned into pUC19.
Mutations in the connector region of the precursor were introduced by
PCR using an 888-base pair Bsu361 fragment from plasmid pgH2
as a template or by site-directed mutagenesis using an 877-base pair
ApaI-ClaI fragment from plasmid pJK2 (Fig.
1A, top diagram). A diagram of the introduced
amino acid substitutions at the connector region of pAGK-AGPR is shown in Fig. 1B. The wild-type
sequence of both the cDNA and genomic constructs is shown at the
top. For simplicity, only the names of the cDNA
constructs are indicated in the figure. Construct pcPG16
contains substitutions of the arginine pair at positions
3 and
2
relative to the N terminus of the AGPR domain to proline and glycine.
Construct pcPG16 was used as the template for the introduction of
additional mutations in several plasmid derivatives. Plasmid pcGTPG5
contains substitutions of arginine residues at positions
15 and
14
to glycine and threonine in a pcPG16 background. Constructs pcG22PG42,
pcP22PG54, and pcG22PG10 contain a substitution of arginine at
22 to
glycine or proline in a pcPG16 background; plasmid pcG22PG10 also has a
stop codon toward the N-terminal region of the mature AGPR domain.
Construct pcA13PG16 substitutes alanine for proline at position
13 in
the pcPG16 background. Construct pcP3 substitutes proline for threonine
at position +3 in a wild-type background. Genomic constructs contain
identical amino acid substitutions as the equivalent cDNA
constructs and include a 56-base intron located toward the 3'-end of
the distal AGPR coding region. The genomic constructs pgPG15-Hph,
pgGTPG4-Hph, pgG22PG42-Hph, and pgP22PG54-Hph resulted from subcloning
the 888-base pair Bsu361 fragment from pcPG16, pcGTPG5,
pcG22PG42, or pcP22PG54 into pRW7.
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Site-directed and PCR-mediated Mutagenesis--
Oligonucleotides
used for site-directed mutagenesis or PCR-mediated mutagenesis were
synthesized using an Applied Biosystems model 391 PCR-mate DNA
synthesizer (Table I). Site-directed
mutagenesis was performed by the Kunkel procedure, modified for the use
of single-stranded DNA derived from any plasmid (21). Single-stranded DNA was obtained by transformation of E. coli RZ1032 and
infection of exponentially growing transformants with helper phage
VCSM13 to a multiplicity of infection of 20. Annealing, extension by T4
DNA polymerase, and ligation were performed as suggested by Promega.
PCR mutagenesis was performed by asymmetric PCR in a Perkin-Elmer
thermocycler (22). Primary PCR reactions contained the template DNA
pgH2 (~10 ng), excess primer BsuAGK or IIB (100 pmol), limiting
primer PG1 or PG2 (1 pmol), 1× PCR buffer (20 mM Tris, pH
8.3, 25 mM KCl, 1.5 mM MgCl2,
0.05% Tween 20, 0.1 mg/ml gelatin), dNTPs (50 µM), and
Taq polymerase (2.5 units) in a final volume of 100 µl.
The PCR conditions for the denaturation, annealing, and extension
reactions were 1 cycle at 94 °C (2 min), 60 °C (2 min), and
72 °C (1 min) followed by 20 cycles at 94 °C (1 min), 60 °C (1 min), and 72 °C (1 min) and 10 cycles at 94 °C (1 min), 60 °C
(1 min), and 72 °C (1.5 min). For the second PCR amplification,
~10 ng of each gel-purified first PCR product was used as a template.
The outside primers, BsuAGK and IIB, were added, and PCR amplification
was carried out using the same conditions as described above for the
first amplification step. A low melting point agarose gel slice
containing the second PCR product (~660 ng) was resuspended in 80 µl of 1× Bsu361 buffer and digested with
Bsu361 in a final volume of 100 µl at 37 °C for 60 min.
The enzyme was inactivated by heating at 65 °C for 10 min. For the ligation reaction, 50 µl of the digestion mixture (~330 ng) was added to 35 ng of Bsu361-linearized pgH2Bsu361
in a final volume of 100 µl of 1× ligation buffer and incubated with
1 unit of T4 ligase at room temperature for 12 h (22). The
ligation mixture was used to transform competent cells of E. coli DH5
(23). Constructs pgGTPG4, pcA13PG16, and pcP3 were
also generated by asymmetric PCR using pgH2 as template DNA. The
presence of the desired mutations was initially screened by digestion
of DNA with SmaI (new site generated by the substitutions of
Arg
2 and Arg
3 to Gly
2 and
Pro
3), KpnI (new site introduced by the
substitutions of Arg
14 and Arg
15 to
Gly
14 and Thr
15), BstX1 (new
site generated by the change Arg
22 to
Gly
22), or HpaI (new site generated by the
change Arg
22 to Pro
22) and subsequently by
sequencing of the entire amplified regions.
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In Vitro Transcription and Translation--
To analyze
processing in vitro, wild-type and mutated precursor
proteins were synthesized by in vitro transcription and
translation. Plasmid DNA was linearized with EcoRV, treated
with proteinase K (50 µg/ml) for 30 min at 37 °C, and precipitated
with 2 volumes of ethanol. DNA was resuspended in DEPC-treated
distilled H2O and stored at 20 °C. Proteinase-treated
template was transcribed with T7 RNA polymerase as suggested by the
manufacturer (Promega). Transcripts were visualized by electrophoresis
using denaturing agarose gels (1.2% agarose containing 17%
formaldehyde in MOPS buffer). Translation reactions in rabbit
reticulocyte lysates were carried out in a final volume of 50 µl as
suggested by the manufacturer (Promega). In vitro
transcribed RNA (2 µl) was mixed with 1 µl of RNasin (10 units/ml),
1 µl of a mixture of amino acids (1 mM) except
methionine, 5 µl of [35S]methionine (1,200 Ci/mmol, 10 mCi/ml), 6 µl of H2O, and 35 µl of reticulocyte lysate.
The reaction was incubated at 30 °C for 1 h. A 2.5-µl aliquot
was resolved by 7.5% SDS-PAGE and analyzed using a PhosphorImager
(Molecular Dynamics). The rest of the sample was brought to a final
concentration of 0.3 M sucrose and 0.05 mM
methionine and stored at
80 °C.
Isolation of Import-competent Mitochondria and in Vitro Import Assays-- Mitochondria were isolated by differential centrifugation from N. crassa wild-type strain 74A grown for 12 h at 30 °C in minimal medium (12). Import reactions were performed by incubating a suspension containing ~70 µg of freshly isolated mitochondria in 100 µl of import buffer (3% bovine serum albumin, 2.5 mM MgCl2, 80 mM KCl, 10 mM MOPS/KOH, pH 7.2, 250 mM sucrose, and 2 mM NADH) with 5 µl of in vitro translated product for 30 min at 25 °C. 1,10-Phenanthroline (2.5 mM) and EDTA (5 mM) were used (if indicated) as inhibitors of metal-dependent peptidases (24, 25). Prior to import, mitochondria were preincubated with the inhibitors at 25 °C for 5 min in import buffer. After the addition of precursor to the reaction mixture, incubation was continued for 30 min. Postimport mitochondria were reisolated by centrifugation at 12,000 × g for 12 min and treated (if indicated) with 50 or 100 µg/ml of proteinase K for 30 min at 0 °C to remove externally bound precursor. 35S-Labeled import products were analyzed by electrophoresis in denaturing 7.5% polyacrylamide gels and fluorography.
Transformation of N. crassa-- The genomic constructs pRW7, pgPG15-Hph, pgGTPG4-Hph, pgG22PG42-Hph, and pgP22PG54-Hph were introduced into the arg-6 recipient strain LA358 by transformation following the procedure of Vollmer and Yanofsky (26). Hygromycin-resistant transformants were selected after 3-4 days of incubation at 30 °C in regeneration agar overlaid on agar plates containing arginine (200 µg/ml) and hygromycin (200 µg/ml). Transformants were tested for arginine prototrophy by screening on plates without arginine but containing 100 µg/ml hygromycin. Four isolates from each transformation were purified to homokaryons by four successive single conidial isolations. Purified transformants were analyzed by Southern and Western blots.
Crude Extracts and Immunoblots--
Wild-type and
ARG+ transformants were grown in 50 ml of minimal medium on
a rotatory shaker at 30 °C and harvested after 12 h by vacuum
filtration. Mycelia (0.5 g, dry weight) were washed three or four times
with deionized H2O, frozen in liquid nitrogen, and ground
in liquid N2 with a mortar and pestle to a fine powder. The
powder was suspended in 1.5 volumes of boiling extraction buffer (3%
SDS, 20 mM Tris-Cl, pH 8.0, 5 mM EDTA, 2 mM -mercaptoethanol, 1 mM
phenylmethylsulfonyl fluoride) and boiled for 5-7 min with vigorous
mixing every 2.5 min. Cell debris was removed by centrifugation, and
supernatants containing solubilized proteins were divided into 1-ml
aliquots and frozen quickly on dry ice. Protein concentrations were
determined with the Bio-Rad protein microassay. Solubilized proteins
(50 µg) were separated by SDS-PAGE and transferred to a
polyvinylidene difluoride membrane (Bio-Rad) by electroblotting at
4 °C in transfer buffer (25 mM Tris-Cl, pH 8.3, 150 mM glycine, 20% methanol). The kinase (AGK) and reductase
(AGPR) proteins were detected using anti-kinase or anti-reductase
antiserum and an enhanced chemiluminescence system with anti-rabbit
IgG-horseradish peroxidase as a secondary antibody (Amersham Pharmacia
Biotech). Blots were exposed to film (Hyper-film type MP, Amersham
Pharmacia Biotech) for time periods of 3-30 s.
N-terminal Sequencing--
Mitochondria from transformants were
purified by sucrose gradient centrifugation, and ~5-10 mg was
sonicated three or four times for 10 s each in a Fisher sonic
dismembrator, model 300, 35% maximal power, microtip probe in 1% TNET
(50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 1% Triton X-100) containing 2 mM phenylmethylsulfonyl
fluoride. Mitochondrial membranes were removed by centrifugation at
15,000 × g for 15 min at 4 °C. Supernatants were
brought to 400 µl with 1% TNET, and a 40-µl aliquot of undiluted
anti-AGPR antiserum was added for each 5 mg of starting mitochondrial
protein. Samples were incubated overnight with gentle rotation at
4 °C. Immunocomplexes were precipitated with the equivalent of two
volumes of Staphylococcus aureus extract as the source of
protein A. Incubation was continued for 60 min on ice. Samples were
centrifuged at 15,000 × g for 1-2 min, and pellets
were washed four times with 1 ml of TX/SDS (25 mM Tris-Cl,
pH 7.0, 150 mM NaCl, 5 mM EDTA, 0.1% SDS,
0.05% Triton X-100) and once with 1 ml of TBS (25 mM
Tris-Cl, pH 7.4, 100 mM NaCl, 1 mM KCl). Washed
pellets were solubilized in 50 µl of SDS/sample buffer without
-mercaptoethanol. Immunocomplexes were resolved by 7.5% SDS-PAGE
and blotted onto polyvinylidene difluoride membranes (Bio-Rad) in CAPS
transfer buffer (10 mM CAPS, 10% methanol, pH 11) at
4 °C (27). Proteins on the membrane were stained with Coomassie blue
R250, the band of interest was cut out of the membrane, and the amino
terminus was sequenced by the UCLA Sequencing Facility.
Enzyme Assays--
N-Acetylglutamate kinase and
N-acetyl--glutamyl-phosphate reductase activities of the
uncleaved precursor were assayed using mitochondria purified by sucrose
step gradient centrifugation (3). Acetylglutamate kinase was assayed by
a radioactive procedure modified from Wolf and Weiss (28). Reactions
contained 0.15 M Tris, pH 8.5, 60 mM
MgCl2, 30 mM ATP, 3.75 mM
[14C]acetylglutamate, and 0.2 M
NH2OH and were incubated for 80 min at 30 °C. Feedback
inhibition was measured in the presence of 18.75 mM
arginine. The reactions were initiated by the addition of 200 µl of
mitochondrial extract and stopped by transferring to a boiling water
bath. The reaction mixtures were acidified by adding 100 µl of 0.5 N formic acid and centrifuged at 14,000 × g to remove insoluble material. Supernatants were applied to an AG1-X8 (Bio-Rad) column (0.7 × 6 cm) equilibrated with 0.1 N HCOOH and eluted with 0.1 N HCOOH. Glutamate
was eluted in the first 10 ml followed by the reaction product,
acetylglutamyl hydroxamate, in the next 10 ml; unreacted
acetylglutamate was eluted with 1.0 N HCOOH. Fractions
containing the product were dried in a vacuum dessicator, redissolved
in 1 ml of 0.1 N HCOOH, and counted. Counts from a control
reaction containing no ATP were subtracted from the values obtained in
the test reactions. One unit of enzyme is defined as the amount
required to produce 1 µmol of product in 1 min at 30 °C. The
activity of acetylglutamate kinase is expressed in microunits and
represents the average of three independent determinations.
Miscellaneous-- Restriction enzymes were purchased from Promega and New England Biolabs. Taq polymerase was purchased from Promega. Sequenase version 2.0 was obtained from U.S. Biochemical Corp. An in vitro transcription kit containing T7 RNA polymerase was obtained from Promega. 35S E. coli hydrolysate labeling reagent (Tran 35S label, 1274 Ci/mmol, 11.74 mCi/ml) was purchased from ICN. A nuclease-treated rabbit reticulocyte lysate kit was obtained from Promega.
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RESULTS |
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Role of the Arginine Pairs at Positions 2 and
3 and Positions
14 and
15 from the N Terminus of the AGPR Domain--
The
importance of an arginine residue at position
2 or
3 relative to
the cleavage site of mitochondrial targeting sequences has been
demonstrated (13-16). Preliminary analysis indicated that replacement
of arginine with glycine at position
2 relative to the N terminus of
the AGPR domain (Arg
2 to Gly
2) did not
prevent cleavage of the polyprotein in
vitro.2 A precursor
containing the changes Arg
2 and Arg
3 to
Gly
2 and Pro
3 (pcPG16) was used as a
substrate for an in vitro import assay (Fig.
2A). Postimport mitochondria
were reisolated and treated or not treated with proteinase K to digest
bound precursor. Processing of the wild-type precursor (lanes
1 and 4) resulted in two protein bands; the upper
protein band corresponds to mature AGK (52 kDa), and the lower protein
band corresponds to mature AGPR (37 kDa) (29). Processing of the mutant
precursor derived from pcPG16 resulted in an AGK protein that
comigrated with wild-type AGK, but the AGPR protein (AGPR*) appeared to
have a higher molecular mass than wild-type AGPR (Fig. 2A,
lanes 2 and 5). This result suggested that
processing at the N terminus of the AGPR domain, between amino acids
1 and +1, was prevented by the amino acid substitutions and that one
or more proteolytic cleavages took place at a location(s) upstream of
the
1/+1 site in the connector region of the precursor.
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Role of the Arginine Residue at Position 22 from the N Terminus
of the AGPR Domain--
Since mutations at the arginine pairs upstream
of the
1/+1 cleavage site identified a second processing site in the
connector region, the role of the arginine at position
22 was
examined. Arg
22 was chosen because it is in a context
that resembles the cleavage site at
1/+1. Analysis of the precursor
derived from pcP22PG54 (Fig. 2B, lane 5), in
which arginine at
22 was changed to Pro in a background where the
arginine pair at
2 and
3 have been changed to Gly and Pro, shows
that processing of the precursor was prevented by these substitutions,
since no mature AGK or AGPR products were observed. The uncleaved
precursor derived from pcP22PG54 was imported into mitochondria, since
it was protected from proteinase K digestion. Some degradation of the
precursor may have occurred in the matrix, suggested by the smear under
the precursor band.
Cleavage of the Connector Region of the Precursor Is Inhibited by 1,10-Phenanthroline and EDTA-- MPP cleaves mitochondrial targeting sequences during or after import of precursor proteins into the mitochondria. In N. crassa, removal of the targeting sequence by MPP can occur in more than one step (30). In some cases in yeast and mammals, processing of mitochondrial targeting sequences that are cleaved in two steps is carried out by two different enzymes; the first cleavage is performed by MPP, and the second cleavage is performed by a mitochondial intermediate peptidase that produces the mature protein (31-33). MPP and mitochondial intermediate peptidase are metal-dependent proteases inhibited by the chelators 1,10-phenanthroline and EDTA (9, 10, 30), which do not affect the import of precursor proteins (24). Thus, it was of interest to investigate a possible role of a mitochondrial metal-dependent peptidase in the processing at the upstream cleavage site in pAGK-AGPR.
Import reactions with wild-type and mutated precursors were performed in the presence of inhibitors of metal-dependent processing peptidases (Fig. 3). To ensure that no precursor remained associated with the mitochondrial outer membrane and to evaluate the efficiency of proteolytic cleavage at the second site, postimport mitochondria were reisolated and treated with proteinase K. Wild-type precursor (Fig. 3, lanes 1 and 6) showed processing into two mature proteins, AGK and AGPR, in the absence of inhibitors (lane 1). Processing of the wild-type precursor was inhibited in the presence of 1,10-phenanthroline and EDTA (lane 6), as inferred from the presence of a protein band corresponding to unprocessed precursor and the almost complete absence of bands corresponding to mature AGK and AGPR. Processing of mutant precursors derived from pcPG16 (lanes 2 and 7) and pcGTPG5 (lanes 3 and 8) was also inhibited by 1,10-phenanthroline and EDTA, indicating that proteolytic cleavage at both processing sites is blocked by inhibitors of metal-dependent peptidases. Wild-type pAGK-AGPR is cleaved by purified MPP in the connector region to generate mature AGK and AGPR proteins (4). The importance of arginine at position
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Effect of Proline after an Arginine Pair on Processing--
The
arginine pair at positions 14 and
15 is located in a sequence motif
that resembles the recognition site for MPP (14-17) but did not appear
to function as a signal for processing. Analysis of amino acid
sequences flanking cleavage sites of several hormone and protein
precursors revealed that most peptides that contain a Pro residue at
position +1 are not cleaved (34, 35). To determine if the presence of a
proline residue at position
13 disrupted a possible MPP cleavage
site, the proline residue at position
13 was changed to an alanine in
a background where the arginine residues at
2 and
3 were changed to
Gly and Pro. Processing of the resulting precursor (pcA13PG16; Fig. 3,
lane 4) resulted in three protein bands: unprocessed
precursor and bands corresponding to wild-type AGK (compare with
lane 1) and a larger AGPR that comigrated with AGPR*
generated from pcPG16 and pcGTPG5 (lanes 2 and
3). Processing was completely inhibited in
1,10-phenanthroline/EDTA pretreated mitochondria (Fig. 3, lane
9). This result indicates that substitution of Pro for Ala in the
wild-type sequence RRPAL is not sufficient to generate a cleavage site
at RRA
AL. We conclude that arginine pairs by themselves are not
sufficient for proteolytic cleavage in the connector region of
pAGK-AGPR.
Role of Threonine in Recognition by MPP-- A threonine residue is often found 2 or 3 residues downstream from the cleavage site of mitochondrial targeting sequences. Interestingly, a threonine residue is also present 3 residues downstream from the cleavage site at the N terminus of the mature domain of the distal AGPR. To investigate the role of Thr+3 in the AGPR domain in processing of the connector region, this residue was changed to a proline in a wild-type background to generate the construct pcP3. In vitro processing of the resulting precursor resulted in three protein bands (Fig. 3, lane 5). The upper band corresponded to remaining unprocessed precursor; the two lower bands corresponded to wild-type AGK and to a larger sized AGPR (AGPR*) containing the N-terminal extension indicative of processing exclusively at the second cleavage site. Processing of the precursor was completely inhibited in 1,10-phenanthroline/EDTA pretreated mitochondria (Fig. 3, lane 10). Thus, proteolytic cleavage at the N terminus of the distal AGPR was prevented by the threonine to proline substitution. These results suggest that threonine is a critical residue of the processing motif in the connector region of the precursor. The large fraction of unprocessed precursor (~35% as measured by scanning densitometry) protected from proteinase K digestion suggests that processing at the second site is much less efficient than processing at the N terminus of AGPR.
Western Blot Analysis of in Vivo Expressed Constructs Containing
Mutations in the Connector Region of the Precursor--
To analyze
processing of pAGK-AGPR in vivo, strain LA358
(AGKAGPR
) was transformed with constructs
containing different amino acid substitutions in the connector region
of pAGK-AGPR. Fig. 4 shows the results of
immunoblot analysis of transformants obtained with the construct
pgGTPG4-Hph (Arg
15, Arg
14,
Arg
3, and Arg
2 to GTPG). This construct is
the genomic equivalent to the cDNA construct pcGTPG5, which was
processed in vivo at only the upstream second cleavage site.
The immunoblot was probed with anti-AGK antisera (Fig. 4, panel
A) and reprobed with anti-AGPR antisera (Fig. 4, panel
B). The recipient strain, LA358, has no detectable AGK or AGPR.
AGK in the transformants (lanes T4-T16) comigrated with
wild-type AGK (74a). This result was consistent with the in
vitro analysis of processing that showed generation of a wild-type AGK for the corresponding construct. The results for AGPR showed three
types of transformants. One type expressed a wild-type AGPR (compare
with 74A). A second type expressed only a larger sized AGPR (AGPR*),
and a third type expressed both wild-type and mutant AGPR*
(mAGPR*). These results were also observed with the
construct pgPG15-Hph (Arg
3 and Arg
2 to Pro
and Gly) (data not shown). The presence of the larger AGPR is
consistent with the results observed in vitro, where
processing only at the upstream cleavage site took place. The presence
of wild-type AGPR can be explained by homologous recombination
following transformation with the break point of the crossover located
upstream of the site of the mutation. The presence of wild-type and
mutant forms in the same isolate would result if the transformant is a
heterokaryon containing both types of nuclei. These results confirm
that processing of precursors containing amino acid substitutions at
positions
2 and
3 from the N terminus of AGPR results in proteolytic cleavage at only the second site.
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Identification of the Second Cleavage Site--
To identify the
second site of proteolytic cleavage, the larger form of AGPR (AGPR*)
was isolated from mitochondria of transformants pgPG15-Hph and
pgGTPG4-Hph by immunoprecipitation, and the amino termini were
sequenced (see "Materials and Methods"). The results are shown in
Fig. 6. The variant AGPR* from both
transformants had the same N-terminal sequence, indicating that
cleavage occurred at the position between Tyr20 and
Ser
19 of the connector region. This resulted in an AGPR
protein with a 20-amino acid N-terminal extension. Comparison of
the sequence RG
YLT (first cleavage) and
RGY
ST (second cleavage) reveals that the
scissile bonds are in a sequence flanked by well conserved arginine and
threonine residues (in boldface type). This suggests important roles
for arginine and threonine as part of the cleavage site. Both
residues appear to be critical for processing, since substitution of
either of them results in misprocessing at the AGPR N terminus (see
above).
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Enzyme Activities and Feedback Inhibition in Uncleaved AGK-AGPR
Precursor--
A question of major interest is whether processing of
pAGK-AGPR into two mature proteins in the mitochondrial matrix is
required for acetylglutamate kinase and acetylglutamyl-phosphate
reductase to be active. Transformants expressing AGPR* with the
N-terminal extension and those expressing an uncleaved precursor were
able to grow in minimal medium at a rate comparable with that of wild type (not shown). This result indicated that processing was not essential for the functioning of these enzymes in the mitochondrial matrix. To obtain more direct evidence that proteolytic processing was
not required, AGK and AGPR activities were measured in purified mitochondria from transformants expressing the uncleaved AGK-AGPR precursor. Transformants expressing uncleaved precursors derived from
pgG22PG42-Hph (Arg22, Arg
3, and
Arg
2 to GPG) and pgP22PG54 (Arg
22,
Arg
3, and Arg
2 to PPG) exhibited activities
equal to or greater than wild type (Table
II). In addition, feedback inhibition of
AGK was not significantly affected by the lack of cleavage. These
results show that the two-step proteolytic processing at the connector
region of the pAGK-AGPR precursor to generate two mature proteins is
not required for the biological activity of the two protein
domains.
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DISCUSSION |
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The 871-amino acid AGK-AGPR polyprotein precursor of N. crassa is organized as two protein domains separated by a 200-amino acid connector region with a 45-amino acid mitochondrial targeting sequence at the N terminus, which is cleaved into two mature proteins in the mitochondria (4). Proteolytic cleavage of leader sequences of mitochondrially targeted proteins has been shown to be performed in one or two steps by MPP (13-17) or in more than one step by two unrelated enzymes: MPP and a mitochondrial intermediate peptidase (31-33). MPP and mitochondial intermediate peptidase recognize and cleave different amino acid sequences. Processing of the internal processing sequence of the AGK-AGPR precursor involves removal of 22 residues upstream of the N terminus of the AGPR domain. Both cleavage events are inhibited by 1,10-phenanthroline and EDTA (this report) and take place upon incubation of in vitro synthesized precursor with purified MPP (4).
Kinetic studies of processing using oligopeptides with different amino
acid sequences and lengths showed the necessity for at least 16 residues consisting of 11 residues upstream and 5 residues downstream
from the cleavage site for effective hydrolysis by MPP (36). Analysis
of amino acid sequences around the cleavage site of mitochondrial
targeting peptides cleaved by MPP has revealed no consensus amino
acids; however, several motifs conserved within various subgroups have
been identified (14, 15). In one subgroup, arginine residues were often
observed at positions 2,
3,
10, and
11, relative to the
scissile bond. In the connector region of pAGK-AGPR, arginine residues
are present at positions
2,
3,
14, and
15 relative to the N
terminus of mature AGPR. No motif resembling a cleavage site for
mitochondial intermediate peptidase was identified. Although both MPP
and mitochondial intermediate peptidase are metal-dependent
proteases inhibited by 1,10-phenanthroline and EDTA, analysis of the
cleavage sites suggests that the two-step cleavage is carried out by
MPP alone. Thus, maturation of pAGK-AGPR involves three proteolytic
cleavage events: cleavage of the leader (targeting) peptide and two
cleavages removing the internal processing sequence.
Cleavage at the connector region of the AGK-AGPR precursor occurs at
sequences RGYST and RG
YLT, 20 amino acids apart. The results of
in vitro import and processing of the mutant precursors support an important role for arginine at
2 or
3 and revealed the
importance of a threonine at +2 or +3 relative to the cleavage site. A
third sequence motif containing two arginines (RRPAL) occurs between
the two cleavage sites. Substitution of alanine for proline, creating
the sequence RRAAL, did not result in a new cleavage site.
The involvement of secondary structure in recognition by processing
peptidases has been reported (34, 35). Processing sites are believed to
be in exposed and flexible regions of the precursors situated in, or
immediately next to, -turns or larger loops. The
-turn may
constitute a key feature in the proteolytic processing reaction by
providing a favorable conformation for optimal substrate-enzyme active
site recognition. An average
-helix is 17 Å long and contains 11 residues, which corresponds to three turns. Individual
-sheets are,
on average, 20 Å long, which corresponds to 6.5 residues (37). Most
loops and turns occur at the surface and contain relatively polar
residues. The most common type of loops link anti-parallel
-strands
(
-turns) or adjacent
-sheets (
-hairpin). Predictions of local
secondary structures in the connector region of the pAGK-AGPR precursor
were performed using the combined algorithms of Chou-Fasman and
Robson-Garnier (Mac Vector protein analysis package). The connector
region of pAGK-AGPR is not predicted to form an amphipathic
-helix.
However, local sheets and turns are predicted.
We examined possible correlations between the predicted perturbations
in the local secondary structure of the connector region of the
precursor and the presence or absence of proteolytic cleavage as shown
by the processing assays. The wild-type precursor (Fig. 7A) shows three short
stretches with the potential to form -turns.
-Turns are apparent
in the N terminus to C terminus direction around positions
40,
19,
and
3. The formation of a
-sheet is apparent around position +2 in
the AGPR domain. Mutations in the arginine pair at positions
2 and
3 from the N terminus of the AGPR domain (pcPG16; Fig. 7B)
decreased the predicted length of the
-turn at
3; the rest of the
structure was not affected. Mutations at the arginine pairs at
positions
2 and
3 and positions
14 and
15 from the C terminus
of the AGPR domain (pcGTPG5; Fig. 7C) eliminated the turn
predicted to be centered at
19. Substitution of Arg
22
with Gly
22, combined with the substitutions at
2 and
3 (pcG22PG42; Fig. 7D) increased the length of the turn
centered at
19. Substitution of Arg
22 with
Pro
22 (pcP22PG54; Fig. 7E) shifted the turn
centered at
19 toward the C terminus. Substitution of
Pro
13 for Ala
13 in combination with changes
at
2 and
3 resulted in the same structure predicted in Fig.
7B, where only changes at
2 and
3 were made (pcA13PG16;
Fig. 7F). Substitution of Thr+3 with a
Pro+3 in a wild-type background (pcP3; Fig. 7G)
resulted in complete loss of the turn centered at
3 and the
-sheet
at +2. Therefore, specific amino acid replacements in the connector
region of the precursor are predicted to affect the local secondary
structure of the region. We found that in general, loss of proteolytic
processing between the
1 and +1 residues could be correlated with a
decrease in the length or the complete loss of the
-turn centered at
3. However, loss of the
-turn centered at
19 did not affect
processing at
20 (Fig. 7C). Loss of processing at position
20 is likely to result from the substitution of the arginine residue
at
22. Thus, the predicted changes in the secondary structure caused by the amino acid substitutions consisted of variations in the length
and position of
-turns, and some of these changes may be correlated
with loss of proteolytic cleavage. Protein secondary structure as well
as specific amino acid residues have been shown to be important for the
processing of mammalian and plant precursors of mitochondrial proteins
(38, 39).
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The biological advantage of targeting a fusion protein versus two independent proteins is not obvious; several possible reasons have been postulated (3, 4). Facilitation of a multienzyme complex formation in the mitochondrial matrix for the channeling of labile intermediates is one explanation. In yeast, the His4 locus encodes a multifunctional protein with three different functional domains (40). Proteolytic processing of the His4 protein is not required for function, since the purified native protein contains the three activities.
AGK and AGPR activity assays using transformants expressing only an
unprocessed pAGK-AGPR revealed that proteolytic cleavage in the
connector region is not required for activity. In addition, the
functions of AGK and AGPR in vivo were not affected as
assessed by the ability of the unprocessed precursor to support growth of AGKAGPR
mutants. We conclude that
conformational changes that may be associated with the lack of
proteolytic processing of the fused proteins did not have a dramatic
effect on the function of the uncleaved enzymes. Moreover, the lack of
processing did not affect feedback inhibition of AGK by arginine.
However, whether interaction with other proteins in the mitochondrial
matrix has been affected by the lack of processing remains to be
studied.
The results shown here indicate that two proteolytic cleavages in the
connector region of the precursor occur to release the two protein
domains. Since cleavage in the connector region of the precursor takes
place at positions 19 and
20 and positions
1 and +1 from the N
terminus of the mature AGPR, we propose that the enzyme responsible,
possibly MPP, scans the connector region as the precursor reaches the
mitochondrial matrix. The enzyme recognizes a first sequence for
cleavage at Arg
22, and it cleaves two residues toward the
C terminus. The scanning continues as import of the precursor
progresses. The enzyme skips the arginine pair at positions
15 and
14, probably due to the absence of a threonine residue in the
recognition sequence. As scanning continues, the enzyme recognizes the
arginine pair at positions
3 and
2 and cleaves 2 residues
C-terminal. Since some unprocessed precursor is still observed after
cleavage at the second site, cleavage at the N terminus of AGPR seems
to be more efficient than the cleavage at the upstream position. At
this point, folding of the processed distal domain into a functional protein begins, and the enzyme falls off the substrate.
What is the role of processing of the pAGK-AGPR precursor in the metabolism of arginine? It has been suggested that this precursor resulted from the fusion of two genes for independently targeted proteins. It has been hypothesized that this arrangement may result in a more efficient delivery of the proteins to the mitochondria or to stabilization of one or both proteins prior to their assembly into their mature functional forms (3, 4, 29). The increased activity of AGPR in transformants expressing the uncleaved polyprotein (Table II) suggests that possible effects on protein stability can be further enhanced by retaining the two enzymes as a polyfunctional protein. Because such transformants grow normally, the advantage of cleavage must be subtle, and its identification will require more extensive analysis of the properties of the polyprotein and independent enzymes.
Identification of the internal processing sites allowed identification of the precise C terminus of AGK. This confirmed the existence of a ~185-amino acid subdomain that is absent from prokaryotic homologs (4). A role for this subdomain may be related to the feedback inhibition properties of AGK in the mitochondrial matrix. Another possibility is that this subdomain plays a role in protein-protein interaction: mutations in arg-6 can affect the activity or feedback sensitivity of acetylglutamate synthase encoded by the unlinked arg-14 gene (28, 41, 42). Processing of the precursor may be needed for this interaction to occur. Kinetic analysis of the uncleaved precursor and examination of its interaction with other proteins may reveal new aspects of the role of proteolytic processing of the AGK-AGPR precursor on the metabolism of arginine.
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ACKNOWLEDGEMENTS |
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We are grateful to Rowland H. Davis for the gift of Neurospora arg-6 mutant strains, to Angela Wandinger-Ness for anti-AGK and anti-AGPR antiserum, and to Silvia Diaz-Perez for the plasmid construct pRW7. We are also grateful to Audree Fowler of the UCLA Protein Sequencing Facility for advice in preparing samples for sequencing and the sequencing of the proteins. We thank Albert J. Courey for valuable suggestions, Terri Moulds for helpful discussions, and Gloria E. Turner and Steven F. Gessert for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM47631 and National Science Foundation Grant DCB9119151 (to R. L. W.).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: Hitachi Chemical Research Center, Inc., Plumwood
House, Irvine, CA 92715.
§ To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, UCLA, Los Angeles, CA 90095-1569. Tel.: 310-825-3621; Fax: 310-206-5213; E-mail: weiss{at}chem.ucla.edu.
1
The abbreviations used are: AGK,
N-acetylglutamate kinase; AGPR,
N-acetyl--glutamyl-phosphate reductase; MPP,
mitochondrial processing peptidase; PCR, polymerase chain reaction;
MOPS, 4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel
electrophoresis; CAPS, 3-(cyclohexylamino)propanesulfonic acid.
2 J.-H. Kim and R. L. Weiss, unpublished results.
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REFERENCES |
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