Processing and Function of a Polyprotein Precursor of Two Mitochondrial Proteins in Neurospora crassa*

Lilian Parra-Gessert, Kenneth KooDagger , Joaquin Fajardo, and Richard L. Weiss§

From the Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095-1569

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In Neurospora crassa, the mitochondrial arginine biosynthetic enzymes, N-acetylglutamate kinase (AGK) and N-acetyl-gamma -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-Glydown-arrow Tyr-Leu-Thr at the N terminus of the AGPR domain and Arg-Gly-Tyrdown-arrow 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.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-gamma -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, alpha -MPP and beta -MPP; alpha -MPP is soluble in the matrix, and beta -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 alpha -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 alpha -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.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Strains and Growth Conditions-- Escherichia coli strains DH5alpha 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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Fig. 1.   A, domain organization of the polyprotein precursor (96 kDa, 871 amino acids) encoded by the arg-6 locus in N. crassa. The mitochondrial targeting sequence at the N terminus of the precursor (44 amino acids) is indicated as the mitochondrial targeting sequence (MTS). The kinase domain (52 kDa, ) and reductase domain (37 kDa, ) are indicated as AGK and AGPR, respectively. The internal processing sequence located in the connector region (~200 amino acids) of the precursor is indicated as the internal processing sequence (IPS). The other portion of the connector region is indicated as the eukaryotic domain. Abbreviations for restriction endonucleases are as follows. B, Bsu361; A, ApaI; C, ClaI. B, diagram of internal processing sequence mutations; amino acid substitutions in each construct are indicated in boldface letters.

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 pgH2Delta Bsu361 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 DH5alpha (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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Table I
Oligonucleotides designed for site-directed and PCR mutagenesis
Bases in bold face type indicate intended mutations and add-ons.

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 beta -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 beta -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-gamma -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.

Acetylglutamyl-phosphate reductase activity was assayed by following the increase in fluorescence as NADP+ was converted to NADPH (3). The reaction mixtures contained 0.1 M glycine, pH 9.3, 1.33 mM acetylglutamate 5-semialdehyde, 25 mM K2HPO4, pH 9.3, 0.67 mM NADP+ (freshly prepared), and 100, 200, or 300 µl of mitochondrial extract in a final volume of 3 ml. The reaction was initiated by the addition of the substrate, acetylglutamate semialdehyde, and the increase in fluorescence was followed using a Gilson Spectra/Glo filter fluorometer (excitation filter, 330-380 nm; emission filter, 430-600 nm). The reactions were carried out at 25 °C. The activity is expressed as the change in fluorescence/min and represents the average from 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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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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Fig. 2.   Import and processing of wild-type pAGK-AGPR and mutated derivatives by isolated mitochondria. A, import and processing of precursors derived from pcPG16 (Arg-2 and Arg-3 to Gly and Pro) and pcGTPG5 (Arg-2 and Arg-3 to Gly and Pro, and Arg-14 and Arg-15 to Thr and Gly). Precursor proteins were generated in a rabbit reticulocyte translation system in the presence of [35S]methionine. Import reactions were carried out as indicated under "Materials and Methods." After import, mitochondria were reisolated and either not treated (lanes 1-3) or treated (lanes 4-6) with 100 µg/ml proteinase K (PTK). Samples were analyzed by SDS-PAGE and fluorography. Mutant AGPR protein is indicated as AGPR*. Plasmids carrying the mutated precursor genes are identified at the top of the lanes. B, import and processing of precursors from pcG22PG10 (Arg-2 and Arg-3 to Gly and Pro, and Arg-22 to Gly) and pcP22PG54 (Arg-2 and Arg-3 to Gly and Pro, and Arg-22 to Pro). Reisolated postimport mitochondria were treated with 50 µg/ml proteinase K (lanes 1-5). Unprocessed precursor protein is indicated by P. Truncated precursor is indicated by T. Mutant AGPR protein is indicated as AGPR*. Plasmids carrying the mutant genes are identified at the top of the lanes. C, import and processing of precursors from pcG22PG42 and pcP22PG54. Postimport mitochondria were reisolated and either not treated (lanes 1-5) or treated (lanes 6-10) with 100 µg/ml proteinase K. Samples were analyzed by SDS-PAGE and fluorography. Names of the constructs examined are indicated at the top of each lane. Precursor protein is indicated by P. D, diagram of the processed and unprocessed products from wild-type AGK-AGPR and mutant derivatives.

To investigate the role of the arginine residues at positions -14 and -15, mutations were introduced at these sites in a background that contained the previous changes at -2 and -3. Fig. 2A (lanes 3 and 6) shows that processing of the precursor derived from pcGTPG5, which contains the changes Arg-15, Arg-14 to Gly-15, Thr-14 and Arg-3, Arg-2 to Pro-3, Gly-2 resulted in an AGK protein band that comigrated with wild-type AGK (compare with lane 1) and an AGPR band with a higher molecular mass than wild-type AGPR. The larger AGPR band comigrated with the AGPR* observed in the processing of the precursor from pcPG16 (lanes 2 and 5). Thus, substitution of the arginine pair at -14 and -15 had no effect on processing at the second cleavage site.

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.

The role of arginine at position -22 can also be observed with construct pcG22PG10 (Fig. 2B, lane 4). The truncated precursor (T) was imported into mitochondria (protected from proteinase K) but was not cleaved (higher molecular mass than wild-type AGK). A full-length precursor containing the substitution at Arg-22 to Gly-22 (pcG22PG42; Fig. 2C, lanes 4 and 9) yielded similar results to those obtained with the precursor derived from pcP22PG54 (Fig. 2C, lanes 5 and 10); both precursors were imported (protected from proteinase K digestion) but were not processed in the mitochondria. However, some proteolytic degradation in the mitochondrial matrix was observed. These results indicate that proteolytic cleavage occurs at two different positions in the connector region of the precursor (Fig. 2D).

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 -2 or -3 relative to the cleavage sites and the sensitivity to metal ion chelators suggest that MPP is likely to be responsible for the two-step proteolytic processing of the connector region of pAGK-AGPR.


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Fig. 3.   Effect of inhibitors of the mitochondrial processing peptidase on the import and processing of wild-type and mutant precursors. Isolated mitochondria were preincubated in import buffer without (lanes 1-5) or with (lanes 6-10) 5 mM EDTA and 2.5 mM 1,10-phenanthroline (o-Phe) for 5 min at 25 °C. 35S-Labeled precursor was added, and incubation was continued for 30 min at 25 °C. Mitochondria were reisolated and treated with proteinase K (50 µg/ml) at 0 °C for 30 min. To stop proteolysis, 1 mM phenylmethylsulfonyl fluoride was added, and the mixture was incubated for 5 min at 0 °C. Postimport mitochondria were reisolated, and labeled proteins were visualized by SDS-PAGE and fluorography. Lanes 1 and 6, wild-type precursor; lanes 2 and 7, precursor from pcPG16; lanes 3 and 8, pcGTPG5; lanes 4 and 9, pcA13PG16; lanes 5 and 10, pcP3.

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 RRAdown-arrow 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 (AGK-AGPR-) 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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Fig. 4.   Expression and processing of precursor derived from construct pgGTPG4-Hph. DNA from pcGTPG5 (Arg-2 and Arg-3 to Gly and Pro, and Arg-14 and Arg-15 to Thr and Gly) was subcloned into a vector containing the Hygr selective marker (see "Materials and Methods"). The resulting construct, pgGTPG4-Hph, was transformed into strain LA358 (arg-6, allele CD118), which lacks AGK and AGPR proteins. A, Western blot analysis of initial heterokaryon transformants probed with anti-AGK antisera. Control lanes are wild-type strain, 74A (lane 1), and recipient strain, LA358 (lane 2). B, the same blot reprobed with anti-AGPR antisera.

Processing of precursors derived from pgG22PG42-Hph (Arg-22, Arg-3, and Arg-2 to Gly-22, Pro-3, and Gly-2) and pgP22PG54-hph (Arg-22, Arg-3, and Arg-2 to Pro-22, Pro-3, and Gly-2), the genomic equivalents of pcG22PG42 and pcP22PG54, was analyzed by Western blotting; no mature AGK was observed (Fig. 5). These results are consistent with those observed in vitro and substantiate the conclusion that substitution at Arg-22 in combination with substitutions at Arg-2 and Arg-3 in the connector region of the precursor generates an uncleavable precursor.


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Fig. 5.   Expression and processing of precursor derived from constructs pgG22PG42-Hph and pgP22PG54-Hph. DNAs from constructs pgG22PG42-Hph (Arg-22, Arg-3, and Arg-2 to Gly, Pro, and Gly) and pgP22PG54-Hph (Arg-22, Arg-3, and Arg-2 to Pro, Pro, and Gly) were used to transform strain LA358 (arg-6, allele CD118), and transformants were selected by hygromycin resistance (see "Materials and Methods"). Crude extracts were prepared from initial hygromycin resistance isolates, and proteins were analyzed by immunoblot with anti-AGK antiserum. Lanes 1 and 8, molecular weight (MW) markers; lane 2, wild-type construct; lane 3, recipient strain LA358. Hygr transformants with pgG22PG42-Hph and pgP22PG54-Hph are shown (as indicated at the top). Wild-type protein is indicated by AGK, and unprocessed precursor is indicated by P.

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 Tyr-20 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 RGdown-arrow YLT (first cleavage) and RGYdown-arrow 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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Fig. 6.   Determination of second cleavage site in the connector region of the precursor by N-terminal sequencing of mutant AGPR proteins. Mutant proteins were purified by immunoprecipitation with anti-AGPR antiserum from crude extracts of the corresponding transformants and blotting to a polyvinylidene difluoride membrane. A second cleavage site was identified upstream of the N terminus of wild-type AGPR. A, wild-type sequence upstream of the N terminus of AGPR. B, mutant sequence from pgPG15-Hph (Arg-3 and Arg-2 to Pro and Gly). C, mutant from pgGTPG4-Hph (Arg-15 and Arg-14 to Gly and Thr, and Arg-3 Arg-2 to Pro and Gly). The positions of cleavages are indicated by arrows. Positively charged residues are indicated in boldface letters. Numbers under the amino acid indicate position relative to the N terminus of the AGPR domain. Italics indicate amino acid sequence data.

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 (Arg-22, 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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Table II
AGK and AGPR specific activities of uncleaved AGK-AGPR precursors
All strains and transformants were grown in minimal medium except LA358, which was supplemented with 0.2 mg/ml arginine. Mitochondria were purified from the indicated strains and transformants, lysed, and assayed for acetylglutamate kinase and acetylglutamyl-phosphate reductase activities as described under "Materials and Methods." The values shown are an average of assays performed in triplicates.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 RGYdown-arrow ST and RGdown-arrow 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, beta -turns or larger loops. The beta -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 alpha -helix is 17 Å long and contains 11 residues, which corresponds to three turns. Individual beta -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 beta -strands (beta -turns) or adjacent beta -sheets (beta -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 alpha -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 beta -turns. beta -Turns are apparent in the N terminus to C terminus direction around positions -40, -19, and -3. The formation of a beta -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 beta -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 beta -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 beta -turn centered at -3. However, loss of the beta -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 beta -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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Fig. 7.   Predicted secondary structure of the connector region of wild-type and mutant precursors based on the combined algorithms of Chou-Fasman and Robson-Garnier. Predictions for alpha -helices (Hlx), beta -sheets (Sht), or beta -turns (Trn) starting at position -51 (T) to position +11 (V), are indicated by shaded and black boxes. A window size of 7 was used. A, wild-type sequence; B, substitution of Arg-2 and Arg-3 by Gly and Pro; C, substitution of Arg-2 and Arg-3 by Gly and Pro, and Arg-14 and Arg-15 by Thr and Gly; D, substitution of Arg-2 and Arg-3 by Gly and Pro, and Arg-22 by Gly; E, substitution of Arg-2 and Arg-3 by Gly and Pro, and Arg-22 by Pro; F, substitution of Arg-2 and Arg-3 by Gly and Pro, and Arg-13 by Ala; G, substitution of Thr+3 by Pro.

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 AGK-AGPR- 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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger 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-gamma -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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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