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J. Biol. Chem., Vol. 277, Issue 46, 43792-43798, November 15, 2002

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A Higher Plant Mitochondrial Homologue of the Yeast m-AAA Protease

MOLECULAR CLONING, LOCALIZATION, AND PUTATIVE FUNCTION^*

Marta Kolodziejczak, Anna Kolaczkowska, Bartosz Szczesny, Adam Urantowka, Carina Knorpp^§, Jan Kieleczawa^¶, and Hanna Janska^**

From the  Institute of Biochemistry and Molecular Biology, University of Wroclaw, Tamka 2, Wroclaw 50-137, Poland, the ^§ Department of Plant Biology, Swedish University of Agricultural Sciences, Box 7080, Uppsala S-750 07, Sweden, and the ^¶ Department of Biology, Brookhaven National Laboratory, Upton, New York 11973

Received for publication, April 19, 2002, and in revised form, September 9, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mitochondrial AAA metalloproteases play a fundamental role in mitochondrial biogenesis and function. They have been identified in yeast and animals but not yet in plants. This work describes the isolation and sequence analysis of the full-length cDNA from the pea (Pisum sativum) with significant homology to the yeast matrix AAA (m-AAA) protease. The product of this clone was imported into isolated pea mitochondria where it was processed to its mature form (PsFtsH). We have shown that the central region of PsFtsH containing the chaperone domain is exposed to the matrix space. Furthermore, we have demonstrated that the pea protease can complement respiration deficiency in the yta10 and/or yta12 null yeast mutants, indicating that the plant protein can compensate for the loss of at least some of the important m-AAA functions in yeast. Based on biochemical experiments using isolated pea mitochondria, we propose that PsFtsH-like m-AAA is involved in the accumulation of the subunit 9 of the ATP synthase in the mitochondrial membrane.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Accumulating evidence suggests that energy-dependent mitochondrial proteases play an important role in the overall regulation of mitochondrial biogenesis and function (1, 2). All of them are derived from bacterial ancestors. Among bacterial ATP-dependent proteases only a zinc metalloprotease called FtsH¹ is embedded in the membrane and is essential for cell growth (3). FtsH is a bifunctional enzyme, in which a metalloprotease domain is fused at the C terminus of a well-conserved AAA module.

Although most bacteria have only one FtsH protease, yeast mitochondria contain three, designated Yta10p (Afg3p), Yta11p (Yme1p), and Yta12p (Rca1p) (4). They form two high molecular weight complexes in the inner mitochondrial membrane. The hetero-oligomeric m-AAA complex composed of Yta10p·Yta12p faces the matrix, whereas the homo-oligomeric i-AAA complex, containing Yta11p, exposes the active site to the intermembrane space.

The m-AAA complex is involved in a number of cellular activities. It mediates the degradation of unassembled membrane polypeptides and is required for assembly of the mitochondrial respiratory and ATP synthase complexes (4-6). Moreover, the m-AAA protease affects the splicing of some mitochondrial gene transcripts (6). Prohibitins were identified as negative regulators of the m-AAA proteolytic activity by stabilization of newly synthesized mitochondrial translation products (7-9). Young and co-workers (10) reported that peptides generated upon proteolysis of inner-membrane proteins by the m-AAA protease are released from mitochondria and may allow communication between mitochondria and their cellular environment.

Mitochondrial m-AAA proteases have been analyzed mainly in yeast. Recently, based on sequence homology, membrane topology, and biochemical characterization, a homologue of Yta10p and Yta12p has been identified in filamentous fungus Neurospora crassa (11). The protein products of human genes AFG3L1, AFG3L2, and SPG7 share significant homology to m-AAA (12). A mutation in SPG7 has been correlated to a hereditary form of spastic paraplegia (13). However, the function of these gene products on the molecular level remains to be elucidated.

Although mitochondrial AAA metalloprotease has not yet been characterized in plants, searches of the sequence data base reveal the existence of at least eleven isomers of the FtsH-like proteases in Arabidopsis thaliana (14).² Seven of them are predicted as being chloroplastic, whereas the two called FtsH3 and FtsH10 are predicted to be located in the mitochondria. To date, two of the chloroplastic isomers, FtsH1 and FtsH2, have been cloned, and their functions in the biogenesis of plastids have been partially characterized (15-17).

In contrast to the chloroplastic FtsH-like proteases, there is no information about plant mitochondrial homologues except the sequence data from A. thaliana and the prediction of their respective location. Here, we report experimental identification and characterization of the AAA metalloprotease (PsFtsH) in pea mitochondria, which is able to complement respiration deficiency in yeast lacking Yta10p and/or Yta12p.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plant Growth and Isolation of Organelles-- Garden pea plants (Pisum sativum) were grown at 20 °C for 12 days under a 12-h photoperiod. Mitochondria were isolated from leaves as described by Hakansson et al. (18), and intact chloroplasts were isolated as described by Szczepaniak et al. (19).

Identification of a Full-length Pea FtsH cDNA-- To identify pea proteins homologous to yeast m-AAA proteases, the computer search was performed within A. thaliana genomic and expressed sequence tag databases. Two genomic sequences, AC005315 and AC022464, highly similar to YTA10 and YTA12 yeast genes, were found. On the basis of their multiple sequence alignments, a pair of primers (forward: 5'-GATGCTAAAATTCCAAAAGGTGC-3' and reverse: 5'-CAAACATTAGCAATATCAGCTCC-3') was designed to PCR-amplify a 525-bp fragment using genomic DNA from the pea as a template. The PCR product was cloned into a TOPO vector (TOPO PCR cloning kit, Invitrogen) and sequenced. Analysis of the sequence confirmed a high similarity to the AC005315 and AC022464 A. thaliana genes.

To obtain the full-length cDNA, the 5' and 3' rapid amplification of the cDNA ends (RACE) procedures were done on pea mRNA using a kit (Marathon Ready, Clontech). Primers derived from the partial pea gene sequence were as follows: for the 3' RACE forward GSP1 (5'-GGAAAAGGGGCCGTGGAGGTTTCTC-3'), for the 5'RACE, an antisense GSP2 (5'-GCCCAGGCCTAAGTAGGGCATTGTC-3'). The resulting 1.2-kb (3'-end) and 1.5-kb (5'-end) fragments were inserted into the TOPO vectors. Five independent clones for each RACE-PCR were sequenced. The whole cDNA sequence composed of the two overlapping clones was 2637 bp long. It comprises a 71-bp 5'-untranslated region, an open reading frame of 2433 bp, and a 133-bp-long 3'-untranslated region. A putative polyadenylation signal (AATAAA) was detected in the 3'-untranslated region.

On the basis of the obtained pea cDNA sequence two specific primers were designed and used for the end-to-end PCR amplification (for the 5'-end of the gene: 5'-ATGATTTTCTCAAGGATTGGGCGC-3'; for the 3'-end of the gene: 5'-CTATGTGGGAACAACCTCTGGCTCTAG-3'; start and stop codons are underlined). The final 2.4-kb PCR fragment was cloned into the TOPO vector yielding TOPO-PsFtsH. DNA sequencing was performed using protocols recommended by ABI, and samples were run on an ABI373 automated DNA sequencer as recommended by the manufacturer (Applied Biosystems).

Sequence Analysis-- Sequencing traces were edited and assembled using Sequencher V3.1.1 (Gene Codes Corp.) software. The cDNA sequence analysis and nucleotide and protein data base searches were performed on NCBI using TBLASTN, BLASTP, BLASTX, and FASTA programs. Multiple sequence alignments were generated by the use of ClustalW, version 1.7. Protein domains were identified on the deduced amino acid sequence by the PFAM software (www.sanger.ac.uk/Pfam/). I-PSORT and MITOPROT algorithms were used to predict the putative mitochondrial transit peptide.

SDS-PAGE and Western Blotting-- Total protein extracted from mitochondrial or chloroplast fractions were solubilized and resolved on SDS-PAGE according to Laemmli (20) (12% gel unless otherwise indicated) and then transferred onto nitrocellulose membranes. Blots were probed with rabbit immune sera raised against a synthetic peptide derived from the A. thaliana FtsH3. The synthetic peptide IYLKKIKLDHEPSYYS, corresponding to amino acid residues 542-558 of FtsH3, was conjugated to keyhole limpet hemocyanin using Sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate, Pierce, Rockford, IL). Agrisera (Umea, Sweden) produced a rabbit polyclonal antiserum raised against this peptide. The homologous peptide from PsFtsH differs only in one amino acid, there is a tyrosine instead of a lysine at position 4. To check whether the antisera could detect the pea protein, PsFtsH containing an N-terminal His tag was expressed in Escherichia coli. The expressed protein was detected by antibodies against the His tag as well as by the FtsH3 antiserum.

Antibodies against mitochondrial (NAD3) and chloroplast (cytochrome f) proteins were used to confirm the purity of the organellar fractions. Antibodies against the F₁-ATPase of spinach mitochondria and a monoclonal antibody directed against pigeon cytochrome c (clone 7H8.2C12, Pharmingen) were used as controls for matrix and intermembrane space fractions of mitochondria. Immunodetections were performed using the Enhanced Chemiluminescence detection system (ECL, Roche Molecular Biochemicals). Protein concentration was determined by the BCA method (21).

In Vitro Translation and Import into Isolated Mitochondria-- The BamHI-NotI fragment containing PsFTSH was subcloned from p[CYC1-PsFTSH], the construct described below, into pBluescript (Stratagene) under the control of the T7 promoter. Precursor protein was synthesized by in vitro transcription and translation using the TNT-coupled reticulocyte lysate system (Promega) in the presence of [³⁵S]methionine, according to the manufacturer's instruction. Import assays were carried out as described by Knorpp et al. (22). Labeled proteins were separated on SDS-PAGE on 8% gel according to Laemmli (20) and visualized by autoradiography. In vitro processing reactions were performed according to Whelan et al. (23).

Functional Expression of PsFtsH in Yeast-- The following yeast strains were used in this study: W303 (MAT a ade2-1 his3-11, 15 trp1-1 leu2, 112 ura3-52), YHA101 (MATa ade2-1 his3-11, 15 trp1-1 leu2, 112 ura3-52 yta10::URA3), YHA201 (MATa ade2-1 his3-11, 15 trp1-1 leu2, 112 ura3-52 yta12::HIS3), YHA301 (MATa ade2-1 his3-11, 15 trp1-1 leu2, 112 ura3-52 yta12::HIS3 yta10::URA3). Cells were grown on YP medium (1% yeast extract, 2% peptone) containing 2% glucose or 2% galactose and 0.5% lactate.

Construction of p[CYC1-PsFTSH] was as follows: DNA sequences encoding PsFtsH were PCR-amplified from the TOPO-PsFtsH construct using Y12a forward (5'-GTTCGGATCCATGATTTTCTCAAGGATTG-3') and YT12b reverse (5'-GAATGCGGCCGCCTATGTGGGAACAACCTC-3') primers (the start and stop codons of PsFtsH are in italic, restriction enzyme sites are underlined). The 2.3-kb PCR product was digested with BamHI and NotI and ligated into BamHI-NotI sites of yeast shuttle vector pCM185 (24).

Yeast strains were transformed by the lithium acetate method (25). For complementation analysis, yeast cells were grown overnight in liquid complete synthetic drop-out medium (0.7% yeast nitrogen base, 2% glucose) lacking appropriate auxotrophic markers. The cultures were then diluted into fresh medium and grown until mid-log phase. Serial dilutions of the same starting number of cells were then replica-plated on YPGlucose or YPGlycerol (1% yeast extract, 1% peptone, 4% glycerol) plates and incubated at 30 °C or 37 °C for 3 days.

In Organello Protein Synthesis and Analysis of Labeled Proteins-- The synthesis of mitochondrially encoded proteins was carried out at 4 °C. Each sample contained a 150-µl synthesis mix (5 mM KH₂PO₄, 2 mM GTP, 0.4 M mannitol, 60 mM KCl, 2 mM dithiothreitol, 50 mM HEPES, 10 mM MgCl₂, 25 µM of each amino acid except methionine, 10 mM malic acid, 1 mM pyruvate, 4 mM ADP, 0.1% bovine serum albumin, pH 7.0) and the mitochondria equivalent of 200 µg of proteins. Labeling of synthesized proteins was initiated by the addition of 30 µCi of [³⁵S]methionine (>1000 Ci/mmol) to each sample. After 3 h of synthesis at 4 °C, the labeling was quenched by adding of unlabeled methionine to a final concentration of 25 mM (pulse). In pulse-chase experiments, the sample was further incubated at 4 °C or higher temperatures for 60 min. After the pulse or the pulse-chase, 350 µl of medium containing 0.4 M mannitol, 10 mM KH₂PO₄, and 0.5% bovine serum albumin was added to the samples. Mitochondria were pelleted in a microcentrifuge for 4 min at a maximum speed. Each pellet was dissolved in 30 µl of electrophoresis sample buffer (4% SDS, 10% glycerol, 62.5 mM Tris-HCl, 0.01% bromphenol blue, 1% dithiothreitol, pH 6.8) and incubated for 30 min at 56 °C. Then, the suspension was pelleted by a short centrifugation, and the supernatant was subjected to discontinuous Tricine-SDS-PAGE according to Schagger and von Jagow (26). The molecular weights of the labeled proteins were determined using protein weight markers (MBI Fermentas). Following electrophoresis, gels were dried onto Whatman 3MM paper, and labeled proteins were visualized using the Typhoon 8600 ImageQuaNT system (Amersham Biosciences).

The pulse-chase experiment was also performed in the presence of different reagents: puromycin (50 µg/ml), o-phenanthroline (25 mM), phenylmethylsulfonyl fluoride (4 mM), N-ethylmaleimide (2 mM) as well as oligomycin (20 µM) with apyrase (40 units/ml) in 350 µl of medium containing 0.4 M mannitol, 10 mM KH₂PO₄, and 0.5% bovine serum albumin.

Fractionation of Mitochondrial Proteins and Purification of ATP9-- Before fractionation, o-phenanthroline was added to mitochondria to prevent the activity of the ATP-dependent metalloprotease. After the pulse or pulse-chase experiments, four samples were pooled and organelles were collected by centrifugation. Mitochondria were resuspended in 600 µl of 0.1 M Na₂CO₃, pH 11.5, and the suspension was sonicated three times for 10 s on ice, followed by centrifugation at 105,000 × g for 60 min. The resulting pellet (membrane fraction) was directly dissolved in the electrophoresis sample buffer. The supernatant (soluble fraction) was precipitated with chloroform/methanol/water (4/1/3, v/v) and pelleted by centrifugation, 20,000 × g, for 15 min. The pellet was washed with 4 volumes of methanol and dissolved in the electrophoresis buffer. Synthesis in organello was performed at 25 °C for 2 h, and subunit 9 of the ATPase synthase was purified from 4 mg of mitochondrial proteins using chloroform/methanol extraction as described by Michon et al. (27).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Sequence Analysis of the Pea FtsH-like cDNA-- Using the strategy described under "Experimental Procedures," we have identified a 2430-bp-long open reading frame encoding a predicted pea protein of 810 amino acids (GenBank^TM accession number AF397903). Sequence analysis of the pea FtsH-like protein revealed several well-conserved regions of the AAA proteases. In particular, as shown in Fig. 1, the pea protein contains AAA signature (amino acids 463-482), Walker A (GPPGTGKT), and Walker B (IIFIDEIDAI) motifs. The sequence motif HEXXH, typical for the metalloproteases, was found at the position 689-693. Two putative transmembrane domains TM1 (139-153) and TM2 (268-279) were predicted with the DAS algorithm. The predicted amino acid sequence of the pea AAA protease compared with the GenBank^TM and EMBL databases by BLASTX and BLASTP programs showed the highest identity with the A. thaliana FtsH3 (70%), followed by FtsH10 (67%). Only the TBLASTN algorithm identified FtsH10 as being the most homologous to pea protein (89% identity; FtsH3, 72% identity). Based on the homology search, it is not clear whether pea AAA metalloprotease is more homologous to A. thaliana FtsH3 or to FtsH10. Therefore, at this moment, we have designated the Pisum sativum protease only as PsFtsH. Pea FtsH protease also exhibits considerable identity to the human counterparts Afg3L2 (52%) and Spg7 (40%); yeast Yta12 (48-49%) and Yta10 (49%); as well as to the bacterial ancestor E. coli FtsH (41%). As shown in Fig. 1, the homology is not restricted to the conserved motifs but spans almost the entire protein.

View larger version (110K): [in this window] [in a new window]   Fig. 1.   Multiple alignment of the deduced amino acid sequences from pea FtsH and the related AAA proteases. The following sequences have been aligned using the ClustalW program (EMBL accession numbers in parentheses): PsFtsH protease from the pea (AF397903), FtsH3 protease from A. thaliana (AC005315), FtsH10 protease from A. thaliana (AC0022464), Yta12p from Saccharomyces cerevisiae (Z49259), Yta10p from S. cerevisiae (X81066). Structural motifs: Walker A, Walker B, AAA signature, and metal binding sequences are underlined. The position of putative processing site is indicated by an arrow. Residues that are identical or similar in at least three proteins are framed in black and gray boxes, respectively.

Localization of PsFtsH-- In eukaryotic cells the AAA metalloproteases seem to be localized exclusively in mitochondria and chloroplasts (2, 12, 14, 17). To verify the predicted localization of PsFtsH, an immunoblot analysis with mitochondrial and chloroplast protein extracts was performed using A. thaliana FtsH3 antiserum. As shown in Fig. 2, a cross-reacting band of ~80 kDa was found exclusively in the mitochondrial fraction. Less-abundant fast migrating bands are the most probable degradation products of the PsFtsH protein. However, we could not exclude the possibility that these signals result from cross-reactions with other mitochondrial proteins. Following the demonstration of the existence of PsFtsH protein in the mitochondrial fraction, post-translational protein import experiments were performed. The size of the precursor protein synthesized in vitro using a coupled transcription/translation system corresponds well to the theoretical value of 89 kDa calculated from the deduced amino acid sequence (Fig. 3A, lane 1). Upon incubation of the labeled precursor protein with mitochondrial extract, we observed a shift to a smaller band of 82 ± 1.5 kDa (Fig. 3A, lane 2). This result agrees not only with the size of the band observed by immunodetection (Fig. 2) but also with the size of the mature protein predicted by sequence analysis using using the i-PSORT algorithm. According to the prediction, the precursor protein of PsFtsH has an N-terminal, 58-amino acid-long, targeting sequence. Therefore, the processing should shorten the precursor by 6.3 kDa. The inhibition of processing by the metal chelator EDTA (Fig. 3A, lane 3) and the presence of a conserved Arg at the 2 position from the cleavage site suggest that the mitochondrial processing peptidase cleaves the PsFtsH precursor. In vitro translated precursor protein was imported into isolated mitochondria. Appearance of the PsFtsH mature form resistant to proteinase K accompanied the import (Fig. 3B). No import was observed in the presence of valinomycin (Fig. 3B, lanes 4 and 5), suggesting that the import of PsFtsH is dependent on a membrane potential and involves the protein translocation system of the inner membrane.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Subcellular localization of PsFtsH. Twenty micrograms of mitochondrial (M) and chloroplast (CH) protein fractions were resolved on 12% SDS-PAGE gel followed by Western blotting with A. thaliana FtsH3 antiserum (A), a mitochondrial protein-NAD3 antiserum (B) and a chloroplastic protein-cytochrome f antiserum (C). B and C are controls for the purity of the organellar fractions. Positions of molecular mass standards (in kDa) are indicated.

View larger version (57K): [in this window] [in a new window]   Fig. 3.   Import and processing of PsFtsH. The positions of precursor (p) and mature (m) PsFtsH proteins are shown by arrows on the right. The migration of molecular weight standards are indicated on the left. A, processing of PsFtsH by pea mitochondrial extract. Lane 1, translation product; lane 2, processing in the presence of 2 mM MnCl2; lane 3, processing in the presence of 2 mM EDTA. B, in vitro import of PsFtsH into isolated mitochondria. Lane 1, translation product; lanes 2-5, in vitro import into isolated mitochondria. Proteinase K (PK, 10 µg/ml) and valinomycin (VAL, 10 µM) were added as indicated.

Following this conclusion, we decided to characterize the topological orientation of the PsFtsH protein in the inner membrane (Fig. 4). Isolated mitochondria were subjected to an osmotic shock to selectively disrupt the outer membrane. The efficiency of outer membrane disruption was tested by immunoblotting with antibodies directed against an intermembrane space protein, cytochrome c. The integrity of the mitoplasts after the proteinase K treatment was monitored with ATP-synthase antibodies. We found that the PsFtsH protein is present in the mitoplast fraction and is fully resistant to proteinase K treatment, as judged by immunodetection with antibodies directed against a peptide derived from the central part of the homologous FtsH3 protease. Taken together, the results clearly show that the PsFtsH protease, like yeast m-AAA, is an integral part of the inner membrane and exposes its central part to the matrix.

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Submitochondrial localization and topology of PsFtsH. Intact mitochondria and mitoplasts were treated with 10 µg/ml proteinase K for 30 min on ice. An equivalent amount of proteins (intact mitochondria (lane 1), intact mitochondria after addition of protease K (lane 2), mitoplasts (lane 3), and mitoplasts after addition of protease K (lane 4)) were separated by SDS-PAGE, transferred, and immunostaining with the appropriate antibodies. A, Western blot with antibodies against PsFtsH (10% SDS-PAGE). B, Western blot with antibodies against cytochrome c (intermembrane space protein, 15% SDS-PAGE). C, Western blot with antibodies against F1-ATP synthase (matrix localized protein, 12.5% SDS-PAGE).

Pea FtsH Complements Loss of Mitochondrial Respiratory Function in Yeast yta10 and yta12 Null Mutants-- If PsFtsH has not only the orientation, but also analogous functions to that of the yeast m-AAA protease, then it may be able to substitute for Yta10p and/or Yta12p. Yeast cells lacking a functional copy of YTA10 or YTA12 have impaired mitochondrial functions and fail to grow on non-fermentable carbon sources. Therefore, full complementation of the yta10 and yta12 knockout mutants would prevent the loss of respiratory function. To determine whether PsFtsH exhibits such functional conservation, the pea FTSH coding sequences were placed under the control of the CYC1 promoter in the yeast centromeric expression vector pCM185 yielding p[CYC1-PsFTSH]. W303 yeast strain and its derivatives with disrupted YTA10 and/or YTA12 loci were transformed with p[CYC1-PsFTSH] or pCM185 vector alone. All strains grew on glucose (Fig. 5). The untransformed or vector-transformed yta disruptants failed to grow on glycerol either at 30 °C or 37 °C. Single yta10 or yta12 disruptants as well as double yta10/yta12 knockout strain transformed with PsFTSH were able to grow on glycerol at 30 °C or 37 °C (Fig. 5). This result strongly indicates that pea FtsH is a functional homologue of yeast Yta12p and Yta10p.

View larger version (85K): [in this window] [in a new window]   Fig. 5.   Pea FTSH restores growth on glycerol of the yta10 and/or yta12 null mutants. Yeast strain W303 (WT) or yta10 and/or yta12 mutants were transformed with recombinant plasmid pCM185 carrying the PsFTSH gene (+ PsFTSH) or with empty vector (+ vector). 10-fold serial dilutions of cells were replica-plated on YP plates containing 2% glucose (YPD) or 4% glycerol (YPG) and incubated at 37 °C for 3 days.

ATP and Metal-dependent Accumulation of ATP9 in the Mitochondrial Membrane-- It is thought that the respiratory defect observed in cells lacking either YTA10 or YTA12 is caused at least in part by a block in the assembly of the ATP synthase and respiratory chain complexes (4-6). In yta10 and yta12 mutant cells, a lower abundance of the membrane-associated subunit 9 of the ATP synthase (ATP9) (5) as well as its 48-kDa assembly intermediate (4) has been observed. By analogy, we demonstrate that the accumulation of ATP9 in the pea mitochondrial membrane could be blocked by known inhibitors of AAA metalloproteases.

Mitochondrial proteins were synthesized in organello at 4 °C in the presence of [³⁵S]methionine. To identify the electrophoretic mobility of pea ATP9, this protein together with subunit 6 of ATP synthase (ATP6) was purified from in organello labeled mitochondrial products using the chloroform/methanol procedure (27). As expected, two abundant bands corresponding to ATP6 and ATP9 were visible in the hydrophobic preparations (Fig. 6, lane 2). Based on the alignment presented in Fig. 6, the most intensive band with molecular mass below 8.2 kDa was identified as the ATP9 protein among the labeled proteins. We analyzed the fate of this subunit in pulse-chase experiments. After inhibition of labeling by the addition of cold methionine, the mitochondria were further incubated at varying temperatures. The data presented in Table I suggest that accumulation of ATP9 during the chase is temperature-dependent. To establish whether this accumulation is connected with the insertion of ATP9 into the mitochondrial membrane, pulse-chase experiments were repeated, but this time the labeled products were separated into soluble and integral membrane proteins by carbonate extraction. Fig. 7 presents the images of radioactive signals of ATP9 in the soluble and membrane fraction obtained after pulse at 4 °C and increasing chase periods at 25 °C. The proportion of newly synthesized ATP9 in the membrane fraction increased during the chase and reached a plateau between 30 and 60 min (Fig. 7A, lanes 1-5). A barely detectable portion of ATP9 can be observed in the soluble fraction (Fig. 7B). The amount of soluble ATP9 did not change during the chase.

View larger version (38K): [in this window] [in a new window]   Fig. 6.   Identification of the ATP9 electrophoretic mobility. SDS-PAGE of total labeled mitochondrial proteins (lane 1) and labeled hydrophobic proteins extracted with chloroform/methanol as described by Michon et al. (27) (lane 2). Positions of ATP6 and ATP9 are identified on the right side of lane 2. Positions of molecular mass standards (in kDa) are indicated on the left.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Accumulation of ATP9 in the mitochondrial membrane (A) and soluble (B) fractions with increasing chase period and inhibition of this accumulation by depletion of ATP and metal ions. Samples after synthesis at 4 °C were pooled. After an increasing period of chase, aliquots were taken out and then mitochondria were subjected to subfractionation. The bands from the respective fraction corresponding to ATP9 after 0, 10, 20, 30, 60 min of chase are presented on lanes 1, 2, 3, 4, and 5, respectively. Lane 6, 60 min of chase in the presence of o-phenanthroline. Lane 7, 60 min of chase in the presence of apyrase and oligomycin.

The ATP-dependent metalloproteases require the presence of ATP and divalent metal ions for their activity. We found that the observed changes in the ATP9 accumulation in the membrane fraction during the chase were inhibited by ATP depletion (Fig. 7A, lane 7) or the addition of o-phenanthroline, a membrane-permeating chelator for divalent metal ions (Fig. 7A, lane 6). Puromycin (an inhibitor of translation) and phenylmethylsulfonyl fluoride (an inhibitor of serine proteases) did not block these changes, however, N-ethylmaleimide (a cysteine-group modifier) had a slightly inhibitory effect (data not shown). It was previously reported that metallopeptidases are sensitive toward thiol-blocking agents (28). We conclude that at a low temperature newly synthesized ATP9 accumulates in the mitochondrial membrane at a significantly reduced level, because the chaperone-like and the proteolytic activities of the ATP-dependent metalloproteases are inhibited. When both activities fail, the ATP9 polypeptides tend to form aggregates. It is well known that highly hydrophobic proteins accumulate as insoluble aggregates, even in the presence of SDS (29, 30). Consequently, when temperature increases, the activities of the recovered ATP-dependent metalloprotease promote disaggregation and subsequent reassembly of ATP9 complexes toward complexes easily disrupted under denaturating conditions.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plant homologues of FtsH protease have been characterized only in chloroplasts (15-17). This paper provides the first experimental evidence for the existence of a FtsH-like protease in plant mitochondria, which we termed PsFtsH. Based on the topology in the membrane and the complementation studies, we conclude that PsFtsH is a functional homologue of yeast m-AAA protease.

The observation that PsFTSH can compensate for the inactivation of YTA10 or YTA12 genes excludes the possibility that the pea metalloprotease simply replaces one of the yeast component of the m-AAA complex. It is possible that the AAA protease in pea mitochondria, analogously to MAP-1 from the filamentous fungus N. crassa, is a homo-oligomeric complex made up only from PsFtsH. All distinctive functions of the two subunits of the yeast m-AAA complex would then be fulfilled by PsFtsH. Phylogenetic analysis of mitochondrial AAA metalloproteases supports the idea that complexes orthologous to yeast m-AAA may be either homo- or hetero-oligomeric depending on the extent of single ancestral gene duplication and diversification that has taken place during evolution (12).

We propose that by performing the in organello experiments at 4 °C we inhibit the activity of the AAA metalloprotease complex in pea mitochondria. Studies in yeast established that substrate binding to the AAA domain of the ATP-dependent metalloprotease is temperature-dependent and hardly detectable at 4 °C (31). Thus, it is possible that in isolated pea mitochondria the activity of the ATP-dependent metalloprotease is significantly reduced at low temperatures.

Although the exact molecular function of PsFtsH has yet to be resolved, our in organello experiments suggest that the pea protease like its yeast homologue affects accumulation of the mitochondrially encoded subunit 9 in the membrane. The amount of newly synthesized ATP9 recovered in the membrane fraction increased significantly when samples after the pulse at 4 °C were further incubated at 25 °C. Known effective inhibitors of AAA metalloproteases (apyrase with oligomycin, o-phenanthroline) blocked this increase, indicating that both the chaperone-like and proteolytic activities of the pea AAA protease are essential in this process. Again, this is not surprising considering the studies in yeast, where it was shown that not only the chaperone-like but also the proteolytic function of mAAA is required for respiration and the assembly of yeast inner-membrane protein complexes (6). The inhibition study using in organello system does not rigorously demonstrate the effect of PsFtsH, because the involvement of other proteases/factors cannot be excluded. However, together with the complementation analysis, which established the functional conservation between PsFtsH and m-AAA, it is very likely that the pea protease like its yeast homologue is involved in the assembly of the membrane-associated subunit 9 of the ATP synthase.

The fact that puromycin does not block the temperature-dependent accumulation of ATP9 in the membrane fraction indicates that translation does not contribute to the appearance of this protein during the chase. Moreover, newly synthesized ATP9 subunits were not found as monomers in the soluble fraction. Based on this observation, a reasonable explanation is that aggregates of ATP9 resistant to solubilization are formed during cold inactivation of the ATP-dependent metalloprotease. When the temperature increases, the recovered AAA metalloprotease activity likely leads to the dissociation of ATP9 aggregates. This can then be followed by reassembly toward a functional ATP-synthase complex. These complexes are disrupted during denaturing conditions and can therefore be monitored during electrophoresis conditions. We propose that the AAA metalloprotease in pea mitochondria not only mediates the assembly process but also promotes disassembly of aggregated proteins.

	ACKNOWLEDGEMENTS

We are very grateful to T. Langer for supplying the yeast strains used in this study and H. Augustyniak, A. Szczepaniak, and E. Glaser for the antibodies against NAD3, cytochrome f, and F₁-ATPase, respectively.

	FOOTNOTES

^* This work was supported in part by Grant 6P04C08018 from the Polish State Committee for Scientific Research (to H. J.) and the Carl Tryggers Foundation (to C. K.).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.

Supported by the Office of Biological and Environmental Research of the U.S. Department of Energy. Present address: Genetics Institute, 35 Cambridge Park Dr., Cambridge, MA 02140.

^** To whom correspondence should be addressed. Tel.: 48-71-3752-710; Fax: 48-71-3752-608; E-mail: Janska@bf.uni.wroc.pl.

Published, JBC Papers in Press, September 11, 2002, DOI 10.1074/jbc.M203831200

² Z. Adam, personal communication.

	ABBREVIATIONS

The abbreviations used are: FtsH, zinc metalloprotease from E. coli; PsFtsH, FtsH-like mitochondrial protease from Pisum sativum; RACE, rapid amplification of cDNA ends; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; TM, transmembrane; ATP6, -9, subunits 6 and 9 of the ATP synthase; m-AAA, matrix AAA protease; i-AAA, intermembrane AAA protease.

	REFERENCES

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