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The Nuclear-Encoded SDH2-RPS14 Precursor Is Proteolytically Processed between SDH2 and RPS14 to Generate Maize Mitochondrial RPS14
Biochemical and Biophysical Research Communications 271, 380 –385 (2000) doi:10.1006/bbrc.2000.2644, available online at http://www.idealibrary.com on
The Nuclear-Encoded SDH2-RPS14 Precursor Is Proteolytically Processed between SDH2 and RPS14 to Generate Maize Mitochondrial RPS14 Pablo Figueroa,* Loreto Holuigue,* Alejandro Araya,† and Xavier Jordana* ,1 *Departamento de Gene´tica Molecular y Microbiologı´a, Facultad de Ciencias Biolo´gicas, P. Universidad Cato´lica de Chile, Casilla 114-D, Santiago, Chile; and †Laboratoire REGER, UMR 5097 CNRS-Universite´ Victor Segalen Bordeaux 2, IFR 66 Pathologies Infectieuses, 146, rue Leo Saignat, 33076 Bordeaux Cedex, France
Received March 30, 2000
In maize, the functional gene encoding mitochondrial ribosomal protein S14 (rps14) has been translocated to the nucleus where it became integrated between both exons of a gene encoding the iron-sulfur subunit of succinate dehydrogenase (sdh2). Two transcripts are generated from this locus by alternative splicing. One transcript encodes a precursor for a functional SDH2 protein, while the second transcript encodes a chimeric SDH2(t)-RPS14 precursor protein. In this paper we show that the same mitochondrial targeting presequence is able to direct the import of both precursors into isolated mitochondria and is removed during import. This processing event generates a 28 kDa protein from the SDH2 precursor, which corresponds to the iron-sulfur subunit of respiratory complex II present in maize mitochondria. In addition to cleavage of the presequence, the chimeric precursor undergoes proteolytical processing between SDH2 and RPS14. This processing generates RPS14, which is found assembled into mitochondrial ribosomes, and a truncated SDH2 protein which is degraded. Therefore, our results support a role of the SDH2 domain in the chimeric precursor only in providing a mitochondrial targeting function for RPS14. © 2000 Academic Press Key Words: evolutionary gene transfer; internal proteolytical processing; iron-sulfur subunit; maize; mitochondrial targeting peptide; plant mitochondria; ribosomal protein S14, RPS14; SDH2; succinate dehydrogenase.
The endosymbiont hypothesis has become widely accepted to explain the origin of organelles in eukaryotic 1 To whom correspondence should be addressed. Fax: 56-22225515. E-mail: [email protected].
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
cells (1). During evolution most of the genetic information originally present in the ancestral endosymbiont has either been lost or transferred to the nuclear genome. Variability in ribosomal protein gene content is found among angiosperm mitochondrial genomes (2), suggesting evolutionary recent transfer events of functional genes to the nucleus in the angiosperm lineage. Experimental evidence for this on-going phenomenon in higher plants has been provided by characterization of nuclear genes for cox2 in legumes, rps12 in Oenothera, rps10 and rps19 in Arabidopsis thaliana, rps11 in rice and rps14 in maize, rice and A. thaliana (3–11). For instance, the functional gene encoding ribosomal protein S14 (rps14) is located in the mitochondrial genomes of broad bean, Oenothera, rapeseed and pea (12–15). In contrast, the functional rps14 gene has been translocated to the nucleus in A. thaliana, maize and rice (9 –11). In both monocots the transferred rps14 genes acquired the signals conferring expression and product targeting to the mitochondrion in a way not previously described. Indeed, rps14 became integrated between both exons of a gene encoding the iron-sulfur subunit (SDH2) of respiratory complex II (succinate dehydrogenase; succinate:ubiquinone oxidoreductase, EC 1.3.5.1). Alternative splicing generates two transcripts from the sdh2-rps14 locus (Fig. 1): one encoding a chimeric protein formed by truncated SDH2 fused to RPS14 [SDH2(t)-RPS14] and the other encoding a whole SDH2 protein (9, 10). In this paper we report the characterization of the complex proteolytical processing which occurs when the maize chimeric SDH2(t)-RPS14 precursor is imported into plant mitochondria. In addition to removal of the SDH2 transit peptide, the protein precursor is cleaved between SDH2(t) and RPS14 to give a free functional RPS14.
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MATERIALS AND METHODS Primer sequences. 1. 5⬘-TGAATTCGCTTCCACCATGGCCGCCGCCCTGCTCCGC-3⬘ (ATG initiation codon underlined, EcoRI site and Kozak’s consensus in italics). 2. 5⬘-CTGGATCCTTACAGTTCACCCTTGTTTGCCAAG-3⬘ (BamHI site in italics, stop codon underlined). 3. 5⬘-CTGGATCCTTAGGCACTTGGGGCACCCAACTG-3⬘ (BamHI site in italics, stop codon underlined). 4. 5⬘-GCGATACCATGGCGAAGACCTTCTCGATCTACCG-3⬘ (NcoI site in italics, ATG codon underlined). 5. 5⬘-GACTGACTCGAGTGGCTCGACGCTCTTGTACTG-3⬘ (XhoI site in italics). 6. 5⬘-GCGATACCATGGAGAAGAGAAACCTGCGGGAC-3⬘ (NcoI site in italics, ATG codon underlined). 7. 5⬘-GACTGACTCGAGCCACGATGCCTTCTTTACGCC-3⬘ (XhoI site in italics). Protein import into mitochondria. Highly purified mitochondria were prepared from potato (Solanum tuberosum cv. Bintje) tubers (16), and resuspended in a buffer containing 300 mM mannitol and 10 mM KH 2PO 4 (pH 7.4). The full-length cDNA of maize sdh2(t)-rps14 cloned behind the T7 promoter of pBluescribe (Stratagene) was obtained previously (9). This plasmid cDNA was amplified by PCR between primers 1 and 2 to construct a cDNA directing the synthesis of a SDH2(t)-RPS14(s) protein shortened by 8 amino acids at the C-terminus. A full-length maize sdh2 cDNA was obtained by RT-PCR: first-strand cDNA synthesis was performed as previously described (9) and PCR amplification was carried out between primers 1 and 3. Both PCR products were cloned behind the T7 promoter of pBluescribe. Construct structures were verified by DNA sequencing. The three precursors (SDH2, SDH2 truncated-RPS14 and SDH2 truncated-RPS14 shortened) were synthesized by a TNT quick coupled transcription–translation reticulocyte lysate system in the presence of [ 35S]methionine, according to the supplier’s instructions (Promega). In vitro import reactions to isolated mitochondria were carried out as described previously (9). In some experiments, imported proteins were immunoprecipitated with either anti-RPS14 or anti-SDH2 antibodies (Fig. 4A). After completion of import reactions and proteinase K treatments, mitochondria (1 mg protein) were repurified by centrifugation through a 20% sucrose cushion at 15,000g for 10 min. Mitochondrial pellets were resuspended in 200 l of RIPA (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 g/ml pepstatin A, 5 g/ml aprotinin, 0.5 mM PMSF and 2 mM benzamidine) and incubated on ice for 30 min (17). Lysates were cleared by centrifugation at 21,000g for 45 min and supernatants were incubated for 1 h at 4°C with 5 l of antisera against RPS14 or SDH2 (see below). Two hundred microliters of 10% (w/v) protein A Sepharose (Sigma) were added to each sample and incubated for 1 h at 4°C. Sepharose beads were collected by centrifugation at 10,000g for 15 s. Supernatants were aspirated, and the protein A Sepharose beads were washed three times with RIPA. Finally, Sepharose beads were resuspended in 20 l of 2⫻ Tricine– SDS loading dye, heated for 10 min at 85°C, and samples were electrophoresed on 12% Tricine–SDS–polyacrylamide gels (18). The radiolabeled products were analyzed by fluorography. Antibody preparation. To obtain anti-SDH2 and anti-RPS14 antibodies, DNA fragments corresponding to either sdh2 exon 1 (codons 47–161, construct 1) or rps14 (codons 251–348, construct 2) were amplified by PCR between primers 4 and 5 or primers 6 and 7, respectively (Fig. 1B). PCR products were digested with NcoI and XhoI and cloned into the pET-32a expression vector (Novagen). Constructs were verified by DNA sequencing. The recombinant proteins (fusions with thioredoxin and a 6⫻ His-tag) were expressed in E. coli
according to the supplier’s instructions (Novagen). They were obtained as inclusion bodies and were purified by HiTrap affinity chromatography under denaturating conditions, according to manufacturer’s protocols (Amersham Pharmacia Biotech). Antibodies against SDH2 and RPS14 were raised by immunization of rabbits with the purified recombinant proteins, according to a standard protocol (19). Isolation of a mitochondrial ribosome-enriched fraction. Maize mitochondria were prepared from etiolated seedlings (cv B73) and purified through discontinuous Percoll gradients (16, 20). Mitochondria were disrupted in lysis buffer (10 mM Tris–HCl, pH 7.5, 0.5 M KCl, 20 mM MgCl 2, 5 mM DTT, 0.6 M PMSF, 2% Triton X-100) for 20 min with agitation. The lysate was centrifuged at 31,000g for 20 min and the supernatant was layered onto a 4 ml cushion of 0.7 M sucrose in 10 mM Tris–HCl, pH 7.5, 27 mM KCl, 20 mM MgCl 2, and centrifuged at 175,000g for 4 h. The ribosome-enriched pellet was then resuspended in 10 mM Tris–HCl, pH 7.5, 6 M urea, and stored at ⫺20°C. Protein blot analysis. Proteins of the ribosomal-enriched fraction were separated by 12% Tricine-SDS–PAGE and electroblotted onto PVDF membranes in a Bio-Rad Trans-Blot cell, using a buffer containing 25 mM Tris–HCl (pH 8.3), 192 mM glycine, 0.1% SDS and 20% methanol. The transfer was performed at 4°C and 56 mA for 12 h. Protein gel blots were incubated with blocking solution (25 mM Tris–HCl, pH 7.4, 150 mM NaCl, 5% non-fat dry milk) at room temperature for 30 min. Anti-RSP14 (1:1000 dilution) or anti-SDH2 (1:1500 dilution) antibodies were added to the gel blots and incubated at room temperature for 1 h. The membranes were washed five times for five min with TTBS solution (25 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20). An anti-rabbit antibody conjugated to horseradish peroxidase (Gibco-BRL) was added in blocking solution (1:2000 dilution). The blots were incubated at room temperature for 1 h, washed, and visualized using the Chemiluminescence Reagent Plus (NEN).
RESULTS SDH2 and SDH2(truncated)-RPS14 precursors are imported into plant mitochondria. Both SDH2 and SDH2(t)-RPS14 precursors contain the same N-terminal extension when compared to the mitochondrially encoded Reclinomonas americana or red algal SDH2 (Fig. 1B; 9, 21, 22). This sequence exhibits several common features of mitochondrial presequences (23), and we have previously shown that SDH2(t)-RPS14 precursor is targeted to isolated plant mitochondria (9). To demonstrate that SDH2 is also targeted to mitochondria and to compare its processing with that of SDH2(t)-RPS14, we imported the in vitro-translated precursors into isolated mitochondria (Fig. 2). The SDH2 precursor (31 kDa, Fig. 2A, lane 1) was converted into a 28 kDa protein (lane 2) which was resistant to added proteinase K (lane 3). The protection was abolished when mitochondria were incubated with Triton X-100 and proteinase K (lane 4), and the import reaction was blocked when membrane potential was abolished by the oxidative phosphorylation uncoupler CCCP (carbonylcyanide m-chlorophenylhydrazone; lane 5). In Fig. 2B, the import of the SDH2 precursor (lane 2) is compared to that of the in vitro translated SDH2(t)-RPS14 precursor (lane 1). As shown previ-
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FIG. 1. (A) Structure of the maize sdh2-rps14 locus. Sdh2 exons are shown by open boxes. The rps14 homologous region is indicated by a gray box and a region of unknown origin (the “connector” region) in the second exon is shown in black. Diagonal lines connect exons in the two alternatively splice transcripts, whose polyadenylation sites are shown by vertical arrows. (B) Schematic representation of the protein precursors utilized for in organello import experiments (not to scale). M.T. and black box correspond to mitochondrial targeting presequence and connector region, respectively. The second protein is 8 amino acids shorter than the first SDH2(t)-RPS14 precursor at the C-terminus. SDH2(t)-RPS14 and SDH2 are the proteins encoded by the two alternatively spliced transcripts shown in A. Numbering represents amino acid positions and vertical arrows the putative cleavage site of the presequence.
ously, the SDH2(t)-RPS14 precursor (39 kDa) is imported and processed in a more complex way, being converted to smaller proteins of 36, 27, 25 and 15 kDa. These results suggest that the same transit peptide of 2–3 kDa is removed during in organello import of both precursors. The sizes of the SDH2(t)-RPS14 imported and processed products suggest proteolytical processing between SDH2 and RPS14, the polypeptide of 15 kDa probably corresponding to RPS14 (9). Characterization of the SDH2(t)-RPS14 processing products. Two different experimental approaches were designed to analyze the complex proteolytical processing of the SDH2(t)-RPS14 precursor. First, the chimeric SDH2(t)-RPS14 precursor was shortened by eight amino acids at the C-terminus (Fig. 1B) to see whether the 15 kDa protein is affected by such a manipulation. Its import into isolated mitochondria was characterized (t-s precursor) and compared to that of SDH2(t)-RPS14 protein (t precursor) (Fig. 3). The t-s precursor (38 kDa) was converted to smaller proteins of 35, 27, 25, and 14 kDa (lane 4), which were resistant to added proteinase K (lane 6). It is clear from the comparison that shortening of the precursor at the C-terminus resulted in size decreases for the 36 and 15
FIG. 2. Import of SDH2 into isolated mitochondria. The in vitro translated SDH2 precursor is imported into plant mitochondria (A), and its import is compared to that of the chimeric SDH2(t)-RPS14 protein (B). (A) Lane 1 shows the in vitro labeled precursor, and lane 2 the import reaction in which the labeled precursor was incubated with mitochondria under conditions that support import. Lane 3, as lane 2, with proteinase K treatment (100 g/ml; PK). Lane 4, as lane 2, with proteinase K treatment in the presence of 1% Triton-X100. Lane 5, as lane 3, but import reaction was carried out in the presence of 50 M CCCP. Size markers are indicated on the right. (B) Lanes 1 and 2 show the import reactions of SDH2(t)-RPS14 and SDH2 precursors, respectively, followed by a 100 g/ml proteinase K treatment. The 25 kDa protein is detected in variable amounts in different experiments.
kDa polypeptides, whereas the 27 and 25 kDa proteins remained unchanged (lanes 5 and 6). Therefore we can conclude that the polypeptide of 15 kDa corresponds to the processed form of RPS14, and that the proteins of 27 and 25 kDa probably represent SDH2(t). This con-
FIG. 3. Proteolytical processing of a SDH2(t)-RPS14 precursor shortened at the C-terminus. The in vitro-translated SDH2(t)-RPS14 precursor (lane 1; t) and that shortened at the C-terminus (lane 2; t-s) were incubated with mitochondria under conditions that support import (lanes 3 and 4, respectively). Lanes 5 and 6, as lanes 3 and 4, respectively, but with proteinase K treatment. Lanes 7 and 8, as lanes 3 and 4, respectively, but with proteinase K treatment in the presence of 1% Triton X-100.
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28 kDa and 27 kDa proteins were clearly separated under the electrophoresis conditions we used. These results strongly suggest that the truncated SDH2 protein resulting from import of the chimeric SDH2(t)RPS14 is not assembled into a functional succinate dehydrogenase complex and is rapidly degraded. DISCUSSION
FIG. 4. Immunological characterization of the SDH2(t)-RPS14 precursor proteolytical processing upon import to mitochondria. (A) Immunoprecipitation of imported labeled proteins. Lane 1 shows an import reaction followed by a 100 g/ml proteinase K treatment. After import and proteinase K treatment, labeled proteins were immunoprecipitated with either anti-SDH2 (lane 2) or anti-RPS14 antibodies (lane 3), and then analyzed by SDS–PAGE electrophoresis. (B) Western blot analysis of a maize mitochondrial fraction enriched in ribosomes. Approximately 30 g of proteins from a mitochondrial fraction obtained as described under Materials and Methods were loaded per lane and the blot probed with either antisera against RPS14 (lane 1) or antisera against SDH2 (lane 2).
clusion was further substantiated by a second approach which consisted in the immunoprecipitation of the in vitro-imported proteins with anti-SDH2 or antiRPS14 antibodies and their analysis by SDS–PAGE (Fig. 4A). Both antibodies precipitated the precursor (39 kDa) and the 36 kDa protein (lanes 2 and 3), a result consistent with our prediction that the 36 kDa protein is generated by removal of a 2–3 kDa transit peptide from the 39 kDa precursor. In contrast, the anti-SDH2 antibodies immunoprecipitated the 27–25 kDa proteins and not the 15 kDa polypeptide (lane 2), whereas the anti-RPS14 antibodies were able to precipitate the 15 kDa polypeptide and not the 27–25 kDa proteins (lane 3). Maize mitochondria contain the mature SDH2 and RPS14 proteins. A ribosome-enriched fraction was prepared from maize mitochondria as described under Materials and Methods and analyzed by immunoblotting. The anti-RPS14 antibodies recognized a 15 kDa polypeptide from this maize mitochondrial fraction (Fig. 4B, lane 1). This size corresponds to that of the smaller polypeptide detected in the in vitro import reactions of the SDH2(t)-RPS14 precursor, confirming its identity as the mature functional ribosomal protein S14. The anti-SDH2 antibodies recognized a 28 kDa protein: this size corresponds to that of the imported and processed SDH2 precursor (Fig. 2, lane 2). In contrast, the 27 kDa protein resulting from import of the SDH2(t)-RPS14 precursor is not detected by these antibodies. It is important to point out that the processed
In both maize and rice the functional rps14 gene has been transferred to the nucleus, where it is integrated between the two exons of a gene encoding the ironsulfur subunit of complex II (9, 10). It has to be pointed out that in these monocots sdh2 or rps14 genes have not been detected by Southern blot analysis in arrangements different from the sdh2-rps14 locus (i.e., independent sdh2 or rps14 genes). Since the nuclear gene encoding mitochondrial RPS14 is essential for mitochondrial function in yeast (24), and SDH2 is essential for electron transport within complex II (25), we presumed that expression of the sdh2-rps14 locus must generate functional SDH2 and RPS14 proteins. It has been previously shown that two transcripts are generated from the sdh2-rps14 locus by a combination of alternative splicing and the use of alternative polyadenylation sites (Fig. 1). One transcript encodes a chimeric SDH2(t)-RPS14 protein, while the second transcript encodes the SDH2 protein. In this paper we have shown that the precursor encoded by this second transcript is targeted to mitochondria. Upon import, a transit peptide of 2–3 kDa is removed to generate a 28 kDa protein (Fig. 2), which corresponds to the iron-sulfur subunit of complex II present in maize mitochondria (Fig. 4B). These sizes are consistent with cleavage at the R-3 motif RXX*(A/S) (T/S) present in the mitochondrial targeting presequences (Fig. 5A; X denotes any amino acid and the asterisk the cleavage site; 26). We
FIG. 5. Comparison of the mitochondrial targeting presequences (A) and connector region sequences (B) in maize and rice (9, 10). Colons (:) indicate identity with the residue in maize. Putative cleavage sites discussed in the text are indicated by a black box (the cleaved bond is marked by a vertical arrow).
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have also performed a detailed investigation of the proteolytic processing pathway of the SDH2(t)-RPS14 protein when it is imported into mitochondria. Our most important conclusion is that, in addition to removal of the same transit peptide as expected, a second cleavage occurs between SDH2 and RPS14 to liberate RPS14, which is found assembled into mitochondrial ribosomes. The truncated form of SDH2 is not present in maize mitochondria, being probably degraded because it is not assembled into functional complexes. Indeed, truncated SDH2 could not be functional as a component of complex II since it lacks the third cysteine-rich cluster encoded by the second exon. This cluster is mainly involved in formation of the third iron–sulfur center, which is essential for electron transport within the subunit. Our evidence strongly support a role for the SDH2 domain of the chimeric SDH2(t)-RPS14 protein only in generating a mitochondrial targeting function for RPS14. In the chimeric protein, SDH2 and RPS14 sequences are connected by a 25-amino acid sequence of unknown origin (Fig. 5B). Based on the size of the products detected by immunoprecipitation, we suggest that cleavage of the precursor occurs in this region. However, identification of the precise processing site remained to be determined. To our knowledge, there are two examples in fungi where different nuclear-encoded mitochondrial proteins were generated by proteolytical processing of a polyprotein precursor (27, 28). In only one instance detailed characterization of this processing has been performed (28, 29). Two precise cleavage sites in the connector region of this precursor have been identified (RGY2ST and RG2YST) and it was shown that their cleavage was carried out by the mitochondrial processing peptidase (MPP) and processing enhancing protein (PEP) which also process the N-terminal mitochondrial targeting sequence. It is tempting to speculate that a similar mechanism works in maize SDH2(t)-RPS14 precursor processing since a sequence RGYHG is found in the connector region, just upstream of the RPS14 homologous sequence (Fig. 5B). Interestingly, this sequence is also similar to the R-3 cleavage motif RX (F/Y)2(A/S) (T/S) proposed for mitochondrial plant presequences (30) and is conserved in maize, rice and wheat (our unpublished results). However, RPS14 would have a molecular weight of 12 kDa, which is lower than the size estimated by SDS–PAGE. We do not know if the processed 15 kDa protein has the connector region (2–3 kDa) as an N-terminal extension, or migrates anomalously on SDS–PAGE due to its high content of basic residues. The analysis of the 27 and 25 kDa processing products, which are both recognized by anti-SDH2 antibodies, gives no clues about the cleavage site, since the 27 kDa protein may correspond to either a transit peptide-SDH2(t) or a SDH2(t)connector region protein (the 25 kDa protein may cor-
respond to truncated SDH2, devoid of both the targeting peptide and the connector region). ACKNOWLEDGMENTS The authors are greatly indebted to Simon Litvak and Laura Tarrago´ for their support, especially during the stage of one of us (P.F.) in his laboratory. We also thank Jean-Claude Farre´ for valuable suggestions concerning immunoprecipitations, and Virginia Garreto´n, Gabriel Leo´n and Alvaro Elorza for fruitful discussions. This work was supported by Research Grants 8980005 (Lı´neas Complementarias) and 2970071 (Ph.D. students) from FondecytChile, C98B01 from ECOS-Conicyt, the French CNRS, and Universite´ Victor Segalen Bordeaux 2.
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The Nuclear-Encoded SDH2-RPS14 Precursor Is Proteolytically Processed between SDH2 and RPS14 to Generate Maize Mitochondrial RPS14 Pablo Figueroa,* Loreto Holuigue,* Alejandro Araya,† and Xavier Jordana* ,1 *Departamento de Gene´tica Molecular y Microbiologı´a, Facultad de Ciencias Biolo´gicas, P. Universidad Cato´lica de Chile, Casilla 114-D, Santiago, Chile; and †Laboratoire REGER, UMR 5097 CNRS-Universite´ Victor Segalen Bordeaux 2, IFR 66 Pathologies Infectieuses, 146, rue Leo Saignat, 33076 Bordeaux Cedex, France
Received March 30, 2000
In maize, the functional gene encoding mitochondrial ribosomal protein S14 (rps14) has been translocated to the nucleus where it became integrated between both exons of a gene encoding the iron-sulfur subunit of succinate dehydrogenase (sdh2). Two transcripts are generated from this locus by alternative splicing. One transcript encodes a precursor for a functional SDH2 protein, while the second transcript encodes a chimeric SDH2(t)-RPS14 precursor protein. In this paper we show that the same mitochondrial targeting presequence is able to direct the import of both precursors into isolated mitochondria and is removed during import. This processing event generates a 28 kDa protein from the SDH2 precursor, which corresponds to the iron-sulfur subunit of respiratory complex II present in maize mitochondria. In addition to cleavage of the presequence, the chimeric precursor undergoes proteolytical processing between SDH2 and RPS14. This processing generates RPS14, which is found assembled into mitochondrial ribosomes, and a truncated SDH2 protein which is degraded. Therefore, our results support a role of the SDH2 domain in the chimeric precursor only in providing a mitochondrial targeting function for RPS14. © 2000 Academic Press Key Words: evolutionary gene transfer; internal proteolytical processing; iron-sulfur subunit; maize; mitochondrial targeting peptide; plant mitochondria; ribosomal protein S14, RPS14; SDH2; succinate dehydrogenase.
The endosymbiont hypothesis has become widely accepted to explain the origin of organelles in eukaryotic 1 To whom correspondence should be addressed. Fax: 56-22225515. E-mail: [email protected].
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cells (1). During evolution most of the genetic information originally present in the ancestral endosymbiont has either been lost or transferred to the nuclear genome. Variability in ribosomal protein gene content is found among angiosperm mitochondrial genomes (2), suggesting evolutionary recent transfer events of functional genes to the nucleus in the angiosperm lineage. Experimental evidence for this on-going phenomenon in higher plants has been provided by characterization of nuclear genes for cox2 in legumes, rps12 in Oenothera, rps10 and rps19 in Arabidopsis thaliana, rps11 in rice and rps14 in maize, rice and A. thaliana (3–11). For instance, the functional gene encoding ribosomal protein S14 (rps14) is located in the mitochondrial genomes of broad bean, Oenothera, rapeseed and pea (12–15). In contrast, the functional rps14 gene has been translocated to the nucleus in A. thaliana, maize and rice (9 –11). In both monocots the transferred rps14 genes acquired the signals conferring expression and product targeting to the mitochondrion in a way not previously described. Indeed, rps14 became integrated between both exons of a gene encoding the iron-sulfur subunit (SDH2) of respiratory complex II (succinate dehydrogenase; succinate:ubiquinone oxidoreductase, EC 1.3.5.1). Alternative splicing generates two transcripts from the sdh2-rps14 locus (Fig. 1): one encoding a chimeric protein formed by truncated SDH2 fused to RPS14 [SDH2(t)-RPS14] and the other encoding a whole SDH2 protein (9, 10). In this paper we report the characterization of the complex proteolytical processing which occurs when the maize chimeric SDH2(t)-RPS14 precursor is imported into plant mitochondria. In addition to removal of the SDH2 transit peptide, the protein precursor is cleaved between SDH2(t) and RPS14 to give a free functional RPS14.
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MATERIALS AND METHODS Primer sequences. 1. 5⬘-TGAATTCGCTTCCACCATGGCCGCCGCCCTGCTCCGC-3⬘ (ATG initiation codon underlined, EcoRI site and Kozak’s consensus in italics). 2. 5⬘-CTGGATCCTTACAGTTCACCCTTGTTTGCCAAG-3⬘ (BamHI site in italics, stop codon underlined). 3. 5⬘-CTGGATCCTTAGGCACTTGGGGCACCCAACTG-3⬘ (BamHI site in italics, stop codon underlined). 4. 5⬘-GCGATACCATGGCGAAGACCTTCTCGATCTACCG-3⬘ (NcoI site in italics, ATG codon underlined). 5. 5⬘-GACTGACTCGAGTGGCTCGACGCTCTTGTACTG-3⬘ (XhoI site in italics). 6. 5⬘-GCGATACCATGGAGAAGAGAAACCTGCGGGAC-3⬘ (NcoI site in italics, ATG codon underlined). 7. 5⬘-GACTGACTCGAGCCACGATGCCTTCTTTACGCC-3⬘ (XhoI site in italics). Protein import into mitochondria. Highly purified mitochondria were prepared from potato (Solanum tuberosum cv. Bintje) tubers (16), and resuspended in a buffer containing 300 mM mannitol and 10 mM KH 2PO 4 (pH 7.4). The full-length cDNA of maize sdh2(t)-rps14 cloned behind the T7 promoter of pBluescribe (Stratagene) was obtained previously (9). This plasmid cDNA was amplified by PCR between primers 1 and 2 to construct a cDNA directing the synthesis of a SDH2(t)-RPS14(s) protein shortened by 8 amino acids at the C-terminus. A full-length maize sdh2 cDNA was obtained by RT-PCR: first-strand cDNA synthesis was performed as previously described (9) and PCR amplification was carried out between primers 1 and 3. Both PCR products were cloned behind the T7 promoter of pBluescribe. Construct structures were verified by DNA sequencing. The three precursors (SDH2, SDH2 truncated-RPS14 and SDH2 truncated-RPS14 shortened) were synthesized by a TNT quick coupled transcription–translation reticulocyte lysate system in the presence of [ 35S]methionine, according to the supplier’s instructions (Promega). In vitro import reactions to isolated mitochondria were carried out as described previously (9). In some experiments, imported proteins were immunoprecipitated with either anti-RPS14 or anti-SDH2 antibodies (Fig. 4A). After completion of import reactions and proteinase K treatments, mitochondria (1 mg protein) were repurified by centrifugation through a 20% sucrose cushion at 15,000g for 10 min. Mitochondrial pellets were resuspended in 200 l of RIPA (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 g/ml pepstatin A, 5 g/ml aprotinin, 0.5 mM PMSF and 2 mM benzamidine) and incubated on ice for 30 min (17). Lysates were cleared by centrifugation at 21,000g for 45 min and supernatants were incubated for 1 h at 4°C with 5 l of antisera against RPS14 or SDH2 (see below). Two hundred microliters of 10% (w/v) protein A Sepharose (Sigma) were added to each sample and incubated for 1 h at 4°C. Sepharose beads were collected by centrifugation at 10,000g for 15 s. Supernatants were aspirated, and the protein A Sepharose beads were washed three times with RIPA. Finally, Sepharose beads were resuspended in 20 l of 2⫻ Tricine– SDS loading dye, heated for 10 min at 85°C, and samples were electrophoresed on 12% Tricine–SDS–polyacrylamide gels (18). The radiolabeled products were analyzed by fluorography. Antibody preparation. To obtain anti-SDH2 and anti-RPS14 antibodies, DNA fragments corresponding to either sdh2 exon 1 (codons 47–161, construct 1) or rps14 (codons 251–348, construct 2) were amplified by PCR between primers 4 and 5 or primers 6 and 7, respectively (Fig. 1B). PCR products were digested with NcoI and XhoI and cloned into the pET-32a expression vector (Novagen). Constructs were verified by DNA sequencing. The recombinant proteins (fusions with thioredoxin and a 6⫻ His-tag) were expressed in E. coli
according to the supplier’s instructions (Novagen). They were obtained as inclusion bodies and were purified by HiTrap affinity chromatography under denaturating conditions, according to manufacturer’s protocols (Amersham Pharmacia Biotech). Antibodies against SDH2 and RPS14 were raised by immunization of rabbits with the purified recombinant proteins, according to a standard protocol (19). Isolation of a mitochondrial ribosome-enriched fraction. Maize mitochondria were prepared from etiolated seedlings (cv B73) and purified through discontinuous Percoll gradients (16, 20). Mitochondria were disrupted in lysis buffer (10 mM Tris–HCl, pH 7.5, 0.5 M KCl, 20 mM MgCl 2, 5 mM DTT, 0.6 M PMSF, 2% Triton X-100) for 20 min with agitation. The lysate was centrifuged at 31,000g for 20 min and the supernatant was layered onto a 4 ml cushion of 0.7 M sucrose in 10 mM Tris–HCl, pH 7.5, 27 mM KCl, 20 mM MgCl 2, and centrifuged at 175,000g for 4 h. The ribosome-enriched pellet was then resuspended in 10 mM Tris–HCl, pH 7.5, 6 M urea, and stored at ⫺20°C. Protein blot analysis. Proteins of the ribosomal-enriched fraction were separated by 12% Tricine-SDS–PAGE and electroblotted onto PVDF membranes in a Bio-Rad Trans-Blot cell, using a buffer containing 25 mM Tris–HCl (pH 8.3), 192 mM glycine, 0.1% SDS and 20% methanol. The transfer was performed at 4°C and 56 mA for 12 h. Protein gel blots were incubated with blocking solution (25 mM Tris–HCl, pH 7.4, 150 mM NaCl, 5% non-fat dry milk) at room temperature for 30 min. Anti-RSP14 (1:1000 dilution) or anti-SDH2 (1:1500 dilution) antibodies were added to the gel blots and incubated at room temperature for 1 h. The membranes were washed five times for five min with TTBS solution (25 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20). An anti-rabbit antibody conjugated to horseradish peroxidase (Gibco-BRL) was added in blocking solution (1:2000 dilution). The blots were incubated at room temperature for 1 h, washed, and visualized using the Chemiluminescence Reagent Plus (NEN).
RESULTS SDH2 and SDH2(truncated)-RPS14 precursors are imported into plant mitochondria. Both SDH2 and SDH2(t)-RPS14 precursors contain the same N-terminal extension when compared to the mitochondrially encoded Reclinomonas americana or red algal SDH2 (Fig. 1B; 9, 21, 22). This sequence exhibits several common features of mitochondrial presequences (23), and we have previously shown that SDH2(t)-RPS14 precursor is targeted to isolated plant mitochondria (9). To demonstrate that SDH2 is also targeted to mitochondria and to compare its processing with that of SDH2(t)-RPS14, we imported the in vitro-translated precursors into isolated mitochondria (Fig. 2). The SDH2 precursor (31 kDa, Fig. 2A, lane 1) was converted into a 28 kDa protein (lane 2) which was resistant to added proteinase K (lane 3). The protection was abolished when mitochondria were incubated with Triton X-100 and proteinase K (lane 4), and the import reaction was blocked when membrane potential was abolished by the oxidative phosphorylation uncoupler CCCP (carbonylcyanide m-chlorophenylhydrazone; lane 5). In Fig. 2B, the import of the SDH2 precursor (lane 2) is compared to that of the in vitro translated SDH2(t)-RPS14 precursor (lane 1). As shown previ-
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FIG. 1. (A) Structure of the maize sdh2-rps14 locus. Sdh2 exons are shown by open boxes. The rps14 homologous region is indicated by a gray box and a region of unknown origin (the “connector” region) in the second exon is shown in black. Diagonal lines connect exons in the two alternatively splice transcripts, whose polyadenylation sites are shown by vertical arrows. (B) Schematic representation of the protein precursors utilized for in organello import experiments (not to scale). M.T. and black box correspond to mitochondrial targeting presequence and connector region, respectively. The second protein is 8 amino acids shorter than the first SDH2(t)-RPS14 precursor at the C-terminus. SDH2(t)-RPS14 and SDH2 are the proteins encoded by the two alternatively spliced transcripts shown in A. Numbering represents amino acid positions and vertical arrows the putative cleavage site of the presequence.
ously, the SDH2(t)-RPS14 precursor (39 kDa) is imported and processed in a more complex way, being converted to smaller proteins of 36, 27, 25 and 15 kDa. These results suggest that the same transit peptide of 2–3 kDa is removed during in organello import of both precursors. The sizes of the SDH2(t)-RPS14 imported and processed products suggest proteolytical processing between SDH2 and RPS14, the polypeptide of 15 kDa probably corresponding to RPS14 (9). Characterization of the SDH2(t)-RPS14 processing products. Two different experimental approaches were designed to analyze the complex proteolytical processing of the SDH2(t)-RPS14 precursor. First, the chimeric SDH2(t)-RPS14 precursor was shortened by eight amino acids at the C-terminus (Fig. 1B) to see whether the 15 kDa protein is affected by such a manipulation. Its import into isolated mitochondria was characterized (t-s precursor) and compared to that of SDH2(t)-RPS14 protein (t precursor) (Fig. 3). The t-s precursor (38 kDa) was converted to smaller proteins of 35, 27, 25, and 14 kDa (lane 4), which were resistant to added proteinase K (lane 6). It is clear from the comparison that shortening of the precursor at the C-terminus resulted in size decreases for the 36 and 15
FIG. 2. Import of SDH2 into isolated mitochondria. The in vitro translated SDH2 precursor is imported into plant mitochondria (A), and its import is compared to that of the chimeric SDH2(t)-RPS14 protein (B). (A) Lane 1 shows the in vitro labeled precursor, and lane 2 the import reaction in which the labeled precursor was incubated with mitochondria under conditions that support import. Lane 3, as lane 2, with proteinase K treatment (100 g/ml; PK). Lane 4, as lane 2, with proteinase K treatment in the presence of 1% Triton-X100. Lane 5, as lane 3, but import reaction was carried out in the presence of 50 M CCCP. Size markers are indicated on the right. (B) Lanes 1 and 2 show the import reactions of SDH2(t)-RPS14 and SDH2 precursors, respectively, followed by a 100 g/ml proteinase K treatment. The 25 kDa protein is detected in variable amounts in different experiments.
kDa polypeptides, whereas the 27 and 25 kDa proteins remained unchanged (lanes 5 and 6). Therefore we can conclude that the polypeptide of 15 kDa corresponds to the processed form of RPS14, and that the proteins of 27 and 25 kDa probably represent SDH2(t). This con-
FIG. 3. Proteolytical processing of a SDH2(t)-RPS14 precursor shortened at the C-terminus. The in vitro-translated SDH2(t)-RPS14 precursor (lane 1; t) and that shortened at the C-terminus (lane 2; t-s) were incubated with mitochondria under conditions that support import (lanes 3 and 4, respectively). Lanes 5 and 6, as lanes 3 and 4, respectively, but with proteinase K treatment. Lanes 7 and 8, as lanes 3 and 4, respectively, but with proteinase K treatment in the presence of 1% Triton X-100.
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28 kDa and 27 kDa proteins were clearly separated under the electrophoresis conditions we used. These results strongly suggest that the truncated SDH2 protein resulting from import of the chimeric SDH2(t)RPS14 is not assembled into a functional succinate dehydrogenase complex and is rapidly degraded. DISCUSSION
FIG. 4. Immunological characterization of the SDH2(t)-RPS14 precursor proteolytical processing upon import to mitochondria. (A) Immunoprecipitation of imported labeled proteins. Lane 1 shows an import reaction followed by a 100 g/ml proteinase K treatment. After import and proteinase K treatment, labeled proteins were immunoprecipitated with either anti-SDH2 (lane 2) or anti-RPS14 antibodies (lane 3), and then analyzed by SDS–PAGE electrophoresis. (B) Western blot analysis of a maize mitochondrial fraction enriched in ribosomes. Approximately 30 g of proteins from a mitochondrial fraction obtained as described under Materials and Methods were loaded per lane and the blot probed with either antisera against RPS14 (lane 1) or antisera against SDH2 (lane 2).
clusion was further substantiated by a second approach which consisted in the immunoprecipitation of the in vitro-imported proteins with anti-SDH2 or antiRPS14 antibodies and their analysis by SDS–PAGE (Fig. 4A). Both antibodies precipitated the precursor (39 kDa) and the 36 kDa protein (lanes 2 and 3), a result consistent with our prediction that the 36 kDa protein is generated by removal of a 2–3 kDa transit peptide from the 39 kDa precursor. In contrast, the anti-SDH2 antibodies immunoprecipitated the 27–25 kDa proteins and not the 15 kDa polypeptide (lane 2), whereas the anti-RPS14 antibodies were able to precipitate the 15 kDa polypeptide and not the 27–25 kDa proteins (lane 3). Maize mitochondria contain the mature SDH2 and RPS14 proteins. A ribosome-enriched fraction was prepared from maize mitochondria as described under Materials and Methods and analyzed by immunoblotting. The anti-RPS14 antibodies recognized a 15 kDa polypeptide from this maize mitochondrial fraction (Fig. 4B, lane 1). This size corresponds to that of the smaller polypeptide detected in the in vitro import reactions of the SDH2(t)-RPS14 precursor, confirming its identity as the mature functional ribosomal protein S14. The anti-SDH2 antibodies recognized a 28 kDa protein: this size corresponds to that of the imported and processed SDH2 precursor (Fig. 2, lane 2). In contrast, the 27 kDa protein resulting from import of the SDH2(t)-RPS14 precursor is not detected by these antibodies. It is important to point out that the processed
In both maize and rice the functional rps14 gene has been transferred to the nucleus, where it is integrated between the two exons of a gene encoding the ironsulfur subunit of complex II (9, 10). It has to be pointed out that in these monocots sdh2 or rps14 genes have not been detected by Southern blot analysis in arrangements different from the sdh2-rps14 locus (i.e., independent sdh2 or rps14 genes). Since the nuclear gene encoding mitochondrial RPS14 is essential for mitochondrial function in yeast (24), and SDH2 is essential for electron transport within complex II (25), we presumed that expression of the sdh2-rps14 locus must generate functional SDH2 and RPS14 proteins. It has been previously shown that two transcripts are generated from the sdh2-rps14 locus by a combination of alternative splicing and the use of alternative polyadenylation sites (Fig. 1). One transcript encodes a chimeric SDH2(t)-RPS14 protein, while the second transcript encodes the SDH2 protein. In this paper we have shown that the precursor encoded by this second transcript is targeted to mitochondria. Upon import, a transit peptide of 2–3 kDa is removed to generate a 28 kDa protein (Fig. 2), which corresponds to the iron-sulfur subunit of complex II present in maize mitochondria (Fig. 4B). These sizes are consistent with cleavage at the R-3 motif RXX*(A/S) (T/S) present in the mitochondrial targeting presequences (Fig. 5A; X denotes any amino acid and the asterisk the cleavage site; 26). We
FIG. 5. Comparison of the mitochondrial targeting presequences (A) and connector region sequences (B) in maize and rice (9, 10). Colons (:) indicate identity with the residue in maize. Putative cleavage sites discussed in the text are indicated by a black box (the cleaved bond is marked by a vertical arrow).
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have also performed a detailed investigation of the proteolytic processing pathway of the SDH2(t)-RPS14 protein when it is imported into mitochondria. Our most important conclusion is that, in addition to removal of the same transit peptide as expected, a second cleavage occurs between SDH2 and RPS14 to liberate RPS14, which is found assembled into mitochondrial ribosomes. The truncated form of SDH2 is not present in maize mitochondria, being probably degraded because it is not assembled into functional complexes. Indeed, truncated SDH2 could not be functional as a component of complex II since it lacks the third cysteine-rich cluster encoded by the second exon. This cluster is mainly involved in formation of the third iron–sulfur center, which is essential for electron transport within the subunit. Our evidence strongly support a role for the SDH2 domain of the chimeric SDH2(t)-RPS14 protein only in generating a mitochondrial targeting function for RPS14. In the chimeric protein, SDH2 and RPS14 sequences are connected by a 25-amino acid sequence of unknown origin (Fig. 5B). Based on the size of the products detected by immunoprecipitation, we suggest that cleavage of the precursor occurs in this region. However, identification of the precise processing site remained to be determined. To our knowledge, there are two examples in fungi where different nuclear-encoded mitochondrial proteins were generated by proteolytical processing of a polyprotein precursor (27, 28). In only one instance detailed characterization of this processing has been performed (28, 29). Two precise cleavage sites in the connector region of this precursor have been identified (RGY2ST and RG2YST) and it was shown that their cleavage was carried out by the mitochondrial processing peptidase (MPP) and processing enhancing protein (PEP) which also process the N-terminal mitochondrial targeting sequence. It is tempting to speculate that a similar mechanism works in maize SDH2(t)-RPS14 precursor processing since a sequence RGYHG is found in the connector region, just upstream of the RPS14 homologous sequence (Fig. 5B). Interestingly, this sequence is also similar to the R-3 cleavage motif RX (F/Y)2(A/S) (T/S) proposed for mitochondrial plant presequences (30) and is conserved in maize, rice and wheat (our unpublished results). However, RPS14 would have a molecular weight of 12 kDa, which is lower than the size estimated by SDS–PAGE. We do not know if the processed 15 kDa protein has the connector region (2–3 kDa) as an N-terminal extension, or migrates anomalously on SDS–PAGE due to its high content of basic residues. The analysis of the 27 and 25 kDa processing products, which are both recognized by anti-SDH2 antibodies, gives no clues about the cleavage site, since the 27 kDa protein may correspond to either a transit peptide-SDH2(t) or a SDH2(t)connector region protein (the 25 kDa protein may cor-
respond to truncated SDH2, devoid of both the targeting peptide and the connector region). ACKNOWLEDGMENTS The authors are greatly indebted to Simon Litvak and Laura Tarrago´ for their support, especially during the stage of one of us (P.F.) in his laboratory. We also thank Jean-Claude Farre´ for valuable suggestions concerning immunoprecipitations, and Virginia Garreto´n, Gabriel Leo´n and Alvaro Elorza for fruitful discussions. This work was supported by Research Grants 8980005 (Lı´neas Complementarias) and 2970071 (Ph.D. students) from FondecytChile, C98B01 from ECOS-Conicyt, the French CNRS, and Universite´ Victor Segalen Bordeaux 2.
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