Portal de Periódicos da CAPES

Plant mitochondria actively import DNA via the permeability transition pore complex

2003; Springer Nature; Volume: 22; Issue: 6 Linguagem: Inglês

10.1093/emboj/cdg128

1460-2075

M. V. Koulintchenko,

Photosynthetic Processes and Mechanisms

Article17 March 2003free access Plant mitochondria actively import DNA via the permeability transition pore complex Milana Koulintchenko Milana Koulintchenko Institut de Biologie Moléculaire des Plantes du CNRS, Université Louis Pasteur, 12 rue du Général Zimmer, 67084 Strasbourg, France Siberian Institute of Plant Physiology and Biochemistry of the RAS, Lermontov Street 132, PO Box 1243, 664033 Irkutsk, Russia Search for more papers by this author Yuri Konstantinov Yuri Konstantinov Siberian Institute of Plant Physiology and Biochemistry of the RAS, Lermontov Street 132, PO Box 1243, 664033 Irkutsk, Russia Search for more papers by this author André Dietrich Corresponding Author André Dietrich Institut de Biologie Moléculaire des Plantes du CNRS, Université Louis Pasteur, 12 rue du Général Zimmer, 67084 Strasbourg, France Search for more papers by this author Milana Koulintchenko Milana Koulintchenko Institut de Biologie Moléculaire des Plantes du CNRS, Université Louis Pasteur, 12 rue du Général Zimmer, 67084 Strasbourg, France Siberian Institute of Plant Physiology and Biochemistry of the RAS, Lermontov Street 132, PO Box 1243, 664033 Irkutsk, Russia Search for more papers by this author Yuri Konstantinov Yuri Konstantinov Siberian Institute of Plant Physiology and Biochemistry of the RAS, Lermontov Street 132, PO Box 1243, 664033 Irkutsk, Russia Search for more papers by this author André Dietrich Corresponding Author André Dietrich Institut de Biologie Moléculaire des Plantes du CNRS, Université Louis Pasteur, 12 rue du Général Zimmer, 67084 Strasbourg, France Search for more papers by this author Author Information Milana Koulintchenko1,2, Yuri Konstantinov2 and André Dietrich 1 1Institut de Biologie Moléculaire des Plantes du CNRS, Université Louis Pasteur, 12 rue du Général Zimmer, 67084 Strasbourg, France 2Siberian Institute of Plant Physiology and Biochemistry of the RAS, Lermontov Street 132, PO Box 1243, 664033 Irkutsk, Russia *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:1245-1254https://doi.org/10.1093/emboj/cdg128 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Plant mitochondria are remarkable with respect to their content in foreign, alien and plasmid-like DNA, raising the question of the transfer of this information into the organelles. We demonstrate the existence of an active, transmembrane potential-dependent mechanism of DNA uptake into plant mitochondria. The process is restricted to double-strand DNA, but has no obvious sequence specificity. It is most efficient with linear fragments up to a few kilobase pairs. When containing appropriate information, imported sequences are transcribed within the organelles. The uptake likely involves the voltage-dependent anion channel and the adenine nucleotide translocator, i.e. the core components of the mitochondrial permeability transition pore complex in animal cells, but it does not rely on known mitochondrial membrane permeabilization processes. We conclude that DNA import into plant mitochondria might represent a physiological phenomenon with some functional relevance. Introduction Systematic sequence analyses predict a highly compact mitochondrial genome in the ancestor of green plants (Turmel et al., 2002). Nevertheless, higher plant, especially angiosperm, mitochondrial genomes have a strikingly large size [usually 300–800 kilobase pairs (kb)], as compared with the DNA of animal or fungal mitochondria (usually 16–85 kb). From that wealth of sequences, only 11–18% correspond to protein or structural RNA genes, >5% originate from plastids, nuclei or viruses and, even more puzzling, about half have no recognizable function and origin (Marienfeld et al., 1999; Kubo et al., 2000). Unlike those of mammals and most other eukaryotes, mitochondria of numerous plant species have also acquired, in addition to the main high molecular weight genome, one or several types of extrachromosomal plasmids or replicons composed of either DNA or RNA, with a size ranging from 0.7 to >20 kb (Brown and Zhang, 1995). In the plant species studied so far, mitochondrial DNA plasmids are arranged as species-specific or even line-specific sets of circular and linear DNA molecules with essentially unknown functions, and replicate independently of the main mitochondrial genomic DNA. Most of the mitochondrial plasmids have no sequence homology to the main mitochondrial genome and seem to be dispensable. Nevertheless, according to the developmental stage, they can be present at a high stoichiometry relative to the main genome: a ca 6:1 ratio was shown in the case of the S1 and S2 linear plasmids in maize mitochondria. This somehow resembles the situation occurring with chromosomal DNA and plasmids in bacteria. Linear mitochondrial DNA plasmids may carry expressed sequences encoding proteins or transfer RNAs (Leon et al., 1989, 1992). The origin of mitochondrial plasmids is unknown. It was suggested that double-strand DNA plasmids might have been introduced into higher plant cells by symbionts or pathogens (Douce and Neuburger, 1989). Supporting this hypothesis, the double-strand linear plasmids with 5′-associated proteins are reminiscent of the structure of some viral genomes. As a whole, all these data suggest that plant mitochondria have a high capacity to capture and integrate foreign sequences. The presence of integrated retrotransposons from the nuclear compartment, of sequences derived from RNA viruses and of RNA plasmids in plant mitochondria emphasizes the possibility of intercompartmental transfers via RNA intermediates (Marienfeld et al., 1999). In line with such considerations, the existence of specific tRNA import into plant mitochondria (Maréchal-Drouard et al., 1993; Glover et al., 2001) has indeed been established, in particular in our laboratory, but possible uptake of larger RNAs has received little attention and cannot account for all genetic information capture events and for the acquisition of DNA plasmids. We therefore asked the question of the existence of a mitochondrial mechanism responsible for controlled DNA transport. Using the well defined maize mitochondrial linear DNA plasmid of 2.3 kb (Leon et al., 1989) as a model substrate, we showed that plant mitochondria are able to take up double-strand linear DNA through a sequence-independent active mechanism, establishing a novel mitochondrial transport process of macromolecules. The incorporated DNA can be transcribed in the organelles and serve as a template for repair synthesis. Inhibition studies of the uptake point to an involvement of the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocator (ANT), which are considered as the core components of the mitochondrial permeability transition pore complex (PTPC) in animal cells (Zamzami and Kroemer, 2001). However, DNA import into plant mitochondria does not seem to be related to mitochondrial permeability transition (MPT). Results Plant mitochondria can incorporate large size DNA Tests for DNA import into isolated plant mitochondria were developed using radioactively labeled, double-strand maize (Zea mays) mitochondrial 2.3 kb linear plasmid (Figure 1A). Functional and intact potato (Solanum tuberosum) mitochondria were used throughout the present studies. Their respiratory control ratio (RCR; see Materials and methods) was 4.5–5 with succinate- supported respiration and their integrity was >97%. Mitochondrial incorporation was characterized by acquired resistance to extensive DNase digestion. Various experimental conditions were tested, and DNA uptake into plant mitochondria was obtained in a minimal medium containing an osmoticum (0.4 M sucrose) and a buffer (40 mM potassium phosphate pH 7.0). The detailed protocol is described in Materials and methods. According to time-course analyses, incorporation of the 2.3 kb plasmid into the mitochondrial fraction was progressive and reached a plateau after ∼30 min (Figure 1B). To test the stability of the incorporated DNA in mitochondria, the import reaction was stopped at 30 min by eliminating the excess of labeled exogenous DNA through DNase treatment and washing before further incubation of the organelles in the same conditions without import substrate. Even when mitochondria were re-isolated and extracted after 16 h of post-incubation, the amount of incorporated DNA lost by export (if any) or degradation was low (Figure 1C), indicating that the process leads to rather stable DNA integration into the organelles. In further assays, the mitochondrial outer membrane was broken by an osmotic shock after the import step, generating mitoplasts and leaving accessible to DNase digestion any labeled DNA that would not have crossed the mitochondrial inner membrane. Such a treatment had no influence on the DNase resistance of the incorporated DNA, as compared with a mock treatment (Figure 1D), implying that the DNA was protected by the mitochondrial inner membrane. Considering the large size of the DNA used in the tests, such a protection was likely to require a complete transfer of the DNA to the matrix side. It therefore appears that DNA is able to cross both mitochondrial membranes. The transport by itself is probably fast, as we never observed the appearence of fragments with intermediate sizes or of a smear upon electrophoretic analysis of the incorporated material. The efficiency was estimated at 1–5 pg DNA incorporated for an amount of mitochondria corresponding to 300 μg protein. The process was saturated at a DNA concentration of ∼20 ng/ml and was dependent on the pH (Figure 1E), with an optimal efficiency arround pH 7.0. Figure 1.Plant mitochondria can incorporate large size DNA. (A) Organization of the 2.3 kb maize mitochondrial plasmid (2.3PL). The plasmid contains two copies of a 170 nt inverted repeat (IR), a 885 nt open reading frame (orf1) and chloroplast-like genes for a tRNAPro (P) and a tRNATrp (T). (B) Time course of DNA uptake. Labeled linear maize 2.3 kb plasmid was incubated for different times in the presence of purified potato tuber mitochondria prior to DNase digestion. Mitochondrial nucleic acids were subsequently extracted, fractionated by agarose gel electrophoresis and transferred onto a nylon membrane, which was autoradiographed. (C) Stability of the incorporated DNA. Uptake of labeled maize 2.3 kb plasmid into potato mitochondria was run for 30 min. Following DNase treatment and washing, a sample was kept for direct analysis of the 30 min import (Imp), whereas the rest of the mitochondria was further incubated (Post-inc) for 3 or 16 h in the absence of added DNA. (D) DNase protection of incorporated plasmid is resistant to outer membrane breaking. Following incorporation of labeled maize 2.3 kb plasmid for 15 or 30 min, mitochondria were mock treated (Mt) or submitted to osmotic shock (Mpl) before DNase treatment. (E) DNA uptake is pH dependent. Incorporation of labeled maize 2.3 kb plasmid in the presence of potato mitochondria was run with a series of potassium phosphate buffers with different pH values. Incubation time for uptake was 30 min. Migration of the incorporated plasmid (2.3PL) is indicated. Download figure Download PowerPoint DNA incorporation into plant mitochondria is not sequence specific To characterize the specificity of DNA incorporation into plant mitochondria, different substrates were tested. When the labeled 2.3 kb maize plasmid was heated for 2 min at 95°C and chilled on ice to separate the DNA strands prior to incubation with plant mitochondria, incorporation into the organelles was abolished (Figure 2A), indicating that mitochondrial import is restricted to double-strand DNA. Replacing the maize mitochondrial 2.3 kb linear plasmid by linearized pBluescript, a bacterial vector (3.0 kb), or by a 2.1 kb linear DNA fragment corresponding to the coding sequence of the Arabidopsis thaliana threonyl-tRNA synthetase cDNA (previously cloned in our laboratory; Souciet et al., 1999), showed that mitochondrial DNA import is not specific either for plant plasmid sequences or for plasmid sequences at all (Figure 2B). Assays run with linearized pBluescript containing the 2.3 kb maize plasmid sequence as an insert (final size 5.3 kb) indicated that the efficiency of mitochondrial incorporation seems to decrease when increasing the length of the substrate DNA (Figure 2B). Finally, labeled circular pBluescript was poorly recovered in the mitochondrial fraction following DNA import assays. As a whole, it appears that DNA uptake into plant mitochondria does not depend on the sequence content, but is most efficient for linear fragments up to a few kilobase pairs. Figure 2.Mitochondrial uptake of different DNA substrates. (A) Single-strand DNA is not taken up by plant mitochondria. The labeled linear maize 2.3 kb plasmid was incubated in its native form (Nat) or heat- denatured form (Denat) in the presence of potato mitochondria (Imp). Incubation time for uptake was 30 min. Aliquots of the initial native or denatured DNA probe were also loaded on the gel (Cont), showing the migration of the double-strand (ds2.3PL) or single-strand (ss2.3PL) plasmid. The migration shift of the incorporated DNA versus the initial probe was due to the presence of the bulk of the mitochondrial nucleic acids, as shown by mixing a probe aliquot with a nucleic acid sample obtained from an import assay run without substrate. (B) DNA uptake is not sequence specific. Labeled linear DNA corresponding to the pBluescript vector (BLSc), the A.thaliana threonyl-tRNA synthetase (ThrRS), the maize 2.3 kb plasmid cloned in pBluescript (BLSc 2.3PL), as well as labeled circular pBluescript DNA (BLSc circ), were incubated in the presence of potato mitochondria (Imp). Incubation time for uptake was 30 min. Aliquots of the initial DNA probes were also loaded on the gel (Cont). The size and migration of the probes are indicated. Download figure Download PowerPoint The imported DNA is transcribed and used as a template for DNA synthesis in mitochondria The broad specificity of mitochondrial DNA incorporation allowed the development of expression tests, both to add more information to the body of evidence supporting the existence of the DNA import process and to establish whether the experimental system can be suitable for molecular analyses. For this purpose, DNA construct pCK/GFP/PRmt was prepared (for details, see the Materials and methods) as a basis to yield, by PCR amplification, a 2.3 kb maize mitochondrial plasmid containing the GFP gene from the jellyfish Aequoria victoria as a reporter unrelated to plant mitochondria. The GFP gene replaced the tRNAPro gene present in the wild-type 2.3 kb maize plasmid (Leon et al., 1989). It was placed under the control of the previously characterized promoter sequence of the potato mitochondrial 18S ribosomal RNA gene (Giese et al., 1996). Unlabeled linear 2.3 kb plasmid containing the GFP gene under the control of the plant mitochondrial promoter sequence (Figure 3A) was incubated with isolated S.tuberosum mitochondria in standard DNA import conditions. Following import and DNase digestion, mitochondria were further incubated for different times in a transcription medium designed to keep the organelles intact (Farré and Araya, 2001). Mitochondrial RNA was finally prepared and used for RT–PCR amplification with primers derived from the GFP sequence. A specific reverse transcriptase-dependent product of the appropriate size was amplified with the RNA extracted from mitochondria pre-incubated in the presence of the 2.3 kb plasmid containing the GFP gene and the mitochondrial promoter (Figure 3B). No RT–PCR product was detected when the wild-type 2.3 kb maize plasmid was used during the import step preceding transcription (Figure 3B). This implied that the GFP-containing 2.3 kb plasmid was imported and the reporter gene transcribed inside mitochondria. For further proof, [α-32P]UTP was substituted for UTP during the transcription step following import, so as to yield labeled transcripts which were hybridized to Southern blots carrying GFP-encoding DNA. The mitochondrial cob gene served as an internal control. Confirming the RT–PCR results, a strong hybridization signal was obtained with the labeled RNA extracted from mitochondria pre-incubated in the presence of the 2.3 kb plasmid containing the GFP gene (Figure 3C), whereas no signal was observed when the import step preceding transcription was run without exogenous DNA (Figure 3C). Interestingly, a clear signal was also detected when the labeled transcripts extracted from mitochondria pre-incubated in the presence of the 2.3 kb plasmid/GFP construct were hybridized against the DNA corresponding to the 885 nucleotide (nt) ORF1 present on this plasmid (Leon et al., 1992), whereas RNAs from the control mitochondria still showed no hybridization (Figure 3C). Hence, two distinct coding sequences with independent promoters could be expressed in mitochondria upon incorporation of exogenous DNA, bringing further evidence for the DNA import process and for the capability of isolated mitochondria to engage transcription of the introduced DNA. Figure 3.The incorporated DNA is transcribed and is a template for DNA synthesis in plant mitochondria. (A) Organization of the construct used as an import substrate for transcription and DNA synthesis studies. The tRNAPro gene present in the wild-type 2.3 kb maize plasmid (2.3PL) was replaced by the GFP gene (gfp) driven by the promoter of the potato mitochondrial 18S ribosomal RNA gene (Pr) in plasmid 2.3PLPrGFP. (B) GFP gene transcription in plant mitochondria as shown by RT–PCR. Potato mitochondria were incubated with PCR-amplified unlabeled 2.3PL or 2.3PLPrGFP DNA. Following DNA uptake, transcription was run for 2 h. Mitochondrial nucleic acids were isolated, treated with DNase and used for RT–PCR (+RT) with primers specific for the gfp sequence; amplification controls were run in identical conditions only omitting the reverse transcriptase enzyme in the corresponding medium (−RT). The expected DNA fragment (nucleotides 98–539 of the GFP gene) was also PCR-amplified directly from the initial construct (GF). (C) ORF1 and GFP gene transcription in plant mitochondria as shown by Southern hybridization. Potato mitochondria were incubated in the absence (Cont) or presence (2.3PLPrGFP) of PCR-amplified unlabeled 2.3PLPrGFP DNA. Following DNA uptake, transcription was run for 3 h in the presence of [α-32P]UTP. Mitochondrial nucleic acids were isolated from the different samples and identical amounts of radioactive transcripts were hybridized to Southern blots carrying DNA probes for the potato mitochondrial cob gene (cob), the ORF1 of the maize 2.3 kb mitochondrial plasmid (orf1) and the GFP gene (gfp). (D) DNA synthesis in plant mitochondria as shown by Southern hybridization. Potato mitochondria were incubated in the absence of exogenous DNA (Cont) or in the presence of PCR-amplified unlabeled 2.3PLPrGFP DNA (2.3PLPrGFP) or A.thaliana threonyl-tRNA synthetase encoding DNA (ThrRS). Following uptake, DNA synthesis was run for 1.5 h in the presence of [α-32P]dCTP. Mitochondrial nucleic acids were isolated from the different samples and identical amounts of radioactive DNA were hybridized to Southern blots carrying the (cob), (orf1) and (gfp) probes as in panel (C), or a threonyl-tRNA synthetase probe (thr). Fragment sizes determined with a DNA ladder [(La) in panel (B)] are indicated. Download figure Download PowerPoint Similar experiments were developed to test whether the mitochondrially incorporated 2.3 kb plasmid/GFP could be a template for DNA synthesis. Following import and DNase digestion, mitochondria were further incubated in essentially the same medium as for transcription, but [α-32P]dCTP and unlabeled dNTPs were substituted for [α-32P]UTP and unlabeled NTPs. The radioactive DNA generated was hybridized to Southern blots carrying cob, maize plasmid orf1 and gfp sequences. A strong hybridization to the orf1 and gfp probes was obtained with the labeled DNA extracted from mitochondria pre-incubated in the presence of the 2.3 kb plasmid containing the GFP gene, whereas no hybridization to these probes was observed when the import step was run without added DNA (Figure 3D). However, the generation of radioactive DNA was likely to reflect some repair processes, rather than replication of the 2.3 kb plasmid/GFP construct, because the coding sequence of the A.thaliana threonyl-tRNA synthetase (see above), a DNA fragment presumably deprived of replication information, was also a good template for DNA synthesis upon uptake into mitochondria (Figure 3D). Mitochondrial import of DNA is an active process To gather some hints about the mechanisms allowing DNA import into plant mitochondria, further functional tests were carried out. Malonate inhibits, in a competitive manner, the oxidation of succinate via complex II, leading to a decrease in electron transport. Increasing amounts of malonate thus gradually decrease the ΔΨ membrane potential (Millar et al., 1995). Titrating out the mitochondrial ΔΨ potential by increasing concentrations of malonate in the presence of succinate, progressively inhibited the incorporation of the labeled 2.3 kb maize plasmid into plant mitochondria (Figure 4A). The protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) allows proton diffusion across the inner membrane and collapses the proton-motive force (ΔpH, ΔΨ). CCCP also impaired the uptake of the 2.3 kb plasmid (Figure 4B). DNA import therefore appears to be an active process that requires both components of the mitochondrial inner membrane electrochemical potential. The inhibition of DNA uptake in the presence of increasing KCl concentrations in the import medium (Figure 4C) can tentatively be interpreted in terms of ΔΨ decrease or pH shift (Garlid and Paucek, 2001). Mg2+-mediated inhibition (Figure 4D) is likely to reflect an interaction with the structure of the DNA, as the same effect was obtained with spermidine. Figure 4.Mitochondrial uptake of DNA is an active process. (A) DNA uptake requires the ΔΨ potential. Mitochondrial incorporation of labeled maize 2.3 kb plasmid was tested in the presence of a constant concentration of succinate and increasing concentrations of malonate. (B) DNA uptake requires the proton motive force. Mitochondrial incorporation of labeled maize 2.3 kb plasmid was tested in the presence of increasing concentrations of the protonophore CCCP. (C and D) DNA uptake is inhibited by KCl and MgCl2. Mitochondrial incorporation of labeled maize 2.3 kb plasmid was tested in the presence of increasing concentrations of KCl (C) or MgCl2 (D). Migration of the incorporated plasmid (2.3PL) is indicated. Download figure Download PowerPoint Mitochondrial import of DNA potentially involves the VDAC and the ANT Trypsin pretreatment of the mitochondria prior to the import assay completely abolished DNA uptake (Figure 5A), implying that surface accessible outer-membrane proteins are involved. The two main constitutive pores known in the mitochondrial outer membrane are the translocase of the outer-membrane (TOM) complex and the VDAC (also called porin). The TOM complex is responsible for the transport of nuclear-encoded preproteins (Werhahn et al., 2001) and thus was not an obvious candidate for DNA translocation. We chose to test a possible involvement of the VDAC in mitochondrial DNA uptake. Ruthenium Red was shown previously to induce closure of the VDAC from rat liver mitochondria (Gincel et al., 2001). Addition of increasing concentrations of Ruthenium Red progressively inhibited DNA uptake by plant mitochondria (Figure 5B). Furthermore, incorporation of the 2.3 kb plasmid was abolished when mitochondria were pretreated with an antiserum against potato mitochondrial VDAC (Heins et al., 1994) prior to the import assay (Figure 5C), whereas no inhibition was observed with mock-treated mitochondria and with mitochondria pretreated with an antiserum against the wheat (Triticum aestivum) subunit 9 (NAD9) of the inner-membrane complex I (NADH dehydrogenase) (Lamattina et al., 1993) or against the A.thaliana cytosolic/mitochondrial alanyl-tRNA synthetase (Mireau et al., 1996). The DNA probe was stable when incubated with the antibodies in the absence of mitochondria, showing that there was no DNase activity in the antisera and implying that the lack of incorporation into mitochondria pretreated with the VDAC antiserum was not due to hydrolysis of the probe during the import step. As the VDAC controls the outer membrane permeability to ADP and ATP (Rostovtseva and Colombini, 1997), ADP-stimulated respiration rates in the presence of succinate were tentatively measured in parallel with DNA uptake to assess antibody inhibition. A 20–30% decrease in the respiratory control ratio was observed with mitochondria pretreated with the VDAC antiserum, as compared with mock-treated mitochondria or mitochondria pretreated with the NAD9 antiserum, which was taken as an indication for impairment of ADP import due to the binding of antibodies to the VDAC. Figure 5.Mitochondrial uptake of DNA is likely to involve VDAC/ANT complexes. (A) DNA uptake requires surface-accessible protein(s). The labeled maize 2.3 kb plasmid was incubated in the presence of mock-pretreated (Cont) or trypsin-pretreated (Tryp) potato mitochondria. (B) DNA uptake is inhibited by Ruthenium Red. Mitochondrial incorporation of labeled maize 2.3 kb plasmid was tested in the presence of increasing concentrations of Ruthenium Red (RuR). (C) DNA uptake is inhibited by antibodies against the VDAC. Incorporation of labeled maize 2.3 kb plasmid was tested in the presence of mock-treated mitochondria (Cont), mitochondria pretreated with BSA, or mitochondria pretreated with a polyclonal antiserum against the VDAC, NAD9 or alanyl-tRNA synthetase (AlaRS). As the antisera used contained 50% (v/v) glycerol, a control assay was also run in the presence of the relevant amount of glycerol (Glyc). (D) DNA uptake is inhibited by ADP. Mitochondrial incorporation of labeled maize 2.3 kb plasmid was tested in the presence of increasing concentrations of ADP and a constant concentration (10 μg/ml) of oligomycin (+). (E and F) DNA uptake is inhibited by atractyloside, but enhanced by bongkrekic acid. Mitochondrial incorporation of labeled maize 2.3 kb plasmid was tested in the presence of atractyloside (E) or bongkrekic acid (F). Migration of the incorporated plasmid (2.3PL) is indicated. Download figure Download PowerPoint The ANT is the major channel in the mitochondrial inner membrane. It imports ADP and exports ATP. In the presence of oligomycin, an inhibitor of the ATP synthase, ADP cannot be converted into ATP and therefore binds to the ANT, but does not translocate into the mitochondrial matrix, thereby blocking the channel. In such conditions, DNA uptake into plant mitochondria was clearly inhibited, even at low ADP concentrations (Figure 5D). Similarly, atractyloside, an ANT inhibitor acting on the intermembrane space side, abolished DNA uptake already at a 10 μM concentration (Figure 5E). On the contrary, bongkrekic acid, a matrix-side ANT inhibitor, stimulated DNA import into plant mitochondria (Figure 5F). In control oxygen electrode assays run in parallel, bongkrekic acid completely inhibited ADP-stimulated respiration depending on the ANT activity ('state III' respiration) in potato mitochondria. Mitochondrial import of DNA does not occur through permeability transition The above results strongly suggest that plant mitochondrial DNA uptake involves the VDAC and the ANT, which are actually the core components of the mitochondrial PTPC as defined in animal cells (Zamzami and Kroemer, 2001). In current models of the animal mitochondrial PTPC, hexokinase is associated to the VDAC on the cytosolic side. Hexokinase binding to mitochondria has also been reported in plants (Wilson, 1997). Addition of yeast hexokinase in DNA import assays with plant mitochondria led to a strong inhibition of the incorporation. These observations altogether, raised the question as to whether DNA import is related to MPT. Permeability transition leads to mitochondrial swelling and ultimately to the rupture of the outer membrane (Zamzami and Kroemer, 2001). Neither incubation in the import medium nor addition of DNA triggered any swelling of the mitochondria, as tested by absorption kinetics at 546 nm, indicating that there was no permeabilization of the membranes. DNA import was inhibited by atractyloside and stimulated by bongkrekic acid (see above), which is just the opposite of the effects these effectors have on MPT. Cyclosporin A had no influence on the incorporation of the 2.3 kb plasmid into plant mitochondria, although it is a strong inhibitor of the animal PTPC. MPT is mainly triggered by high Ca2+ and oxidative stress, including thiol oxidation (Vieira et al., 2000; Martinou and Green, 2001). Our experimental data show that DNA uptake by plant mitochondria does not require addition of Ca2+. When increasing amounts of CaCl2 were added to the import assays, the process was gradually inhibited (Figure 6A). Similarly, oxidative stress provided by hydroxyl radical (HO·) generation in the presence of H2O2 and FeSO4 (Fenton's reagent) strongly impaired mitochondrial DNA uptake (Figure 6B). The same effect was observed upon thiol oxidation mediated by diamide (Figure 6C). As a whole, plant mitochondrial DNA import does not seem to be related to known mitochondrial membrane perme