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A novel in vitro system for simultaneous import of precursor proteins into chloroplast and mitochondria Article in The Plant Journal · May 2002 DOI: 10.1046/j.1365-313X.2002.01280.x · Source: PubMed
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The Plant Journal (2002) 30(2), 213±220
TECHNICAL ADVANCE
A novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts Charlotta Rudhe1, Orinda Chew2, James Whelan2,* and Elzbieta Glaser1 1 Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, 106 91 Stockholm, Sweden 2 Plant Molecular Biology Group, School of Biomedical and Chemical Sciences, University of Western Australia, 35 Stirling Highway, Crawley 6009, WA, Australia Received 28 November 2001; accepted 8 January 2002. *For correspondence (fax +618 9380 1148; e-mail
[email protected])
Summary Most chloroplast and mitochondrial precursor proteins are targeted speci®cally to either chloroplasts or mitochondria. However, there is a group of proteins that are dual targeted to both organelles. We have developed a novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts (dual import system). The mitochondrial precursor of alternative oxidase, AOX was speci®cally targeted only to mitochondria. The chloroplastic precursor of small subunit of pea ribulose bisphosphate carboxylase/oxygenase, Rubisco, was mistargeted to pea mitochondria in a single import system, but was imported only into chloroplasts in the dual import system. The dual targeted glutathione reductase GR precursor was targeted to both mitochondria and chloroplasts in both systems. The GR pre-sequence could support import of the mature Rubisco protein into mitochondria and chloroplasts in the single import system but only into chloroplasts in the dual import system. Although the GR pre-sequence could support import of the mature portion of the mitochondrial FAd subunit of the ATP synthase into mitochondria and chloroplasts, mature AOX protein was only imported into mitochondria under the control of the GR pre-sequence in both systems. These results show that the novel dual import system is superior to the single import system as it abolishes mistargeting of chloroplast precursors into pea mitochondria observed in a single organelle import system. The results clearly show that although the GR pre-sequence has dual targeting ability, this ability is dependent on the nature of the mature protein. Keywords: Dual targeting, targeting speci®city, mitochondrial import, chloroplast import.
Introduction Most mitochondrial and chloroplast proteins are nuclear encoded and synthesized on cytosolic polyribosomes with an N-terminal extension called signal peptide that directs precursor proteins to the correct organelle. A presequence and a transit peptide are commonly used terms to describe cleavable signal peptides of mitochondrial and chloroplast precursors, respectively. Signal peptides are recognised by import receptors on the organellar outer membrane, and precursors are imported into the organelle through translocase complexes located on the outer and inner membranes of the organelles called Tom and Tim in ã 2002 Blackwell Science Ltd
mitochondria, and Toc and Tic in chloroplasts. After translocation into the mitochondrial matrix or chloroplast stroma, pre-sequences are cleaved off by the mitochondrial processing peptidase (MPP) and transit peptides by the stromal processing peptidase (SPP) (Braun and Schmitz, 1999; Glaser et al., 1998; Keegstra and Cline, 1999; Schleiff and Soll, 2000). Mitochondrial and chloroplast protein import was demonstrated to be highly speci®c (Boutry et al., 1987; Whelan et al., 1990). In vitro import of several mitochondrial proteins, e.g. F1b and FAd subunits of the ATP 213
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synthase, alternative oxidase and chloroplast proteins, e.g. 33 kDa, PsaK, chlorophyll a/b binding proteins indicates that import is organelle speci®c both in higher plants and in Chlamydomonas reinhardtii (Glaser et al., 1998; Nurani et al., 1997; Soll and Tien, 1998). A number of in vivo studies using transgenic approaches also showed high targeting speci®city into mitochondria and chloroplasts that was targeting signal dependent (Boutry et al., 1987; Schmitz and Lonsdale, 1989; Silva Filho et al., 1997). Missorting of mitochondrial proteins into chloroplasts has not been reported, however, several studies have reported mis-sorting of chloroplast proteins into mitochondria. The Rubisco small subunit transit peptide from C. reinhardtii could direct mouse dihydrofolate reductase and yeast cytochrome oxidase subunit IV into yeast mitochondria (Hurt et al., 1986). The mitochondrial pre-sequence of the yeast cytochrome oxidase subunit Va can function both as a mitochondrial and chloroplastic targeting peptide in transgenic tobacco (Huang et al., 1990). The PsaF protein from C. reinhardtii was shown to be imported in vitro into spinach and C. reinhardtii mitochondria (Hugosson et al., 1995). The transit peptides of the chloroplast outer envelope proteins, triose-3-phosphoglycerate phosphate translocator (TPT) and the 37 kDa protein coupled to CAT were imported into plant mitochondria in vitro but not in vivo (Brink et al., 1994; Silva Filho et al., 1997). More recent studies report mistargeting of pea plastocyanin into spinach mitochondria (von Stedingk, 1999). Furthermore, several pea chloroplast proteins such as the small subunit of Rubisco (Lister et al., 2001), plastocyanin, the 33 kDa photosystem II protein (E. von Stedingk E. Glaser, C. Robinson, unpublished results) could be imported into pea mitochondria. These reports indicate that mistargeting can be observed in a homologous in vitro import system. In the last few years a number of dual targeted proteins have been characterised that can be imported in vivo both into mitochondria and chloroplasts. The dual targeted proteins include pea glutathione reductase (GR), a number of aminoacyl-tRNA synthetases, and Arabidopsis RNA polymerase 2 (Akashi et al., 1998; Creissen et al., 1995; Duchene et al., 2001; Hedtke et al., 2000; Menand et al., 1998; Peeters et al., 2000; Small et al., 1998). All these proteins have been shown to have a dual targeting signal using in vivo approaches where the signal peptide was linked to a marker gene in stable or transient transgenic systems. A number of other proteins have also been reported to be dual targeted, but by means of different targeting signals produced from a single gene by posttranscriptional mechanisms (Chabregas et al., 2001; Small et al., 1998). Arabidopsis ferrochelatase-I was reported to be dual targeted on the basis of in vitro imports into isolated pea mitochondria (Chow et al., 1997), however, it is not imported into puri®ed Arabidopsis or several other plant mitochondrial preparations (Lister et al., 2001).
The targeting of proteins to mitochondria and chloroplasts can be studied using both in vitro or in vivo approaches. In vivo approaches use an intact cellular system and obviously re¯ect the in vivo targeting capacity of a signal. However, in vivo approaches have several limitations: (i) chimeric constructs usually contain passenger proteins and therefore the role of the mature protein is ignored; (ii) the investigated proteins are over-expressed at very high levels with a great variations in expression that may affect interpretation of results; (iii) no kinetics or ef®ciency of targeting can be assessed; and (iv) dissection of the mechanisms involved in protein recognition and import is not possible. In vitro import approaches can overcome these limitations but they have other disadvantages due to a lack of an intact cellular system: (i) it is possible to get incorrect targeting which is not seen in vivo; and (ii) competition between organelles is absent. In order to overcome the limitations of the in vitro import system, to elucidate the mechanisms involved in dual targeting, and to ensure that the correct speci®city of targeting we have developed a novel in vitro dual import system for simultaneous targeting of precursor proteins into mitochondria and chloroplasts. We use puri®ed organelles that are mixed, incubated with precursors and re-puri®ed after import. This allows the determination of the targeting speci®city into either organelle. This also allows the use of authentic precursors so that the role of the mature protein in import can be assessed. Results Development of dual in vitro import system Puri®ed import-competent mitochondria and chloroplasts were mixed and incubated with in vitro synthesized radiolabelled precursor proteins under conditions that supported import into both organelles. After completion of the import assay and thermolysin treatment in order to digest the non-imported precursor, organelles were re-isolated on Percoll gradient and the imported products were analysed (Figure 1). The purity of the re-isolated organelles was investigated by Western blots with antibodies against two chloroplast and two mitochondrial proteins. Figure 2 presents blots of the isolated and re-isolated pea leaf mitochondria and chloroplasts (Figure 2, lanes 1±4) with antibodies against the large subunit of Rubisco, the thylakoid light harvesting complex b2 protein (LHCb2), the mitochondrial inter membrane space protein cytochrome c (cyt c) and the inner membrane uncoupling protein (Ucp). The results clearly show high purity of both the isolated and re-isolated organelles, as antibodies against the chloroplast proteins cross-react only with chloroplasts and antibodies against the mitochondrial proteins only with mitochondria. Only a minor negligible cross-reactivity ã Blackwell Science Ltd, The Plant Journal, (2002), 30, 213±220
Dual targeting of proteins to mitochondria and chloroplasts
Figure 1. Overview of the procedure of the dual import assay. Precursor proteins, chloroplasts and mitochondria are produced using standard protocols. 100 mg of mitochondrial protein are mixed with 25 mg chlorophyll in a single tube and assay allowed to proceed for 20 min. Immediately after import the tube is placed on ice and divided into two equal aliquots, one treated with thermolysin. After the protease treatment the chloroplasts and mitochondria are re-puri®ed using a 4% (v/v) Percoll gradient. The import reactions are analysed on SDS-PAGE.
was observed using Rubisco antibodies with pea and soybean mitochondria, likely due to a very high abundance of Rubisco in leaf cells. A similar pattern was evident when soybean cotyledon mitochondria were used instead of pea leaf mitochondria.
Import of mitochondrial, chloroplast and dual targeted precursor proteins in the single and dual import system In order to examine the ®delity of protein import we carried out in vitro import experiments into pea leaf mitochondria and chloroplasts and soybean cotyledon mitochondria with precursor proteins of the mitochondrial soybean alternative oxidase (pAOX), chloroplast pea small subunit of RUBISCO (pSSU) and dual targeted pea glutathione reductase (pGR). Import was investigated in the single and dual import systems (Figure 3). In the single import system pSSU and pGR were imported into isolated chloroplasts and cleaved to mature size products whereas pAOX was not imported (Figure 3a, lanes 1±3). Import assays into pea mitochondria indicated that pAOX and pGR were imported in a membrane potential dependent manner and processed (Figure 3a, lanes 4±8). Additionally, pSSU was imported into isolated pea mitochondria, parã Blackwell Science Ltd, The Plant Journal, (2002), 30, 213±220
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Figure 2. Western blot analysis of mitochondria and chloroplasts. Antibodies to ribulose bisphosphate carboxylase/oxygenase (Rubisco), the light harvesting complex b2 (LHCb2), cytocrome c (Cyt c) and the inner membrane uncoupling protein (Ucp) were used to probe the organelle preparations. Each panel contains four lanes as follow. Lane 1, puri®ed pea leaf mitochondria. Lane 2, puri®ed pea leaf mitochondria that has been mixed with puri®ed pea leaf chloroplasts and re-isolated. Lane 3, puri®ed pea leaf chloroplasts. Lane 4, isolated pea leaf chloroplasts that have been mixed with pea leaf mitochondria and re-isolated. Lanes 5±8, as lanes 1±4 but with soybean cotyledon mitochondria instead of pea leaf mitochondria. The molecular weight of the proteins is indicated with arrows.
tially in a membrane potential dependent manner and cleaved to the same apparent molecular mass product as in chloroplasts (Figure 3a, lanes 4±8). This import was transit peptide dependent, as mature protein mSSU alone could not be imported (not shown). The isolated soybean mitochondria showed similar import patterns for pAOX and pGR, but no import of pSSU was observed (Figure 3a, lanes 9±13). Thus it was concluded that isolated pea mitochondria did not maintain the expected speci®city in contrast to soybean cotyledon mitochondria. We have previously reported similar results with other chloroplast precursor proteins where pea mitochondria, but not mitochondria from soybean or Arabidopsis, displayed import of chloroplast precursor proteins (Lister et al., 2001). In contrast, in the dual import system, in the presence of both chloroplasts and mitochondria, mitochondrial pAOX was imported only into mitochondria (Figure 3b, lanes 4± 13), chloroplast pSSU was only imported into chloroplasts (Figure 3b, lanes 1±3) and the dual targeted pGR was imported into both chloroplasts and mitochondria (Figure 3b, lanes 1±13). This implies that the dual targeting system is superior in comparison to the single organelle import system as it abolishes mistargeting of the chloroplast precursor observed to pea leaf mitochondria. Note
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Charlotta Rudhe et al. Figure 3. Import of precursor proteins into chloroplasts and mitochondria. (a) Import of AOX, SSU and GR into puri®ed pea leaf chloroplasts, pea leaf mitochondria and soybean cotyledon mitochondria. Lane 1, precursor protein alone. Lane 2, precursor protein incubated with pea leaf chloroplasts. Lane 3, as lane 2 but with the addition of protease (thermolysin) after the import assay. Lane 4, precursor protein alone. Lane 5, precursor protein incubated with pea leaf mitochondria. Lane 6, as lane 5 but with the addition of protease (thermolysin) after the import assay. Lane 7, as lane 5 but with the addition of valinomycin prior to import. Lane 8, as lane 7 but with the addition of protease (thermolysin) after the import assay. Lanes 9±13, as lanes 4±8 but using soybean cotyledon mitochondria. (b) Import of AOX, SSU and GR into the dual organelle import system. Lanes as described above except that the organelles were re-puri®ed after protease treatment.
that in the dual import assay excess precursor protein is added. Removal of both mitochondria and chloroplasts after the incubation period, and addition of the supernatant (containing unimported precursor protein) to mitochondria and chloroplasts alone resulted in imported products into both organelles, indicating that import competent precursor remained in the supernatant. Targeting ability of the dual targeting GR pre-sequence In order to investigate the targeting ability of the dual targeting GR pre-sequence, GR(p), we have constructed chimeric constructs between the GRp and mature proteins of AOX(m), FAd(m) and SSU(m) and investigated import of these constructs in the single and dual import system (Figure 4). The mature protein alone displays no targeting activity to either organelle (data not shown). In both import systems, the GR targeting signal supported import of mature FAd into mitochondria and chloroplasts (Figure 4a,b, lanes 1±13). The AOX(m) protein could be imported only into mitochondria under the direction of the
GR targeting signal (Figure 4a,b, lanes 1±13). There was ef®cient targeting of the SSU(m) protein under the control of the GR pre-sequence into chloroplasts in both import systems (Figure 4a,b, lanes 1±3). GR(p)-SSU(m) was imported into mitochondria in a single organelle import system (Figure 4a, lanes 4±13), but no import of the GR(p)SSU(m) was observed into mitochondria in the dual import system (Figure 4b, lanes 4±13). These results show that the GR pre-sequence has targeting ability for directing proteins into both chloroplasts and mitochondria. However, this ability seems to be selective and restricted by the nature of the mature protein. Furthermore, the dual import system presents additional restrictions in order to ensure the correct sorting of proteins as evidenced by the lack of import ability of SSU into pea leaf mitochondria in the dual import system. Discussion The correct sorting of nuclear encoded organellar proteins is fundamental for biogenesis of mitochondria and ã Blackwell Science Ltd, The Plant Journal, (2002), 30, 213±220
Dual targeting of proteins to mitochondria and chloroplasts
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Figure 4. Import of chimeric precursor proteins into chloroplasts and mitochondria. (a) Import of AOX, SSU and GR into puri®ed pea leaf chloroplasts, pea leaf mitochondria and soybean cotyledon mitochondria. Lane 1, precursor protein alone. Lane 2, precursor protein incubated with pea leaf chloroplasts. Lane 3, as lane 2 but with the addition of protease (thermolysin) after the import assay. Lane 4, precursor protein alone. Lane 5, precursor protein incubated with pea leaf mitochondria. Lane 6, as lane 5 but with the addition of protease (thermolysin) after the import assay. Lane 7, as lane 5 but with the addition of valinomycin prior to import. Lane 8, as lane 7 but with the addition of protease (thermolysin) after the import assay. Lanes 9±13, as lanes 4±8 but using soybean cotyledon mitochondria. (b) Import of AOX, SSU and GR into the dual organelle import system. Lanes as described above except that the organelles were re-puri®ed after protease treatment.
chloroplasts in the plant cell. A high sorting speci®city has been observed in extensive experimental work from many laboratories, and signal peptides were shown to contain information for the correct targeting. Both the mitochondrial and chloroplast signal peptides show a remarkable similarity in amino acid composition although there is no conservation at the primary structure level. The signal peptides are rich in hydroxylated hydrophobic and positively charged amino acid residues, and de®cient in acidic amino acids. In contrast to mitochondrial pre-sequences in non-plant organisms, plant pre-sequences have the same high abundance of serines as chloroplast transit peptides (Bruce, 2000; SjoÈling and Glaser, 1998). Nearly all presequences and transit peptides contain hsp70 binding motifs (Zhang and Glaser, 2002; Zhang et al., 1999). Mitochondrial pre-sequences have the potential to form amphiphilic a-helices, which is important for targeting and ã Blackwell Science Ltd, The Plant Journal, (2002), 30, 213±220
interactions with import receptors (Abe et al., 2000; von Heijne et al., 1989). Chloroplast transit peptides were initially thought to be random coils but, as more sequences of transit peptides have become available, computer predictions have showed that chloroplast transit peptides might also form secondary structures (Bruce, 2000). In most cases of the previously observed mistargeting it was concluded that the mistargeting was due to unusual properties of the signal peptide or the mature protein. The Rubisco transit peptide from C. reinhardtii that could direct proteins into yeast mitochondria (Hurt et al., 1986) contains an amphiphilic a-helix, characteristic of mitochondrial targeting peptides (Franzen et al., 1990). The import of chimeric constructs containing cytochrome oxidase subunit Va into chloroplasts was not receptor mediated. Import of PsaF into mitochondria was pre-sequence inde-
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pendent (Hugosson et al., 1995). We have shown that the mistargeting of pea pSSU into pea mitochondria observed in the single in vitro import system was partially membrane potential dependent and resulted in processing of the precursor to mature size product. The presence of the `competing' organelle, chloroplasts, during the mitochondrial import assay in the dual import system, entirely abolished mistargeting of pea SSU protein to pea mitochondria. In the dual import system with a choice between the organelles pSSU will bind and become imported only into chloroplasts. It has to be emphasised that under the same experimental conditions the dual targeted precursor of GR is imported into both mitochondria and chloroplasts. Thus the higher speci®city observed in the dual system is not simply a result of chloroplasts importing all import competent precursor protein faster than mitochondria due to the faster import kinetics observed in chloroplasts compared to mitochondria (May and Soll, 2000; Schleiff and Soll, 2000). The dual targeting ability of the GR signal is dependent on the nature of the passenger protein. All passenger proteins tested can be targeted to their `home' organelle under the direction of the GR signal, i.e. AOX and FAd to mitochondria and SSU to chloroplasts. However, only FAd could be re-directed to chloroplast under the in¯uence of the GR signal. With AOX and SSU the passenger protein modulates the dual targeting potential of the GR signal, so that it only targets to a single organelle, albeit to both organelles with different passengers. The passenger protein has previously been reported to play a role in the import of precursors proteins into mitochondria and chloroplasts. Mitochondrial protein import requires ATP in the matrix, and most precursors require ATP outside (Herrmann and Neupert, 2000). Although initially the role of external ATP was thought to play a role in unfolding precursors for mitochondrial import, it has been demonstrated that mitochondria can import folded precursors without the assistance of cytosolic factors. The mitochondrial import motor can actively unfold precursor proteins on the mitochondrial surface. The role of external ATP in mitochondria is now proposed to be related to prevention of aggregation (Matouschek et al., 2000). In contrast to mitochondria there is no requirement for external ATP for chloroplast import (Schleiff and Soll, 2000). Regions in the mature portion of SSU have been shown to enhance the interaction with components of the chloroplast import apparatus and the protein import related anion channel in chloroplasts. However, the mechanistic basis of the stimulatory effect of the mature protein is unknown (Dabney-Smith et al., 1999). The ability of the GR pre-sequence to direct FAd but not AOX to chloroplasts may be related to the fact that the FAd mature protein does not require external ATP for import into mitochondria, whereas AOX does (Tanudji
et al., 2001). Moreover, the fact that the GR pre-sequence cannot support the mitochondrial import of SSU mature protein in the dual system but can in the single organelle system, indicates that some factor(s) present in the chloroplast preparation can prevent the import of GR(p)SSU(m) and that these factors act on the mature portion of SSU. In conclusion we have developed a dual import system that maintains a high ®delity of import and allows the investigation of the role of mature protein in import and sorting between mitochondria and chloroplasts. Experimental procedures Plant materials Soybeans (Glycine max [L] Merr. cv. Stevens) and peas (Pisum sativum [L] Green feast) were grown in a controlled environment at 28°C. The growth cabinets were ®tted with arti®cial lights at 600 mmol-2 s-1 and set to a 16-h light and 8-h dark cycle.
Mitochondrial and chloroplastic isolation Mitochondria were isolated from 7-day-old soybean cotyledons and 10-day-old pea leaves using the published method of Day et al. (1985) (Day et al., 1985). Intact chloroplasts were isolated from 10-day-old pea leaves using published procedures (Bruce et al., 1994; Waegemann and Soll, 1995). The mitochondrial protein concentrations were determined using the Coomassie Plus Protein Assay Reagent (Pierce, IL, USA) and the chloroplastic chlorophyll concentrations as outlined previously (Arnon, 1949).
Precursor proteins Pea glutathione reductase (GR) (Creissen et al., 1992), soybean alternative oxidase (AOX) (Whelan et al., 1993), pea, the small subunit of ribulose bisphosphate carboxylase/oxygenase (SSU) (Anderson and Smith, 1986) and soybean subunit FAd of the mitochondrial ATPsynthase (FAd) (Smith et al., 1994) were produced using the rabbit reticulocyte TNT in vitro transcription/ translation kit (Promega, Madison, WI, USA). Chimeric constructs consisting of the GR targeting signal fused to the mature part of AOX, SSU and FAd were created using standard molecular biology techniques. Restriction sites were inserted at appropriate location and fragments were re-ligated using T4 DNA ligase (Sambrook et al., 1989). For all constructs sequences were con®rmed by sequencing using an ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit and analysed using an ABI 310 genetic analyser according to the manufacturer's instructions (Applied Biosystems, Melbourne, Australia).
In vitro import assays Mitochondrial and chloroplastic import experiments were carried out as previously described (Waegemann and Soll, 1995; Whelan et al., 1996). For imports into the dual import system (see Figure 1) mitochondria and chloroplasts were isolated separately, mixed and incubated together with precursor protein in an import buffer supporting import into both mitochondria and chloroplasts ã Blackwell Science Ltd, The Plant Journal, (2002), 30, 213±220
Dual targeting of proteins to mitochondria and chloroplasts in a ®nal volume of 100 ml (0.3 M sucrose, 15 mM HEPES-KOH pH 7.4, 5 mM KH2PO3, 0.2% BSA, 4 mM MgCl2, 4 mM methionine, 4 mM ATP, 1 mM GTP, 0.2 mM ADP, 5 mM succinate, 4.5 mM DTT, 10 mM potassium acetate and 10 mM NaHCO3) for 20 min at 25°C with gentle agitation. The samples were transferred to ice and separated into two equal aliquots, one containing 120 mg ml±1 thermolysin supplemented with 0.1 mM CaCl2 and incubated for 30 min. The thermolysin activity was inhibited with the addition of 10 mM EDTA and each sample was carefully loaded onto a 300-ml 4% (v/v) Percoll gradient in a 400-ml elongated microfugetube (Eppendorf, Hamburg, Germany) and centrifuged for 30 sec in a ®xed angle Microfuge rotor (Sigma 12029) at 4000 g. The organelle fractions were collected separately, chloroplasts at the bottom of the gradient as pellet and mitochondria at the top, and washed in 1 ml of wash buffer (0.3 M sucrose, 15 mM Hepes-KOH pH 7.4, 5 mM KH2PO3, 0.2% BSA) (Figure 1). The chloroplasts and mitochondria were recovered by centrifugation at 830 g and 20 800 g, respectively, and imported products analysed by SDSPAGE and imaged using a BAS2500 (Fuji, Tokyo, Japan).
Western blots Mitochondrial and chloroplastic proteins were treated as for a dual import experiment, but without the addition of radiolabelled precursor proteins. The proteins were resolved using SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, Sydney, Australia) using a semidry blotting apparatus (Millepore, Sidney, Australia). The organelle fractions were probed with chloroplastic antibodies against the stromal protein ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the thylakoid protein light harvesting complex b2 (LHCb2) and mitochondrial antibodies against the intermembrane space protein cytochrome c (cyt c) (BD Bioscience Pharmingen, CA, USA) and the inner membrane uncoupling protein (Ucp) (Considine et al., 2001). Chemiluminescence was used for detection and the bands were visualised and quanti®ed using a LAS 1000 (Fuji, Tokyo, Japan).
Acknowledgements We are greatful to Dr A.H.Millar for useful discussions. Dr Iwona Adamska (Stockholm University) and Prof Per GardestroÈm (UmeaÊ University) are thanked for the gifts of the LHCb2 and Rubisco antibodies, respectively. This work was supported by grants from The Swedish Foundation for the International Cooperation in Research and Higher Education and the Australian Research Council to EG and JW.
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