Báo cáo Y học: Functional integration of mitochondrial and hydrogenosomal ADP/ATP carriers in the Escherichia coli membrane reveals different biochemical characteristics for plants, mammals and anaerobic chytrids pdf

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Báo cáo Y học: Functional integration of mitochondrial and hydrogenosomal ADP/ATP carriers in the Escherichia coli membrane reveals different biochemical characteristics for plants, mammals and anaerobic chytrids pdf

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Functional integration of mitochondrial and hydrogenosomal ADP/ATP carriers in the Escherichia coli membrane reveals different biochemical characteristics for plants, mammals and anaerobic chytrids Ilka Haferkamp 1 , Johannes H. P. Hackstein 2 , Frank G. J. Voncken 2 , Guillaume Schmit 1 and Joachim Tjaden 1 1 Pflanzenphysiologie, Universita ¨ t Kaiserslautern, Germany; 2 Department of Evolutionary Microbiology, Faculty of Science, Catholic University of Nijmegen, the Netherlands The expression of mitochondrial and hydrogenosomal ADP/ATP carriers (AACs) from plants, rat and the anaerobic chytridiomycete fungus Neocallimastix spec. L2 in Escherichia coli allows a functional integration of the recombinant proteins into the bacterial cytoplasmic membrane. For AAC1 and AAC2 from rat, apparent K m values of about 40 l M for ADP, and 105 l M or 140 l M , respectively, for ATP have been determined, similar to the data reported for isolated rat mitochon- dria. The apparent K m for ATP decreased up to 10-fold in the presence of the protonophore m-chlorocarbonyl- cyanide phenylhydrazone (CCCP). The hydrogenosomal AAC isolated from the chytrid fungus Neocallimastix spec. L2 exhibited the same characteristics, but the affinities for ADP (165 l M )andATP(2.33m M )were significantly lower. Notably, AAC1-3 from Arabidopsis thaliana and AAC1 from Solanum tuberosum (potato) showed significantly higher external affinities for both nucleotides (10–22 l M ); they were only slightly influenced by CCCP. Studies on intact plant mitochondria confirmed these observations. Back exchange experiments with preloaded E. coli cells expressing AACs indicate a preferential export of ATP for all AACs tested. This is the first report of a functional integration of proteins belonging to the mito- chondrial carrier family (MCF) into a bacterial cytoplasmic membrane. The technique described here provides a relat- ively simple and highly reproducible method for functional studies of individual mitochondrial-type carrier proteins from organisms that do not allow the application of sophisticated genetic techniques. Keywords: ADP/ ATP carriers; mitochondria; hydrogeno- somes; heterologous expression; Escherichia coli. A high degree of compartmentalization is characteristic of eukaryotic cells. The transport of metabolic intermediates between organelles is necessary for complex metabolism and is mediated by membrane proteins which function as carriers or channels. Mitochondria and certain hydrogeno- somes evolved peculiar ADP/ATP carriers (AACs) that efficiently export ATP, whereas plastids acquired a different type of nucleotide transporter that seems to be specialized in ATP uptake [1–3]. In many multicellular animals and plants as well as in yeast (Saccharomyces cerevisiae),twotofourAACisoforms have been identified [4–8]. Mammalian AACs seem to have a tissue specific expression [4–6,9], whereas in yeast, the expression of the various isoforms is believed to be characteristic for certain metabolic states, or to participate in vacuolar metabolism [10,11]. However, the reasons for the repeated evolution of AAC isoforms and the intrinsic biochemical differences between the various isoforms remained largely unknown until now. Isolated mitochondria of plants and mammals are likely to represent a mixture of mitochondrial variants that possess different isoforms of AACs. Consequently, a biochemical characterization of the various isoforms of AACs is difficult, if not impossible using conventional methods of cell fractionation. The function of the different AACs in S. cerevisiae could be studied taking advantage of the sophisticated genetic tools available for this organism: deletion mutants for all three isoforms have been generated that allowed a detailed analysis of particular AACs in the absence of other isoforms [10,11]. Trivially, comparable studies are only possible in organisms with a highly developed Ôgenetic tool kitÕ (cf. Drosophila [12]). Therefore, alternative methods are required to study particular iso- forms of mitochondrial-type AACs in organisms that do not allow the application of sophisticated genetic tech- niques. The aim of the present study is the development of a simple and reproducible approach to analyse the biochemi- cal properties [substrate affinities, proton motive force (PMF) dependency] of individual mitochondrial-type AACs in order to identify differences in the function of the various isoforms and to understand the repeated evolution of AAC isoforms in a variety of organisms. We have shown earlier that it is possible to express adenine nucleotide transporters of plastids and certain parasites, i.e. Chlamydia and Rickettsia, in Escherichia coli and to test their function in vivo [13–16]. This technique Correspondence to J. Tjaden, Pflanzenphysiologie, Universita ¨ t Kaiserslautern, Erwin-Schro ¨ dinger-Str., D-67663 Kaiserslautern. Fax: + 631 2052600, Tel.: + 631 2052505, E-mail: tjaden@rhrk.uni-kl.de Abbreviations: AAC, ADP/ATP carrier; CCCP, m-chlorocarbonyl- cyanide phenylhydrazone; IPTG, isopropyl thio-b- D -galactoside; MCF, mitochondrial carrier family; PMF, proton motive force. (Received 13 February 2002, revised 29 April 2002, accepted 9 May 2002) Eur. J. Biochem. 269, 3172–3181 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02991.x might be a matter of choice also for other nucleotide carriers, but recently published data suggested that mito- chondrial-type AACs might be deposited preferentially as inclusion bodies in E. coli; for a functional biochemical analysis these inclusion bodies have to be solubilized and reconstituted in vitro [17–19]. Here, we describe a technique that allows the functional expression of a variety of single mitochondrial-type AACs in E. coli. After IPTG-induction we were able to obtain functional integration of the various mitochondrial-type AACs into the cytoplasmic membrane of E. coli. Measuring the uptake of the various adenylates into intact E. coli cells expressing eukaryotic AACs, we were able to determine the biochemical properties of the different mitochondrial-type AACs. We studied two AAC isoforms from rat (Rattus norwegicus) and a mitochondrial-type AAC from the hydrogenosomes of the anaerobic chytrid Neocallimastix. As the knowledge about plant mitochon- drial nucleotide exchange is so far quite limited, we investigated three AAC isoforms from Arabidopsis thaliana and one AAC from potato (Solanum tuberosum). The results obtained by expression in E. coli were justified by comparison with the adenylate uptake into intact plant mitochondria isolated from Arabidopsis and Solanum leaves or potato tubers, respectively. EXPERIMENTAL PROCEDURES DNA constructs for heterologous expression of AACs in E. coli DNA manipulations were performed essentially as des- cribed in Sambrook et al. [20]. The expression plasmids (pet16b, Novagen, Heidelberg, Germany) encoding the recombinant AAC proteins with an additional N-terminal tag of 10 histidine residues were constructed as follows: the cDNA coding the entire AACs were generated by PCR from first-strand cDNA of plants and mammals (Arabid- opsis thaliana, Solanum tuberosum and Rattus norwegicus)or from a full-length cDNA clone (Neocallimastix spec. L2) with Pfx DNA polymerase (Gibco BRL, Eggenheim, Germany), which possesses proof reading activity. Sense primers incorporating an NdeI restriction site and antisense primers were used for the PCR reaction (Table 1). The obtained PCR products were purified (QIAquick PCR Purification Kit, Qiagen, Hilden, Germany), subcloned into the EcoRV restriction site of the plasmid pBSK (Stratagene, Heidelberg, Germany) and checked by sequencing both strands by chain-termination reaction (MWG-Biotech, Ebersberg, Germany). For the construction of the E. coli expression plasmids (encoding His 10 –AAC), the NdeI–XhoI (NdeI–BamHI or NdeI–BglII) DNA inserts of the pBSK- plasmids were introduced in-frame into the corresponding restriction sites of the isopropyl thio-b- D -galactoside (IPTG)-inducible T7-RNA polymerase bacterial expression vector pet16b (Novagen, Heidelberg, Germany). Transfor- mations of E. coli were carried out according to standard protocols. The nucleotide sequences of the AACs reported in this paper are available under the accession numbers (EMBL database): AY042814 (aac1, A. thaliana), AY050857 (aac2, A. thaliana), AL021749/gene ¼ ÔF20O9.60Õ (aac3, A. thaliana), X62123 (aac1, S. tubero- sum), D12770 (aac1, R. norwegicus), D12771 (aac2, R. norwegicus), AF340168 (hdgaac, Neocallimastix spec. L2). Heterologous expression of AACs in E. coli The E. coli strain BL21 (DE3) was used for heterologous expression. The several cDNAs encoding the correspond- ing AAC proteins under control of the T7-promoter were transcribed after IPTG induction of the T7-RNA poly- merase [21]. E. coli cells transformed with the AAC expression plasmids (or control expression plasmid pet16b) were inoculated with a fresh overnight culture and grown at 37 °CeitherinYT Amp/Clm medium (YT: 5gÆL )1 yeast extract, 8 gÆL )1 peptone, 2.5 gÆL )1 NaCl, pH 7.0; for hydrogenosomal AAC) or in TB Amp/Clm medium (TB: 2.5 gÆL )1 KH 2 PO 4 ,12.5gÆL )1 K 2 HPO 4 , 12 gÆL )1 peptone, 24 gÆL )1 yeast extract, 0.4% glycerin, pH 7.0) supplemented with 10 m M malate and 10 m M pyruvate (rat and plant AACs) [20]. A D 600 value of 0.5–0.6 was required for the initiation of T7-RNA polymerase expression by addition of IPTG (final con- centration 0.012%). Cells were grown for 1 h after Table 1. Oligonucleotides used for construction of the expression plasmids of plant, mammalian and chytridic AACs. Thelower-caseletterindicates the introduced base exchange to create restriction sites (NdeIorBglII). aac Oligonucleotide sequence aac1 (A.t.) Sense 5¢-TGCAGAGTTCcAtATGGTTGATCAAG-3¢ Antisense 5¢-CGAAAAAAGGAGGAAGAAGCAATGC-3¢ aac2 (A.t.) Sense 5¢-TGTAGAGGTTcAtATGGTTGAACAGACTC-3¢ Antisense 5¢-CTTAATGACTGCGGGATTTGGTGGTAC-3¢ aac3 (A.t.) Sense 5¢-CTGATTTGTACAAcAtATGGATGGATC-3¢ Antisense 5¢-GGGCTATTCTTTCATCATCCTCATCG-3¢ aac1 (S.t.) Sense 5¢-TTAAACGTTcatATGGCAGATATGAACC-3¢ Antisense 5¢-GGAAGTTACGAGGCTGACTTAGGC-3¢ aac1 (R.n.) Sense 5¢-GCGCCCGCGTTTCcatATGGGGGATCAG-3¢ Antisense 5¢-CCACACAATGGATCTGTGAACCTGTG-3¢ aac2 (R.n.) Sense 5¢-CTTTTTTGCTTTCcAtATGACAGATGCCG-3¢ Antisense 5¢-TACAACATGCCAGAtCtCGGGGAGAAC-3¢ hdgaac (N.spec.) Sense 5¢-TTCCCCATATCCcAtATGGCCCAAAAG-3¢ Antisense 5¢-GCATTCGTTTAGTTCTTAATTCTCCAG-3¢ Ó FEBS 2002 Functional integration of AACs in E. coli (Eur. J. Biochem. 269) 3173 induction and collected by centrifugation for 5 min at 5000 g (8 °C, Sorvall RC5B centrifuge, rotor type SS34; Sorvall-Du Pont, Dreieich, Germany). The sediments were resuspended to D 600 ¼ 5 using potassium phosphate buffer (50 m M , pH 7.0) [16] and stored at 8 °C until use. Uptake of radioactively labeled ATP and ADP IPTG-induced E. coli cells (100 lL) harbouring the AAC expressing plasmids (or the given controls) were added to 100 lL potassium phosphate buffer (50 m M ,pH7.0) containing radioactively labeled ATP or ADP. [a- 32 P] adenine nucleotides were used at specific activities between 100 and 3000 lCiÆlmol )1 .[a- 32 P]ADP was synthesized enzymatically from [a- 32 P]ATP (NEN, Bad Homburg, Germany), as described by Tjaden et al.[13],andthe purity of the [a- 32 P]ADP preparation was confirmed by a thin-layer chromatography [13]. Uptake of nucleotides was carried out at 30 °C in an Eppendorf reaction vessel incubator and terminated after the indicated time periods by transferring the cells to a 0.45-lmfilter(25mm diameter, Orange Scientific, Waterloo, Belgium) under vacuum previously moistened with potassium phosphate buffer (50 m M , pH 7.0) [22]. Cells were further washed to remove unimported radioactivity by addition of three times 4-mL potassium phosphate buffer (50 m M ,pH7.0). The filter was subsequently transferred into a 20-mL scintillation vessel and filled with 10 mL of water. Radioactivity in the samples was quantified in a Canberra–Packard Tricarb 2500 scintillation counter (Canberra–Packard, Frankfurt, Germany). For back exchange (efflux) experiments, the E. coli cells were preincubated for 2 min at 30 °C with potassium phos- phate buffer (50 m M , pH 7.0) containing [a- 32 P]ADP or [a- 32 P]ATP (specific activity 100 lCiÆlmol )1 ). Preloading was stopped by centrifugation (5000 g, 45 s, room tem- perature). The washed sediment (four times with potas- sium phosphate buffer) was subsequently resuspended in incubation buffer containing the indicated additions and incubated for 1.5 min at room temperature. The cells were sedimented for 2 min at room temperature in an eppen- dorf centrifuge (5000 g). The supernatents were trans- ferredtonewreactionvesselsandheatedto95°Cfor 10 min to prevent any further reaction. An aliquot of the exported radioactive solution was used for separation by thin-layer chromatography [23]. Thin-layer chromatography of radioactively labeled adenine nucleotides To identify the type of adenine nucleotide exported from [a- 32 P]ADP or [a- 32 P]ATP preloaded E. coli cells, we employed a thin-layer chromatography system according to the method of Mangold [23]. Radioactively labeled samples were loaded onto a 0.5-mm poly(ethylene amine) cellulose thin-layer chromatography plate and dried with a fan. Separation was carried out for 0.5 min using 0.5 M sodium formiate (pH 3.4), for 2 min using 2 M sodium formiate (pH 3.4) and the front was allowed to run for 15 cm with 4 M sodium formiate (pH 3.4). R f values of radioactively labeled adenine nucleotides were determined after autoradiography and correspond to R f values of unlabeled nucleotides visualized under UV light [23]. Radiolabeling of AAC proteins synthesized in E. coli and enrichment of the histidine-tagged chimeric proteins Ten milliliters of E. coli cells harbouring the indicated plasmids were grown to exponential phase, collected by centrifugation at D 600 ¼ 0.5, and resuspended in 1 mL of a methionine assay medium containing 42 m M Na 2 HPO 4 , 20 m M KH 2 PO 4 ,18m M NH 4 Cl, 8.5 m M NaCl, 1 m M MgSO 4 ,0.1m M CaCl 2 ,20m M glucose, and 0.1 mgÆmL )1 thiamine (Difco Laboratories, Heidelberg, Germany). T7- RNA polymerase synthesis was induced by adding 1 m M IPTG. After shaking the culture for 15 min at 37 °C, 20 lL rifampicin (stock 20 mgÆmL )1 , dissolved in methanol) was added to inhibit the E. coli RNA polymerase. E. coli cells were shaken for additional 15 min after which 5 lL [ 35 S]methionine (50 lCi) were added to label newly syn- thesized proteins for 20 min at room temperature. Cells were sedimented by centrifugation and transferred to liquid nitrogen to destroy cell intactness. After resuspension in a medium consisting of 10 m M Tris/HCl (pH 7.5), 1 m M EDTA, 0.1 m M pefabloc and 15% (v/v) glycerol, cells were further disrupted by ultrasonication (250 W, 3 · 30 s, 4 °C) and the suspension was centrifuged (10 min, 15 800 g,4°C) to remove unbroken cells and inclusion bodies. Membranes extracted in the supernatent were sedimented for 45 min at 100 000 g (TFT 80 rotor, Kontron Instruments, Munich, Germany), resuspended in binding buffer A consisting of 10 m M imidazole, 300 m M NaCl, 100 m M Na 2 HPO 4 (pH 8.0, HCl), and 0.1% dodecylmaltoside. After addition of dodecylmaltoside (3.3% final concentration) and incu- bation on ice for 15 min, the detergent was 10 times diluted with buffer A and centrifuged for 2 min (15 800 g,4°C). The solubilized histidine-tagged AACs in the superna- tent were purified by Ni-chelating chromatography accord- ing to the supplier’s instructions (Novagen, Heidelberg, Germany). Eluted proteins were precipitated by adding acetone (80% f.c.), incubated at )70 °Cfor2hand sedimented by centrifugation (15 800 g,10min,4°C). For SDS/PAGE, the air dried protein sediments were resuspended in 40 lL of double concentrated SDS/PAGE sample buffer medium and incubated on ice for 60 min. Finally, the preparation was applied to a polyacryl- amide gel (3% stacking gel, 15% running gel) for electro- phoresis in the presence of 0.1% sodium dodecyl sulfate (SDS/PAGE). After drying, the gels were autoradiographed for 3 days. Preparation and uptake experiments of plant mitochondria Mitochondria were isolated from potato and Arabidopsis leaves and from potato tubers by isopycnic centrifugation in density gradients of Percoll following the procedure of Neuburger et al. [24]. The plant material was ground in prechilled extraction medium. The homogenate obtained after the mincing process was squeezed through eight layers of muslin. The flow-through was subject to several centrif- ugation steps. The resulting pellet was layered on Percoll gradient. To obtain a discrete fraction, we increased the Percoll concentration up to 32% dependent on the plant material. To obtain energized mitochondria, we added 5m M glycine, 10 m M malate, 10 m M pyruvate and 2 m M NAD + during the isolation procedure as well as in the 3174 I. Haferkamp et al. (Eur. J. Biochem. 269) Ó FEBS 2002 storage medium. The mitochondrial fraction was washed and resuspended in 20 m M Tris/HCl (pH 7.4), 2 m M MgCl 2 and 200 m M sucrose. Purity and intactness were analysed using marker enzymes, as described previously [25–27]. Uptake of [a- 32 P]ADP and [a- 32 P]ATP was carried out using a rapid filtration technique, as described by Winkler et al. [28]. RESULTS Heterologous expression of AACs in E. coli cells Seven different mitochondrial-type AACs from plants, rat and the anaerobic chytridiomycete fungus Neocallimastix spec. L2 were cloned into the plasmid pet16b and expressed in E. coli (BL21, DE3). Induction by IPTG was carried out in the presence of radioactive [ 35 S]methionine in order to detect even low amounts of newly synthesized proteins. The membrane fractions of the induced E. coli cells were isolated and the recombinant AAC proteins were further purified by Ni-nitrilotriacetic acid chromatograpy. The autoradiography of the corresponding SDS/PAGE pre- sented in Fig. 1 confirmed that all AACs studied had been inserted into the bacterial membrane. The overexpressed AAC proteins migrate according to their calculated apparent molecular masses (Fig. 1, lanes 2–8). Notably, the expression levels of AAC2 from rat, AAC1 from potato and AAC3 from Arabidopsis are much higher than those of the AAC1 and AAC2 isoforms from Arabidopsis and AAC1 from rat. The hydrogenosomal AAC from Neocallimastix spec. L2 exhibited the lowest expression level. Furthermore, Western blot analyses of purified membranes of the induced E. coli cells using an anti-His Ig have corroborated the integration of the AACs into the bacterial membrane (data not shown). Not even traces of the recombinant proteins were detectable in the corresponding membrane fraction isolated from induced E. coli cells harbouring the empty plasmid pet16b (Fig. 1, lane 1). Interestingly, when E. coli cells hosting plant and mam- malian AACs are induced for protein expression in ÔnormalÕ growth medium (YT, see Experimental procedures), the growth was retarded with respect to control. These E. coli cells reached a stationary phase at a D 600  1.0 after approximately 1–2 h induction. This observation led to the conclusion that E. coli cells did not possess, under these conditions, a high energy state which is considered as a prerequisite for the proper investigation of the AACs under different PMF levels across the E. coli membrane. There- fore, we optimized growth conditions (TB, see Experimental procedures) for E. coli cells producing the higher expressed plant and mammalian AAC proteins (Fig. 1). The unde- fined TB medium and the addition of pyruvate and malate stimulated the bacterial metabolism and led to the genera- tion of a high proton motive force across the bacterial membrane. Under these conditions, no retardation in cell growth over a time span up to 24 h following IPTG- induction was observed. The D 600 reached a value of about 12. It was tempting to analyse whether the heterologously expressedAACsintegratedintheE. coli membrane were functional. As phosphatidylglycerol and cardiolipin in the mitochondrial membranes are believed to be essential for many mitochondrial functions [29], the deviating lipid content of the E. coli membranes might hamper the function of the transgenic AACs. In particular, cardiolipin is well investigated and considered as very important for mitochondrial AACs [30–32]. As revealed by high-resolu- tion 31 P-NMR, the AAC from beef heart mitochondria has high amounts of tightly bound cardiolipin and the removal of these lipids renders the carrier inactive [33]. The phospholipid composition of the E. coli inner membrane is clearly different from the mitochondrial membranes. It corresponds to about 30% of the acidic phospholipids phosphatidylglycerol and cardiolipin (phosphatidylglycerol, 20–25%; cardiolipin, 5–10% [34]), which might be crucial for the functional integration of the AACs. Notwithstand- ing, we were able to demonstrate a time dependent nucleotide uptake for all seven investigated AACs (Fig. 2, Table 2). Figure 2 shows representative diagrams for the plant (Fig. 2A; AAC2, A.t.), mammalian (Fig. 2B; AAC2, R.n.) and hydrogenosomal (Fig. 2C; hdgAAC, N. spec. L2) AACs expressed in E. coli. The import of 32 P-labeled ADP or ATP into intact E. coli cells expressing the plant and mammalian AACs was linear with time for about 30 min, when the cells were harvested under optimal growth conditions. Interestingly, the weakly expressed hydrogeno- somal AAC (Fig. 1) showed much higher specific uptake rates. However, the rates are linear only for about 10 min. The ratios of ADP to ATP uptake into E. coli cells expressing the plant AACs were significantly lower (Fig. 2A) than the ratios of ADP to ATP uptake of the expressed rat and hydrogenosomal AACs (Fig. 2B,C). Notably, the addition of the specific inhibitors of mitoch- ondrial nucleotide exchange bongkrekic acid and carboxya- tractyloside [35] led to about 50% reduction of transport activity in E. coli expressing the recombinant AACs (data not shown). The treatment of E. coli cells with lysozyme was crucial for this decrease to occur because the outer bacterial membrane obviously prevents the penetration of the mentioned inhibitors. Induced E. coli cells harbouring the Fig. 1. Heterologous expression and membrane purification of [ 35 S]methionine-labeled His-tagged AAC proteins. E. coli cells har- bouring plasmid encoding several AACs and E. coli control cells (pet16b without any insert) were IPTG-induced for protein synthesis in the presence of [ 35 S]methionine. Details of induction, purification and autoradiography are given in ÔExperimental proceduresÕ. Lane 1, Ecoli controlcells;lane2,E. coli cells expressing the hydrogenosomal AAC from Neocallimastix spec. L2; lane 3, E. coli cells expressing the AAC1 from Rattus norwegicus;lane4,E. coli cells expressing the AAC2 from Rattus norwegicus;lane5,E. coli cells expressing the AAC1 from Arabidopsis thaliana;lane6,E. coli cells expressing the AAC2 from Arabidopsis thaliana;lane7,E. coli cells expressing the AAC3 from Arabidopsis thaliana;lane8,E. coli cells expressing the AAC1 from Solanum tuberosum. Ó FEBS 2002 Functional integration of AACs in E. coli (Eur. J. Biochem. 269) 3175 pet16b control plasmid (without any insert) as well as E. coli wildtype cells are not able to transport ADP or ATP [13]. Theimportof[a- 32 P]ADP and [a- 32 P]ATP by the recombinant AACs into E. coli displays typical Michaelis–Menten kinetics. The results were plotted as Lineweaver–Burk and Eadie–Hofstee diagrams, as sum- marized in Table 2. The K m values were determined with or without addition of the protonophore CCCP because nucleotide uptake into well-coupled mitochondria (high energy state) from mammals was shown to be controlled by the membrane potential (proton motive force; PMF) across the inner mitochondrial membrane [36,37]. The addition of CCCP also causes a decrease of the PMF across bacterial membranes [15].The apparent K m values for AAC1 and AAC2 from rat mitochondria are about 40 l M for ADP and about 105 and 140 l M for ATP, respectively. The presence of the protonophore CCCP increases the ATP affinity for both AACs up to 10-fold, whereas the ADP affinity is not affected. In contrast, the four plant AACs show significantly higher affinities for both, ADP and ATP. The corresponding K m values range between 10 and 22 l M . They are only slightly influenced by CCCP. On the other hand, the kinetic data of the hydrogenosomal AAC from Neocallimastix reveals apparent affinities that are four times lower for ADP and about 20 times lower for ATP compared to those of the rat AACs. Nevertheless, the influence of CCCP on ADP and ATP affinities is similar to that observed with the rat AACs. In our approach, the maximal velocity (V max ) of nucleo- tide import into E. coli cells harbouring the AAC expressing plasmids does not appear to be a direct function of the amount of recombinant nucleotide carriers present in the bacterial membrane (Fig. 1, Table 2). We believe that only a certain fraction of the recombinant protein is integrated as a functional homodimer into the bacterial membrane. More- over, isoforms from different organisms are being compared in these experiments. Back exchange experiments with nucleotide preloaded E. coli cells Mitochondrial AACs operate in a counter exchange mode permitting influx or efflux of ADP and ATP in a 1 : 1 stoichiometry [38]. To investigate the internal affinities for both nucleotides, we carried out several back exchange experiments with E. coli cells preloaded with either ADP or ATP. If a counter exchange mechanism is maintained in induced E. coli cells, then the export of adenylates by the recombinant AACs must depend on the presence of the appropriate externally applied substrate. Therefore, we preloaded IPTG-induced E. coli cells (expressing different AACs) with radioactively labeled ADP (or ATP) and performed back exchange experiments in the presence of the various unlabeled external substrates. The radioactively labeled nucleotides, which were released after preloading, were analyzed by thin-layer (poly(ethylene amine) chro- matography. To investigate to what degree incorporated nucleotides are metabolized by the E. coli cells, we disrupted the cells after preloading with ADP (or ATP) Fig. 2. Time dependency of [a 32 P]ADP (j), [a 32 P]ATP (s) uptake into intact E. coli cells. IPTG-induced E. coli cells harbouring plasmid encoding several AACs were incubated with (A) 7.5 l M ADP or ATP (AAC2, Arabidopsis thaliana)(B)12.5l M ADP or ATP (AAC2, Rattus norwegicus)and(C)500l M ADP or ATP (hdgAAC Neocal- limastix spec. L2) for the indicated time periods. Data is the mean of three independent experiments. SE <7% of the mean values. 3176 I. Haferkamp et al. (Eur. J. Biochem. 269) Ó FEBS 2002 alone. After subjecting the cell extracts to thin layer chromatography, both components ATP and ADP could be detected with a similar ATP/ADP ratio of about 0.5 (Fig. 3A–C, lane 1; for ATP-preloading data not shown). Figure 3 shows representative back exchange experiments for AAC2 from Arabidopsis (Fig. 3A), AAC1 from rat (Fig. 3B) and hydrogenosomal AAC (Fig. 3C) expressed in E. coli. As indicated in lane 2 (Fig. 3A–C), no significant release of radoactivity after incubation of preloaded E. coli cells in potassium buffer (without any substrates) was noticed. The radioactive components exported after addition of ATP (Fig. 3A–C, lane 3) or ADP (Fig. 3A–C, lane 4) exhibit a radioactive pattern with a similar ATP/ADP ratio of about 2 for the rat and the hydrogenosomal AACs and about 4 for the plant AAC. This indicates that E. coli cells expressing the plant, mammalian or hydrogenosomal mitochondrial-type AACs exhibit a preferential export of ATP under energized conditions independent of the given counter exchange molecule (ATP or ADP). Interestingly, the plant AAC seems to possess a much higher internal affinity for ATP compared to the hydrogenosomal and mammalian AACs. Moreover, the change of the ratio of exported ATP to ADP in the presence of the uncoupler CCCP is remark- able (Fig. 3A–C, lane 5 and 6). This holds true particularly for the plant AAC (Fig. 3A, lane 5 and 6). The significant decrease of the export affinity for ATP compared to ADP indicates that this transport is strongly dependend on the PMF across the bacterial cytoplasmic membrane. Mitochondria isolation and nucleotide uptake experiments Plant mitochondria were isolated according to the method of Neuburger et al. [24] with some improvements, as described in the Experimental procedures. The percentage of ÔintactnessÕ ranged from 85 to 96%. Contaminations with intact peroxisomes and plastids were not detectable. To obtain energized mitochondria, we added glycine, malate, pyruvate and NAD + to the media used for isolation and storage [39,40]. To analyse uptake of radioactively labeled nucleotides into intact plant mitochondria a rapid filtration technique was used [28]. At the end of the incubation period, mitochondria were filtered through membrane filters previ- ously set under vacuum. An advantage of this accurate and reproducible method in comparison to the silicon oil centrifugation is the complete removal of the pool of nucleotides which are presumably present in the space between the inner and outer membranes and thus no corrections for ÔexternalÕ pool variations are required [28]. The time course of [a- 32 P]ADP or [a- 32 P]ATP uptake into potato tuber mitochondria and Arabidopsis leaf mitochon- dria at 0 °C is shown in Fig. 4. Uptake is linear for about 10 s and reaches saturation after 20–30 s. Nucleotide uptake was significantly faster for ADP than for ATP into potato tuber mitochondria (Fig. 4A) whereas the difference between ADP and ATP uptake for the Arabidopsis leaf mitochondria was less pronounced (Fig. 4B). These results are in close agreement with data observed in rat liver and bovine heart mitochondria [28,41]. The adenine nucleotide uptake measured under these conditions was almost totally inhibited by carboxyatrac- tyloside and bongkrekic acid (Fig. 4). For the determin- ation of the K m values, the mitochondria were incubated with radioactively labeled nucleotides for 7 s. Interest- ingly, the apparent affinities of mitochondria from different plant tissues of potato and Arabidopsis were about 1–2 l M (Table 3) for both, ADP and ATP, and thus significantly higher than those described for rat mitochondria [28,36]. DISCUSSION The functional expression of recombinant AACs in E. coli provides a unique possibility to study the biochemical properties of a homogeneous population of AACs in vivo. Although the unfavourable codon usage of E. coli had been assumed to hamper the expression of recombinant AACs [19], the AACs from plants, mammals and anaerobic chytrid fungus studied here, were expressed without any significant retardation of E. coli cell growth. Under these conditions, we could obtain functional integration of several AACs into the E. coli cell membrane which allowed to measure the uptake of radioactively labeled ADP and ATP into intact E. coli cells. Two AACs from rat, which were studied in a similar way, could be expressed, and the nucleotide exchange data could be compared with those published by different research groups for isolated rat mitochondria. The K m values are described to be around 10–30 l M for ADP and about 100– 150 l M for ATP in energy-rich mitochondria [28,36]. These values closely resemble those obtained with the energized E. coli expressing the two rat AACs which show an Table 2. K m and V max values for ATP and ADP of several heterologously expressed AACs determined on intact E. coli cells under various energy conditions (coupled and uncoupled). K m is given in [l M ], V max is given in (nmolÆmg protein )1 Æh )1 ), E. coli cells were preincubated with 100 l M CCCP for 2 min for uncoupling. ADP ATP ADP + CCCP ATP + CCCP AAC K m V max K m V max K m V max K m V max AAC1 (Rattus norwegicus) 38 0.51 105 0.31 35 0.63 30 0.58 AAC2 (Rattus norwegicus) 40 0.55 140 0.65 26 0.92 12 0.45 AAC1 (Arabidopsis thaliana) 10 0.18 15 0.1 10 0.42 10 0.35 AAC2 (Arabidopsis thaliana) 14 4.41 22 1.34 8.5 2.23 6.5 0.94 AAC3 (Arabidopsis thaliana) 10 0.18 12 0.23 5 0.08 5 0.12 AAC1 (Solanum tuberosum) 14 0.29 18 0.12 10 0.15 8 0.14 AAC1 (Neocallimastix spec. L2) 165 7.11 2325 6.9 155 2.58 226 3.87 Ó FEBS 2002 Functional integration of AACs in E. coli (Eur. J. Biochem. 269) 3177 apparent K m value of 38 l M (AAC1) and 40 l M (AAC2) for ADP and an apparent K m value of 105 l M (AAC1) and 140 l M (AAC2) for ATP (Table 2). In energy-depleted rat mitochondria, the affinity for ADP is nearly unchanged in contrast to the highly increased affinity for ATP [36]. Similar findings were made by uncoupling E. coli cells expressing the rat AACs with CCCP. The affinity for ADP of both rat AACs was slightly changed in contrast to the significantly affected ATP affinity which increased up to 12–30 l M (Table 2). Importantly, E. coli cells must be kept energized after induction in order to obtain biochemical characteristics for recombinant AACs, comparable with the data observed for energized mitochondria. We conclude that a direct corre- lation exists between the nonretarded growth of E. coli and the energization of the cells, which is necessary for building up the required proton motive force across the membrane. Thus, the expression in E. coli allows an easy and reproducible method to assess the influence of PMF on mitochondrial or hydrogenosomal AACs from different organisms. In contrast to mammalian AACs, the heterologous expression in E. coli of the three AAC isoforms from Arabidopsis thaliana and one AAC from potato shows significantly higher affinities for ADP and ATP that are less influenced by the protonophore CCCP (Table 2). Uptake experiments with intact plant mitochondria strengthen these observations. The apparent affinities of different plant mitochondria were about 1–2 l M for both ADP and ATP Fig. 3. Thin-layer chromatography of exported radioactively labeled adenine nucleotides. E. coli cells expressing several AACs were pre- loaded with radioactively-labeled [a- 32 P]ADP at following concentra- tions: (A) 15 l M (AAC2, Arabidopsis thaliana); (B) 25 l M (AAC1, Rattus norwegicus); (C) 100 l M (hdgAAC, Neocallimastix spec. L2). Preloaded cells were used for back exchange under indicated condi- tions: Lane 1 (A–C), separation of radioactive compounds of disrupted E. coli cells after preloading; lane 2 (A–C), separation of radioactive compounds exported by E. coli in counter exchange without any exogenous substrate (control); lane 3 (A–C), separation of radioactive compounds exported by E. coli in counter exchange to exogenous ATP [(A): 75 l M ;(B):125l M ;(C):500l M )]; lane 4 (A–C), separation of radioactive compounds exported by E. coli in counter exchange to exogenous ADP [(A): 75 l M ;(B):125 l M ;(C):500 l M )]; lane 5 (A–C), separation of radioactive compounds exported by E. coli in counter exchange to exogenous ATP (concentrations see lane 3) in the presence of CCCP (100 l M ); lane 6 (A–C), separation of radioactive com- pounds exported by E. coli in counter exchange to exogenous ADP (concentrations see lane 4) in the presence of CCCP (100 l M ). Fig. 4. Time dependency of [a- 32 P]ADP, [a- 32 P]ATP uptake into iso- lated mitochondria. Isolated potato tuber mitochondria (A) and Ara- bidopsis leaf mitochondria (B) were incubated at 0 °Cwith1l M radioactively labeled ADP (j)or1l M radioactively labeled ATP (d) for the indicated time periods. For inhibition of ADP (h)orATP(s) uptake, the mitochondria were preincubated with 50 l M bongkrekic acid and 200 l M carboxyatractyloside. Data is the mean of three independent experiments. SE <8% of the mean values. 3178 I. Haferkamp et al. (Eur. J. Biochem. 269) Ó FEBS 2002 and thus, about 10 times higher compared to the apparent affinities of the single plant AACs in the E. coli system (Table 3). A major problem for studies with intact plant mitochondria is the potential loss of the PMF during the isolation procedure. As pointed out by Walker et al. [42], glycine uptake into isolated mitochondria is PMF-depend- ent. The isolated mitochondria used in our experiments for nucleotide uptake showed a strong CCCP inhibition of glycine uptake down to 8% (data not shown). However, it remains unclear whether the PMF of isolated plant mitochondria is sufficient to allow measurements of external ADP/ATP affinities of energized mitochondria in vivo. Nevertheless, these results indicate different characteristics for plant AACs compared to mammalian AACs. In mammals, the uptake into well-coupled mitochondria (high energy state) of cytosolic ADP in exchange for organellar ATP is controlled via the membrane potential [36,37]. This suggests an asymmetrical exchange of external ADP against internal ATP although the ATP/ADP ratio is greater outside the rat mitochondria than inside [43–46]. Keeping in mind that plants have also a high cytosolic ATP/ADP ratio of about 10 [47,48], it remains unclear how plant mitochon- dria control their nucleotide exchange between cytosolic ADP and intramitochondrial ATP if the external affinities for both nucleotides are similar under energized conditions. However, back-exchange experiments after preloading E. coli cells expressing different AACs demonstrate clearly that under energized conditions the preferred export nuc- leotide is ATP rather than ADP, especially for the plant AACs (Fig. 3). Investigations of plant nucleotide transport using reconstituted mitochondrial membranes from pea leaves exhibit apparent affinities (94 l M for ADP; 53 l M for ATP), which do not appear to be in accordance with our data [49]. This difference may be attributed to several reasons: (a) the lack of cardiolipin might create a quite artificial environment for mitochondrial membrane pro- teins; (b) in addition, the presence of a PMF in this proteoliposome system could not be considered; (c) unfortunately, the mitochondrial-type AAC did not exhibit the unidirectional insertion in proteoliposomes [50]. The orientation of the reconstituted pea AACs were not examined so that the determination of ADP and ATP affinities is a mixture of external and internal K m values. In reconstitution experiments it is difficult to control the orientation of mitochondrial-type AACs; a mutation of a single amino acid could lead to a change of orientation from about 50 : 50 (right side-out/inside-out) for the wild- type to a ratio of 80 : 20 for some mutants [19]. Another approach was the reconstitution of a chroma- tography-purified AAC from maize mitochondria [51]. The deduced K m values for ADP (26 l M )andATP(17l M )are close to our data obtained in the E. coli system. The choice of phospholipids, detergents and buffer concentrations as well as the presence of PMF dramatically influence the nucleotide exchange rates [19]. In conclusion, the E. coli expression system reveals similar biochemical properties for the three AAC isoforms from Arabidopsis thaliana. Therefore, it would be interesting to find out whether the expression of these isoforms is tissue specific or occurs at certain stages of plant development or under special environmental conditions. Future studies with promotor–glucuronidase fusions might give an insight into these open questions. The anaerobic chytrid fungus Neocallimastix spec. L2 is the first anaerobic eukaryote in which a mitochondrial-type AAC has been identified in its hydrogenosomes by protein sequencing and phylogenetic analysis of the corresponding (nuclear) gene [3]. Here, we have shown that the nucleotide affinities of this fungal AAC are significantly lower than those of plant and mammalian AACs (Table 2). This fact might be due to the substantially different metabolism of hydrogenosomes (anaerobic) and mitochondria (aerobic), and the composition of the organelle membranes. Notably, the hydrogenosomal AAC possesses similar transport characteristics as the mitochondrial AACs from mammals concerning the PMF dependency (Table 2). Back exchange experiments demonstrate clearly that the nucleotide trans- port mediated by the hydrogenosomal AAC takes place in a counter exchange mode (Fig. 2). It is noteworthy that the affinity for ATP import is strongly dependent on the PMF over the bacterial membrane. Upon addition of the membrane uncoupler CCCP, the affinity for ATP increased about 10-fold (Table 2). The indicated PMF-dependence of energy transport mediated by the hydrogenosomal AAC postulates also the presence of a PMF across the inner membrane of hydrogenosomes. Indeed, there is ample evidence that a PMF is present in hydrogenosomes of Neocallimastix spec. L2 [52,53]. The functional heterologous expression in E. coli of various mitochondrial-type AACs and the easy determin- ation of biochemical features that are comparable to native properties enables an interesting array of structure–function studies based on site-directed mutagenesis to commence. ACKNOWLEDGEMENTS This work was financially supported by the Deutsche Forschungs- gemeinschaft (TJ 5/1-1, TJ 5/1-2). We thank Dipl. Ing. Zeina Mezher (Pflanzenphysiologie, Universita ¨ t Kaiserslautern, Germany) for dis- cussing and critically reading the manuscript. We also thank Michaela Leroch for the help with the nucleotide uptake experiments. The support and helpful discussions of Prof H. E. Neuhaus (Pflanzenphys- iologie, Universita ¨ t-Kaiserslautern) are gratefully acknowledged. Table 3. K m and V max values for ATP and ADP of isolated plant mitochondria under different energy conditions. K m isgivenin[l M ], V max is given in (nmolÆmg protein )1 Æh )1 ). Mitochondria were preincubated with 100 l M CCCP for 2 min for uncoupling. ADP ATP> ATP + CCCP K m V max K m V max K m V max Arabidopsis leaf mitochondria 0.6 25 0.5 20 – – Potato leaf mitochondria – – 1 32 0.2 19 Potato tuber mitochondria 1.5 42 1.5 28 – – Ó FEBS 2002 Functional integration of AACs in E. coli (Eur. J. Biochem. 269) 3179 REFERENCES 1. Klingenberg, M. (1976) The state of ADP or ATP fixed to the mitochondria by bongkrekate. Eur. J. Biochem. 65, 601–605. 2. Winkler, H.H. & Neuhaus, H.E. (1999) Non-mitochondrial ade- nylate transport. TIBS 24, 64–68. 3. Voncken, F. (2001) Hydrogenosomes: eukaryotic adaptations to anaerobic environments. PhD Thesis, University of Nijmegen, the Netherlands. 4. 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