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Originally published In Press as doi:10.1074/jbc.M412462200 on February 28, 2005

J. Biol. Chem., Vol. 280, Issue 18, 17992-18000, May 6, 2005
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Identification and Characterization of a Novel Plastidic Adenine Nucleotide Uniporter from Solanum tuberosum*

Michaela Leroch, Simon Kirchberger, Ilka Haferkamp, Markus Wahl, H. Ekkehard Neuhaus, and Joachim Tjaden{ddagger}

From the Pflanzenphysiologie, Technische Universität Kaiserslautern, Erwin-Schroedinger-Strasse 22, D-67663 Kaiserslautern, Germany

Received for publication, November 4, 2004 , and in revised form, February 17, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Homologs of BT1 (the Brittle1 protein) are found to be phylogenetically related to the mitochondrial carrier family and appear to occur in both mono- and dicotyledonous plants. Whereas BT1 from cereals is probably involved in the transport of ADP-glucose, which is essential for starch metabolism in endosperm plastids, BT1 from a noncereal plant, Solanum tuberosum (StBT1), catalyzes an adenine nucleotide uniport when functionally integrated into the bacterial cytoplasmic membrane. Import studies into intact Escherichia coli cells harboring StBT1 revealed a narrow substrate spectrum with similar affinities for AMP, ADP, and ATP of about 300–400 µM. Transiently expressed StBT1-green fluorescent protein fusion protein in tobacco leaf protoplasts showed a plastidic localization of the StBT1. In vitro synthesized radioactively labeled StBT1 was targeted to the envelope membranes of isolated spinach chloroplasts. Furthermore, we showed by real time reverse transcription-PCR a ubiquitous expression pattern of the StBT1 in autotrophic and heterotrophic potato tissues. We therefore propose that StBT1 is a plastidic adenine nucleotide uniporter used to provide the cytosol and other compartments with adenine nucleotides exclusively synthesized inside plastids.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Plastids are semiautonomous plant organelles existing in a wide range of differential forms depending on the developmental stage and function of the cells in which they reside. They are the sites of photosynthesis and harbor exclusively many biosynthetic pathways such as synthesis of starch and amino and fatty acids (1). The surrounding envelopes, especially the inner envelope, effectively separate plastid metabolism from the cytosol.

Communication between plastids and the surrounding cytosol is mediated by a large group of phylogenetically related phosphate translocators (2, 3). The daily fixed carbon is exported from chloroplasts as triose phosphates and 3-phosphoglycerate via the triose phosphate/phosphate translocator (4). The phosphoenolpyruvate/phosphate translocator provides the plastids with phosphoenolpyruvate as immediate precursors for the shikimic acid pathway (5). The pentose phosphate/phosphate translocator (xylulose 5-phosphate) represents a link between the cytosolic and plastidic pentose phosphate pathway (6). Plastids of nonphotosynthetic tissues have to import carbon to fuel biosynthetic pathways (for example, fatty acid, amino acid, or starch synthesis) via the glucose 6-phosphate/phosphate translocator (7). All of the members of the plastidic phosphate translocators(s) function as antiport systems and belong to the group of translocators with a 6 + 6 helix (homodimer) folding pattern (8).

In this respect, the plastidic phosphate translocators differ from several other transporters of the plastidic envelope membrane, which function as monomers that contain 12 transmembrane helices. These include a maltose transporter that exports maltose, the product of hydrolytic starch degradation (9), two dicarboxylate translocators that are involved in ammonia assimilation (10), and a H+/Pi symporter that affects Pi allocation within the plant (11). Another prominent plastidic 12-helix transporter is the ATP/ADP transporter (NTT)1 that supplies plastids with energy for the biosynthesis of starch, fatty acids, and other compounds (12, 13).

In storage plastids of mono- and dicotyledonous plants, starch synthesis is dependent on cytosolic precursors. In intact amyloplasts from potato tubers, cauliflower buds, or developing pea and rape seed embryos, starch synthesis occurs via the uptake of hexose phosphates (1) mediated by the above mentioned hexose phosphate/phosphate antiporters. The further conversion to starch is energy-dependent (14–16). Inside the plastid ADP-Glc pyrophosphorylase (AGPase) converts Glc-1-P and ATP into ADP-Glc, the glucose donor for starch synthases (17–19). The ATP required for the AGPase reaction is imported into these plastids via the plastidic NTT in counter exchange for ADP (12, 20).

In cereal endosperms AGPase is mainly localized in the cytosol, which accounts for 85–95% of total activity (21–26). Therefore, cytosolic ADP-glucose is the main precursor for starch synthesis in cereal endosperm plastids and is transported across the inner envelope membrane most likely in exchange for AMP (27). Physiological and immunological studies on the low starch Brittle1 mutant of maize (Zmbt1) lead to the common assumption that Zmbt1 encodes an ADP-Glc transporter, residing in the inner membrane of maize endosperm plastids (23). In this mutant ADP-Glc accumulates in the endosperm of Zmbt1 kernels, although enzymatic activities of AGPase, UGPase, soluble starch synthase, soluble starch-branching enzyme, and sucrose synthase are not reduced compared with normal kernels (28). In addition, intact amyloplasts of Zmbt1 mutant endosperms could only synthesize starch from exogenous ADP-Glc at 25% of the rate of wild type (29), most probably because of the loss of four major amyloplastidic envelope polypeptides (39–44 kDa) detected by antibodies raised against ZmBT1 in normal kernels (30).

Similar physiological evidence was recently observed for low starch mutants of barley (lys5). These barley mutants also carry a defect in a major plastidic envelope protein, HvNST1, showing a high homology of ~75% to the ZmBT1 (31). Both BT1 homologs have no obvious sequence similarities at the amino acid level to nucleotide sugar transporters that are found in the endoplasmic reticulum and in the Golgi apparatus. Structurally, BT1 proteins are members of the mitochondrial carrier family (MCF) comprising transporters for many different substrates (31, 32).

Interestingly, BT1 homologs also occur in noncereal plants such as Solanum tuberosum (StBT1) and Arabidopsis thaliana (AtBT1) with a homology of about 65% to ZmBT1 and HvNST1. The lack of a cytosolic isoform of AGPase in the storage tissues of noncereal species (26, 33–36) strongly indicates a distinct physiological role of the BT1 homologs in dicotyledons.

The aim of this work was to gain insight into the nature and importance of the potato BT1 homolog (StBT1) from a dicotyledonous plant. For functional characterization of StBT1 we have used Escherichia coli for heterologous expression; this system has been previously shown to functionally integrate several plastidic, mitochondrial, and hydrogenosomal membrane proteins into the bacterial cytoplasmic membrane (20, 37–39). We also investigated the tissue-specific expression and subcellular localization of the StBT1.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Growth—Potato plants (S. tuberosum L., cv Désirée) were grown in a greenhouse at 22–26 °C and watered once a day. The ambient light period was extended to 16 h/day with Philips Sont-Agro lights (200 µmol quanta x m-2 x s-1). To analyze the accumulation of mRNA encoding StBT1, potato tissues were collected 12–14 weeks after sprouting and immediately frozen in liquid nitrogen until use.

Quantitative Real Time RT-PCR—Total RNA was prepared from various potato tissues using the RNeasy plant mini kit (Qiagen). To remove any contaminating DNA, the samples were treated with deoxyribonuclease (RNase-free DNase kit; Qiagen). Quantitative PCR was performed on the LightCycler (Roche Applied Science) using the QuantiTectTM SYBR Green RT-PCR kit (Qiagen) according to the manufacturer's instructions with the following cycler conditions: 20 min at 50 °C; 15 min at 95 °C; and 55 cycles of 15 s at 95 °C, 25 s at 58 °C, and 40 s at 72 °C. The sequences of the gene-specific oligonucleotides used for real time RT-PCR are the following: BT1-sense, 5'-CTGAGCTCTATAGAGGTCTCACTC-3'; BT1-antisense, 5'-AACATGAATCCCATCCTGTTCAAG-3'; EF-1{alpha}-sense, 5'-GTTTCACTGCC-CAGGTCATCATC-3'; and EF-1{alpha}-antisense, 5'TGGGCTTGGTGGGAATCATC-3'. The housekeeping gene in S. tuberosum encoding the elongation factor EF-1{alpha} (STU536671) was used for quantitative normalization. The specificity of the obtained RT-PCR products was controlled on 1.8% agarose gels.

cDNA Constructs—DNA manipulations were performed essentially as described by Sambrook et al. (40). The Stbt1 cDNA sequence (X98474 [GenBank] ) available at the EMBL data base (www.ebi.ac.uk/embl/) was used to design primers for PCR amplification in a way to cover the complete coding region. First strand cDNA from potato leaf tissue was used as PCR template.

For construction of the E. coli expression plasmid (encoding His10-Stbt1), a sense primer including a NdeI restriction site (5'-GACTGAcatATGGCGGCGACAATG-3'; the lowercase letters indicate the introduced base exchange to create a NdeI restriction site) as well as an antisense primer (5'-CTACCTTCTTGGCCAAGAAC-3') were selected, and the PCR took place in the presence of Pfu DNA polymerase (Stratagene, Heidelberg, Germany), which possesses a proof reading activity. The obtained PCR product was purified (QIAquick PCR purification kit; Qiagen) and subcloned into EcoRV-restricted pBSK (Stratagene). The NdeI/BamHI DNA insert of the pBSK plasmid was further introduced "in-frame" into the corresponding restriction sites of the isopropyl {beta}-D-thiogalactopyranoside (IPTG)-inducible T7 RNA polymerase bacterial expression vector pET16b (Novagen, Heidelberg, Germany).

The green fluorescent protein (GFP) fusion construct was prepared by amplification of the complete coding region of Stbt1 in the presence of Pfu DNA polymerase using 5'-GATTGATGGCTcGAgGGGATATAC-3' and 5'-CTTCATTTTCTGCCTCgAgCAATATCCTCTTG-3' as forward and reverse primers, respectively. Both primers included XhoI restriction sites for an in-frame insertion into pGFP2 (41). The resulting plasmids were sequenced on both strands by chain termination reaction (Seqlab, Göttingen, Germany).

Heterologous Expression of Stbt1 in E. coli—The E. coli strain BL21 (DE3) was used for heterologous expression. The cDNA sequence encoding StBT1 under control of the T7 promoter was transcribed after IPTG induction of the T7 RNA polymerase (42). E. coli cells harboring the Stbt1 expression plasmid (or control expression plasmid pET16b) were grown at 37 °C in YTAmp/Clm medium (5 g/liter yeast extract, 8 g/liter peptone, 2.5 g/liter NaCl, pH 7.0). An A600 value of 0.5–0.6 was required for the initiation of T7 RNA polymerase expression by addition of IPTG (final concentration, 0.02%). The cells were grown for 1 h after induction and collected by centrifugation for 5 min at 5,000 x g (8 °C, Sorvall RC5B centrifuge, rotor type SS34; Sorvall-Du Pont, Dreieich, Germany). The pellet was resuspended to an A600 value of 8 using potassium phosphate buffer (50 mM, pH 7.0) (43) and promptly used for uptake experiments.

Uptake of Radioactively Labeled ATP, ADP, and AMP into Intact E. coli Cells—IPTG-induced E. coli cells (100 µl) harboring the Stbt1 expression plasmid (or the given controls) were added to 100 µl of potassium phosphate buffer (50 mM, pH 7.0) containing radioactively labeled ATP, ADP, or AMP. [{alpha}-32P]ATP or ADP were used at specific activities between 50–500 µCi/µmol. [{alpha}-32P]ADP was enzymatically synthesized from [{alpha}-32P]ATP (PerkinElmer Life Sciences) as given in Tjaden et al. (20). [14C]AMP was used at specific activities between 20 and 250 µCi/µmol. 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-µm membrane filter (mixed cellulose ester, 25-mm diameter; Schleicher & Schuell) under vacuum (44). The cells were further washed to remove unimported radioactivity by addition of 3 x 4 ml of potassium phosphate buffer (50 mM, pH 7.0). The filter was subsequently transferred into a 20-ml scintillation vessel and filled with either 10 ml of water or 10 ml of scintillation mixture (Quicksafe A; Zinsser Analytic, Frankfurt/Main, Germany). Radioactivity in the samples was quantified in a Canberra-Packard Tricarb 2500 scintillation counter (Canberra-Packard, Frankfurt/Main, Germany). For efflux assays, E. coli cells were incubated with potassium phosphate buffer (50 mM, pH 7.0) containing 5 µM [{alpha}-32P]ADP as a transport substrate. After indicated time periods, the uptake medium was diluted with several unlabeled nucleotides to a final concentration of 5 mM, and the efflux of [{alpha}-32P]ADP was monitored at given time points. Efflux was measured by membrane filtration as described above. To analyze the degree of metabolic conversion of imported [{alpha}-32P]ADP, we carried out a thin layer chromatography according to the method of Mangold (45).

Transient Expression of GFP Fusion Constructs—Protoplasts were prepared from tobacco plants (Nicotiana tabacum cv. W38) grown under sterile conditions as given in Wendt et al. (46). The protoplasts were transformed with column-purified plasmid DNA (30 µg/0.5 x 106 cells). After 18 h of incubation in the dark at room temperature, the protoplasts were checked for green fluorescence using the Carl Zeiss Laser scanning system LSM510 (Carl Zeiss, Jena, Germany). GFP was excited at 488 nm, and emission was detected by a photomultiplier through a 505–530-nm bandpass filter using an Achroplan 40x/0.75 W objective.

Targeting Experiments—The plasmid with the complete coding region of Stbt1 was used as a template for synthesis of the radioactively labeled precursor protein. Circular plasmid DNA was added to Promega TNT® kit reticulocyte lysate allowing coupled transcription and translation in the presence of [35S]methionine (Promega, Heidelberg, Germany). The in vitro synthesized 35S-labeled StBT1 precursor protein was used for import studies on isolated spinach mesophyll chloroplasts according to the method of Weber et al. (47). Uptake of radioactively labeled StBT1 precursor protein was terminated after 20 min of incubation by removal of the incubation medium and subsequent thermolysin treatment as described by Weber et al. (47). Fractionation of spinach chloroplasts into envelope membranes, stroma, and thylakoids was carried out as reported by Flügge et al. (4). The samples were subsequently analyzed by SDS-PAGE (48) and autoradiography.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Sequence Analysis of the StBT1—A Stbt1 cDNA clone was isolated from first strand cDNA of potato leaves as described under "Experimental Procedures." The deduced amino acid sequence was aligned with BT1 homologs from maize (Zea mays), barley (Hordeum vulgare), and A. thaliana (Fig. 1). A comparison of the mature BT1 homologs revealed a high similarity of the StBT1 to the AtBT1, HvNST, and ZmBT1 of 76, 71, and 65%, respectively. For the N-terminal region of StBT1, the ChloroP 1.1 prediction server identified a putative plastidic transit peptide of 22 amino acids as indicated in Fig. 1 (49). The amino acid sequence of StBT1 consists of three tandem repeats of ~100 residues showing six putative transmembrane helices. Three conserved mitochondrial energy transfer signatures were also identified that are characteristic for membrane proteins belonging to the MCF (50, 51). Phylogenetic analyses classify BT1 homologs as members of the MCF forming a monophyletic cluster (39).



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  		FIG. 1.
Alignment of the predicted amino acid sequence of StBT1 with BT1 homologs. The residues identical or similar among all family members are indicated by black shading, and the residues conserved by three proteins are indicated by gray shading. The predicted N-terminal plastidic transit peptides (ChloroP 1.1 prediction server) are indicated by black squares. The six putative membrane-spanning regions are shown as boxes (boxes I–IV). The conserved mitochondrial energy transfer signature (METS; PX(D/E)X(L/I/V/A/T)(R/K)X(L/R/H)(L/I/V/M/F/Y)(Q/G/A/I/V/M)) following each odd membrane-spanning domain is marked by black bars. The dashes represent gaps introduced to improve the similarity between the proteins. The numbers indicate the amino acid positions. StBT1, Brittle1 from S. tuberosum (NCB accession number CAA67107 [GenBank] ; AtBT1, Brittle1 from A. thaliana (NCB accession number CAB79957 [GenBank] ; ZmBT1, Brittle1 from Z. mays (NCBI accession number AAA33438 [GenBank] ; and HvNST1, nucleotide sugar transporter from H. vulgare (NCBI accession number AAT12275 [GenBank] .

 
Subcellular Localization of the StBT1—The ADP-Glc transporter of maize, the ZmBT1, is targeted in vitro to the inner chloroplast envelope membrane (29). Moreover, immunocytolocalization revealed that ZmBT1 resides in the envelope membrane of amyloplasts in endosperm tissues (52). However, the prediction of the subcellular membrane integration of the StBT1 homolog is still unclear. Computer programs, such as TargetP (53), ChloroP, or other search algorithms (49) suggest either plastidic or mitochondrial localization of the StBT1. We therefore cloned a reporter gene encoding GFP at the C-terminal site of the StBT1 so that the putative N-terminal transit peptide would remain functional. For determination of the cellular localization in planta, the Stbt1-GFP fusion was transiently expressed in tobacco protoplasts under control of the cauliflower mosaic virus 35S promoter. As controls, we used GFP alone (present in the cytosol and in the nucleus) as well as a voltage-dependent anion channel located in the inner mitochondrial membrane (54).



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  		FIG. 2.
Localization of StBT1-GFP in tobacco leaf protoplasts. Tobacco protoplasts were prepared as described under "Experimental Procedures" and transformed using polyethylene glycol. Fluorescence was visualized after incubation for 20 h with confocal laser scanning microscopy. The chloroplasts were visualized by their autofluorescence. VDAC, voltage-dependent anion channel localized in mitochondria (54).

 
Confocal laser scanning microscopy confirmed a plastidic localization of StBT1. The fluorescent images shown in Fig. 2 (A–C) reveal a co-localization of the transient expressed Stbt1-GFP (green fluorescent spots) with the tobacco chloroplasts (red fluorescence = autofluorescence of plastids), exhibiting a strikingly distinct pattern compared with the controls (Fig. 2, D and E).

To determine whether StBT1 is localized in the plastidic envelope membrane, we conducted uptake experiments in which isolated spinach leaf chloroplasts were incubated with the in vitro synthesized radioactively labeled StBT1 precursor protein. As demonstrated in Fig. 3, in vitro translation of StBT1 yielded a protein with an apparent molecular mass of about 41 kDa (Fig. 3, lane 1). In the presence of light, incubation of isolated spinach chloroplasts with the radioactively labeled StBT1 precursor protein led to the incorporation of this protein into the envelope membranes (Fig. 3, lane 3). The additional presence of ATP slightly increased the amount of the StBT1 precursor protein targeted to the envelope membranes (Fig. 3, lane 2). In darkened chloroplasts, no incorporation of the StBT1 precursor protein into the envelope membranes was observed (Fig. 3, lane 5). However, the presence of exogenous ATP resulted in an incorporation of the StBT1 precursor protein showing, thus, that ATP is able to replace light in this system (Fig. 3, lane 4). Comparison of the size of the in vitro synthesized StBT1 precursor protein (Fig. 3, lane 2) with the apparent molecular mass of the protein after incorporation into the envelope membranes (about 39 kDa) indicates that StBT1 is processed to its mature form on import. This is in accordance with the prediction of a short plastidic transit peptide of StBT1 (22 amino acids) identified by ChloroP 1.1 prediction server (49). Isolated spinach chloroplasts were routinely treated with thermolysin after incorporation of StBT1 (see "Experimental Procedures"). The resistance against thermolysin (Fig. 3, lanes 2–4) suggests that StBT1 is deeply embedded into the envelope membranes. Lanes 6 and 7 in Fig. 3 show clearly that StBT1 was not targeted to the stroma or the thylakoid fraction.



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  		FIG. 3.
Import of radioactively labeled StBT1 precursor protein into isolated spinach mesophyll chloroplasts. Isolated spinach chloroplasts were incubated for 20 min in import medium containing [35S]methionine-labeled StBT1 precursor protein. Lane 1, aliquot of in vitro synthesized StBT1. Lane 2, envelope membranes of chloroplasts illuminated during incubation with radioactively labeled StBT1 protein in the additional presence of ATP (2 mM). Lane 3, envelope membranes of chloroplasts illuminated during incubation with radioactively labeled StBT1 protein. Lane 4, envelope membranes of darkened chloroplasts incubated with radioactively labeled StBT1 protein in the additional presence of ATP (2 mM). Lane 5, envelope membranes of darkened chloroplasts incubated with radioactively labeled StBT1 protein. Lane 6, stroma fraction of illuminated chloroplasts incubated with radioactively labeled StBT1 protein in the additional presence of ATP (2 mM). Lane 7, thylakoid fraction of illuminated chloroplasts incubated with radioactively labeled StBT1 protein in the additional presence of ATP (2 mM).

 
Heterologous Expression of Stbt1 in E. coli Cells—A high degree of sequence similarity among homologs belonging to the same protein family does not necessarily implicate the same function. It is important to identify for each putative homolog the substrates transported as well as their kinetic properties. The overexpression of plant carrier proteins in E. coli and the subsequent reconstitution of the purified proteins into liposomes offer a possibility for successful analysis (55, 56), but the low expression levels obtained for many heterologously synthesized carrier proteins can be a major drawback to this approach. However, we showed previously that heterologous synthesis of plastidic ATP/ADP transporters in E. coli leads to their functional integration into the bacterial cytoplasmic membrane with transport properties similar to carriers in their authentic membranes (20, 57).



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  		FIG. 4.
Time dependence of adenine nucleotide uptake into intact E. coli cells. IPTG-induced E. coli cells harboring the plasmid encoding the StBT1 were incubated with 150 µM [14C]AMP ({blacktriangleup}), [{alpha}-32P]ADP ({blacksquare}), or [{alpha}-32P]ATP ({circ}) for the indicated time periods. The data are the means of three independent experiments. The S.E. values were less than 10% of the mean values.

 
After heterologous synthesis of StBT1 in E. coli, we observed import of radioactively labeled AMP, ADP, and ATP into intact bacterial cells. Uptake of all three nucleotides was linear with time for at least 20 min with a lower uptake rate for ATP compared with those for AMP and ADP (Fig. 4). For the correct determination of the rate of AMP uptake, it is important to remember that E. coli is able to cleave exogenous AMP to adenosine and inorganic phosphate by a 5'-nucleotidase located between the cytoplasmic and outer membrane (58). Therefore, to reduce the rate of cleavage of [14C]AMP, uptake experiments were performed in the presence of high concentrations of inorganic phosphate (50 mM) and adenosine (1 mM).

Interestingly, we could not detect any uptake of labeled ADP-Glc (at a concentration of 150 µM) into E. coli cells harboring the StBT1 (data not shown). Thus, the substrate of ZmBT1 is not a transport substrate of the StBT1 carrier protein.

We analyzed the affinities for the above mentioned nucleotides at different substrate concentrations within a period of 10 min. The results are plotted in Fig. 5. Increased exogenous radioactively labeled AMP, ADP, and ATP induced increased rates of nucleotide transport into E. coli cells harboring the StBT1. Lineweaver-Burk analyses revealed relatively similar apparent Km values (AMP, 372.7 ± 41.5 µM; ADP, 345.3 ± 37.3 µM; ATP, 374.3 ± 27.3 µM). Interestingly, the calculated Vmax for ATP import was about 50% lower compared with both AMP and ADP, which seems to be a particular feature of this carrier (Fig. 5).

To investigate the substrate specificity of the StBT1, we measured the effect of various nonlabeled metabolic intermediates on the rate of [{alpha}-32P]ADP uptake (Table I). The strongest inhibition of [{alpha}-32P]ADP import could be observed with nonlabeled ADP reducing the transport rate below 36% of the control (without effector). Similar inhibition (about 60%) was caused by nonlabeled AMP and, to a lower extent, by nonlabeled ATP (about 40%). All 24 other metabolic intermediates tested did not show any substantial influence on the rate of uptake, which shows the high substrate specificity of StBT1 (Table I).



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  		FIG. 5.
Substrate saturation of adenine nucleotide uptake into intact E. coli cells. IPTG-induced E. coli cells harboring the plasmid encoding the StBT1 were incubated for 10 min with the indicated concentrations of [14C]AMP (A), [{alpha}-32P]ADP (B), or [{alpha}-32P]ATP (C). [14C]AMP (A) uptake was carried out in the presence of 1 mM adenosine to prevent AMP cleavage (see text). The data are the means of three independent experiments. Background rates of the control (pet16b without inset) have been subtracted. The insets represent a double reciprocal plot of the data for uptake indicating a Km of 372.7 ± 41.5 µM and a Vmax of 1.33 ± 0.25 nmol x mg-1 protein x h-1 for AMP (A), a Km of 345.3 ± 37.3 µM and a Vmax of 1.18 ± 0.21 nmol x mg-1 protein x h-1 for ADP (B) and a Km of 374.3 ± 27.3 µM and a Vmax of 0.57 ± 0.37 nmol x mg-1 protein x h-1 for ATP (C).

 


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  		TABLE I
Effects of various metabolites on [{alpha}-32P]ADP transport activities of StBT1 Metabolic effectors were given at a concentration of 1 mM. [{alpha}-32P]-ADP was present at a concentration of 100 µM. Uptake was carried out for 15 min and stopped by rapid filtration (see "Experimental Procedures"). The data are the means of three independent experiments. The S.E. values were less than 8% of the mean values.

 
We also determined the influence of common inhibitors of [{alpha}-32P]ADP uptake (Table II). To improve the penetration of the tested inhibitors through the outer membrane, we treated the Stbt1-expressing E. coli cells with lysozyme prior to uptake analysis. We showed elsewhere that treatment of E. coli cells with lysozyme had no influence on the nucleotide uptake rate into the cells (37). The highly specific inhibitors of mitochondrial ADP/ATP carriers, bongkrekic acid and carboxyatractyloside (59, 60), showed no inhibitory effect at the given concentrations. In addition, no considerable inhibition was observed with mersalyl and N-ethylmaleimide, known as powerful inhibitors of the peroxisomal adenine nucleotide transporter from Saccharomyces cerevisiae (Ant1p) (61). Interestingly, pyridoxal 5'-phosphate, a potential inhibitor of plastidic phosphate translocators (62), significantly inhibited the [{alpha}-32P]ADP transport rate of StBT1 to below 40% of the control (Table II).


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  		TABLE II
Effects of various inhibitors on [{alpha}-32P]ADP transport activities of StBT1 ADP uptake was measured at a concentration of 100 µM. E. coli cells were preincubated for 10 min with lysozyme (1.25 mg/ml) to allow penetration of the reagents across outer membrane. Uptake was carried out for 10 min and stopped by rapid filtration (see "Experimental Procedures"). The inhibitors were used in following concentrations: bongkrekic acid, 10 µM; carboxyatractyloside, 1 mM; N-ethylmaleimide, 1mM; pyridoxal 5'-phosphate, 2 mM; and mersalyl, 200 µM. The data are the means of three independent experiments. The S.E. values were less than 8% of the mean values.

 
The occurrence of well characterized plastidic ATP/ADP counter exchange transporters (NTTs) in all plants studied so far (63) led us assume a possible difference in the transport mechanism of the StBT1. The E. coli system can also be used to demonstrate the counter exchange properties of a heterologously synthesized carrier protein (43). The principle of this approach is to initiate a putative efflux of imported radioactively labeled nucleotides through a high dilution (chase) with nonlabeled substrates at a certain time point during an uptake experiment (43). To make sure whether imported [{alpha}-32P]ADP is metabolized by E. coli cells harboring either StBT1 or AtNTT1 (see below), we disrupted the cells after preloading with [{alpha}-32P]ADP at several time points and analyzed the cytosolic fraction by thin layer chromatography. Most of [{alpha}-32P]ADP (about 95%) was found not to be metabolized by these E. coli cells over a time span of about 30 min, which suggests the involvement of [{alpha}-32P]ADP in a putative nucleotide exchange (Fig. 6C).

First, we tested this experimental approach on the well characterized plastidic NTT1 from A. thaliana (AtNTT1), which mediates a nucleotide exchange (20). Fig. 6A shows a typical time course for [{alpha}-32P]ADP uptake (at 5 µM) into E. coli cells harboring the AtNTT1. Directly after start of the chase with nonlabeled ADP (5 mM), a rapid efflux led to a total release of labeled nucleotides of about 90% (7 min after start of the chase). UDP, used as a control, is known not to be a substrate for AtNTT1 and therefore showed no influence on the uptake of radioactively labeled ADP after the chase. These results clearly confirm that AtNTT1 operates in a counter exchange mode. Surprisingly, nonlabeled ADP-Glc, although reported not to be a substrate for AtNTT1, significantly inhibited the ADP uptake mediated by AtNTT1. However, we were able to attribute this phenomenon clearly to the fact that commercially available ADP-Glc is contaminated by other adenylates, namely AMP, ADP, and ATP. We determined the contamination of ADP-Glc by HPLC analysis (1.8% ADP; 0.6% ATP; 0.5% AMP) and carried out the same experiment using the calculated nucleotide contaminations as a nonlabeled nucleotide mix (mix: 90 µM ADP, 30 µM ATP, and 25 µM AMP) for the chase during [{alpha}-32P]ADP uptake. The chase with this nucleotide mix led to a competitive inhibition of [{alpha}-32P]ADP uptake comparable with that of commercially available ADP-Glc (5 mM) alone.

For the chase experiments with E. coli cells expressing Stbt1, we selected AMP, ADP, ATP, and ADP-Glc as putative counter exchange substrates. After a 6-min import of [{alpha}-32P]ADP (5 µM), we diluted 1000-fold with the substrates mentioned above (5 mM). In clear contrast to the AtNTT1, none of these nucleotides initiated a significant efflux of radioactivity over a time span of 24 min (Fig. 6B). The 1000 times concentrated substrates AMP, ADP, and ATP immediately inhibited any further uptake of [{alpha}-32P]ADP as competitive inhibitors. Thus, we conclude that the StBT1 is a novel adenine nucleotide uniporter.

The slight inhibition of ADP-Glc observed in Fig. 6B is most probably due to the minimal contaminations with ADP, ATP, and AMP. To elucidate whether ADP-Glc at high concentrations is a possible transport substrate for the StBT1, we performed additional effector studies during [{alpha}-32P]ADP import into E. coli cells harboring the StBT1. The inhibition caused by different concentrations of ADP-Glc between 1 and 5 mM was comparable with the inhibitory effect of the corresponding calculated "contamination mix." In summary, we exclude ADP-Glc as a transport substrate for the StBT1 (Fig. 7).

Expression Analyses of the Stbt1 in Different Potato Tissues—To obtain further insight into the physiological role of the StBT1 as an adenine nucleotide uniporter, we analyzed the accumulation of the corresponding mRNA in various autotrophic and heterotrophic tissues. Northern blot analyses of total RNA failed because of the low expression level of the Stbt1, which is often observed for transport proteins. However, for low abundant proteins, gene expression levels can be determined by real time RT-PCR, which is more specific and sensitive than Northern blot analyses (64). The use of the total RNA mass (or 18 S rRNA) for normalization might be misleading because it consists predominantly of rRNA molecules, which are often not representative for the mRNA fraction in different plant tissues (65). A reliable internal control should be similarly expressed in different cell types (66). Therefore, we chose the elongation factor EF-1{alpha}, which catalyzes the first step of protein synthesis by binding the aminoacyl-tRNA to the aminoacyl site of ribosomes. EF-1{alpha} is described to be a suitable housekeeping gene commonly used in plants as an internal control for real time RT-PCR (66–68).



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  		FIG. 6.
Exchange-mediated efflux studies of intracellular radioactivity. E. coli cells harboring AtNTT1 (A) or the StBT1 (B) were incubated in the presence of 5 µM [{alpha}-32P]ADP. At indicated time points unlabeled nucleotides were added (chase) to a final concentration of 5 mM (1000-fold), and a possible induced efflux was monitored for the given time spans. The data are the means of three independent experiments. The S.E. values were less than 8% of the mean values. 1*, the contamination of 5 mM ADP-Glc was determined by HPLC analysis; the corresponding concentrations of 90 µM ADP, 30 µM ATP, and 25 µM AMP were added and monitored as described above for all unlabeled nucleotides. C, E. coli cells harboring the AtNTT1 were disrupted after preloading with 5 µM [{alpha}-32P]ADP for a time period of 1.45 min (lane 1) or 8.45 min (lane 2), and the cytosolic radioactively labeled compounds were separated by thin layer chromatography. E. coli cells harboring the StBT1 were disrupted after preloading with 5 µM [{alpha}-32P]ADP for a time period of 6 (lane 3) or 30 min (lane 4), and the cytosolic radioactively labeled compounds were separated by thin layer chromatography.

 



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  		FIG. 7.
Inhibition of [{alpha}-32P]ADP uptake into intact E. coli cells at different ADP-Glc concentrations. IPTG-induced E. coli cells harboring the plasmid encoding the StBT1 were incubated for 15 min in the presence of 100 µM [{alpha}-32P]ADP. Set 1, relative uptake of [{alpha}-32P]ADP given as 100%; set 2, inhibition of [{alpha}-32P]ADP uptake by 1 mM ADP-Glc (black bar) and the corresponding contamination (see text) with AMP, ADP, and ATP (gray bar); set 3, inhibition of [{alpha}-32P]ADP uptake by 2.5 mM ADP-Glc (black bar) and the corresponding contamination (see text) with AMP, ADP, and ATP (gray bar); set 4, inhibition of [{alpha}-32P]ADP uptake by 5 mM ADP-Glc (black bar) and the corresponding contamination (see text) with AMP, ADP, and ATP (gray bar). The data are the means of three independent experiments. The S.E. values were less than 5% of the mean values.

 
The real time RT-PCR quantification indicates a general role for StBT1 in plant metabolism because its expression pattern is quite similar in all of the tissues investigated and with the highest level in flowers (Fig. 8). In contrast to the BT1 homologs from maize and barley, which are exclusively expressed in cereal endosperms and are strictly coupled to starch synthesis in these heterotrophic tissues (23, 31), the StBT1 is also expressed in autotrophic tissues such as stems and leaves (Fig. 8).

A comprehensive expression analysis of genes from another dicotyledonous plant, A. thaliana, are available at ETH Zürich (www.genevestigator.ethz.ch) (69) based on data from Affymetrix and ATH1 GeneChip® arrays. Detailed studies of the AtBT1 homolog (At4g32400) show a ubiquitous expression with minor changes in autotrophic and heterotrophic tissues as reported for the StBT1 (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have investigated the biochemical characteristics of a hitherto unknown member of the MCF, the StBT1 (Fig. 1). Functional heterologous synthesis of StBT1 in E. coli clearly reveals a high substrate specificity of StBT1 for AMP, ADP, and ATP (Figs. 4 and 5 and Table I) that classifies StBT1 to the group of adenylate transporters in eukaryotic cells. The StBT1 is not able to transport ADP-Glc (Fig. 7) in contrast to the cereal BT1 homologs from maize and barley, which are involved in ADP-Glc import into endosperm plastids during starch synthesis (23, 31). Starch synthesis in heterotrophic dicotyledonous plastids is dependent on internally produced ADP-Glc because this substrate cannot be synthesized in the cytosol because of the lack of a cytosolic AGPase isoform in noncereal plants (1, 26, 33–36). Therefore, the physiological significance of the StBT1 must be different from comparable cereal BT1 homologs.

A plastidic localization of StBT1 was revealed by transient expression of a Stbt1-GFP fusion in tobacco leaf protoplasts (Fig. 2). Recently, it has been shown by immunolocalization that the thylakoid membrane contains a membrane protein belonging to the MCF. This unknown MCF transporter was proposed to mediate a transthylakoid nucleotide transport based on transport studies across thylakoid membranes (70). However, we proved a localization of StBT1 in the inner envelope membrane of plastids, because the radioactively labeled StBT1 precursor protein was specifically targeted into the envelope membranes of isolated spinach chloroplasts (Fig. 3).



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  		FIG. 8.
Expression levels of Stbt1 in different potato tissues. The transcript levels were determined by quantitative real time RT-PCR using gene specific primers. Three independent preparations of total RNA (100 ng) from potato tissues were assayed in duplicate (S.E. were less than 10%). The relative transcript level was calculated as the reciprocal of 1.85Ct; Ct, cycle threshold. Ct values above 40 (2.06 x 10-11) were considered as not expressed. Elongation factor 1{alpha} as a housekeeping gene from S. tuberosum was selected for normalization.

 
Transport of ATP in exchange with ADP across the inner plastidic envelope membrane is accomplished by a group of 12 helix transporters (NTT), which is phylogenetically and structurally distinct from carrier types belonging to the MCF (71). The major physiological roles of NTT are the provision of storage plastids with ATP used for starch and fatty acid synthesis (1, 13, 72) as well as the supply of chloroplasts with energy that allows high rates of starch degradation at night (73). In comparison with the plastidic NTT (74), the StBT1 exhibits a much lower affinity (20-fold) for adenine nucleotides, but it accepts AMP as an additional substrate (Fig. 5). Another important feature of the StBT1, in contrast to the NTT, is the unidirectional transport mechanism as shown by the chase experiments (Fig. 6).

The ubiquitous expression pattern of Stbt1 in all of the potato tissues investigated indicates a basic function in plant metabolism (Fig. 8). We suggest that the physiological role of StBT1 in autotrophic and heterotrophic plastids is the export of purine nucleotides that are exclusively synthesized in plastids and fulfills an essential task in plant metabolism (75–77). The net export of adenine nucleotides is absolutely necessary for a variety of metabolic pathways outside of the plastids. This function cannot be catalyzed by the plastidic ATP/ADP antiporter (NTT), which explains a co-existence of BT1 and NTT in the plastidic inner envelope membrane of dicotyledonous plants. A correlation between dicotyledonous BT1 expression and purine nucleotide metabolism in planta is currently under investigation. Transgenic plants with reduced BT1 transport activities (antisense repression and knock-out plants) might enable us to determine the physiological influence of this plastidic adenine nucleotide uniporter on de novo purine nucleotide metabolism. First investigations of AtBT1 Arabidopsis knockout plants revealed that homozygous seeds of these knock-out lines are unable to germinate on soil (data not shown). Interestingly, it has been shown that de novo synthesis of purine nucleotides plays an important role during embryo maturation and germination of white spruce somatic embryos (Picea glauca) (78), as well as during the early phases of germination in soybean axes (79).

The StBT1 presents a new type of eukaryotic adenine nucleotide transporter belonging to the MCF (Fig. 1). StBT1 is able to mediate an unidirectional net transport of adenine nucleotides (Fig. 6) in contrast to the majority of carriers belonging to the MCF that are known to function as antiporters (80, 81). However, an uniport mechanism has been reported for other carriers of the MCF: (i) The uncoupling protein mediates an unidirectional transport of protons across the inner mitochondrial membrane (82). (ii) The mitochondrial ADP/ATP and the aspartate/glutamate antiporters switch from obligate counter exchange to unidirectional transport after modification by SH reagents (83). These data underline that moderate transitions on protein level of members belonging to the MCF could easily lead to big differences in the mode of transport.


   FOOTNOTES
 
* This work was supported in part by Deutsche Forschungsgemeinschaft Grant TJ 5/1-3. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 49-631-2053040; Fax: 49-631-2052600; E-mail: tjaden{at}rhrk.uni-kl.de.

1 The abbreviations used are: NTT, nucleotide transporter; ADP-Glc, ADP-glucose; MCF, mitochondrial carrier family; GFP, green fluorescent protein; RT, reverse transcription; AGPase, ADP-Glc pyrophosphorylase; IPTG, isopropyl {beta}-D-thiogalactopyranoside; HPLC, high pressure liquid chromatography. Back


   ACKNOWLEDGMENTS
 
We thank Claude Urbany and Hans-Henning Kunz for the help with the nucleotide uptake experiments and Dr. Christian Lohr and Guido Neumann for expert help during the confocal analysis. The voltage-dependent anion channel-GFP construct was kindly provided by Prof. J. Soll (München, Germany). We gratefully acknowledge critical reading of the manuscript by Prof. W. P. Quick and the invaluable suggestions for improvements.



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