Characterization of a novel eukaryotic ATP/ADP translocator located in the plastid envelope of Arabidopsis thaliana L.

Recently, we have sequenced a cDNA clone from Arabidopsis thaliana L. encoding a novel putative ATP/ADP translocator (AATP1). Here, we demonstrate that the radioactively labeled AATP1 precursor protein, synthesized in vitro , is targeted to envelope membranes of isolated spinach chloroplasts. Antibodies raised against a synthetic peptide of AATP1 recognized a single polypeptide of about 62 kDa in chloroplast inner envelope preparations. The cDNA coding for the AATP1 protein was functionally expressed in Saccharomyces cerevisiae and Escherichia coli . In both expression systems, increased rates of ATP transport were observed after reconstitution of the extracted protein into proteoliposomes. To our knowledge, this is the first report on the functional expression of an intrinsic plant membrane protein in E. coli . To yield high rates of ATP transport, proteoliposomes had to be preloaded with ADP, indicating a counter-exchange mode of transport. Carboxyatractyloside did not substantially interfere with ATP transport into proteoliposomes containing the plastidic ATP/ADP translocator. An apparent K_M for ATP of 28 µM was determined which is similar to values reported for isolated plastids. The data presented here strongly support the conclusion that AATP1 represents a novel eukaryotic adenylate carrier and that it is identical with the so far unknown plastidic ATP/ADP translocator.     

Altered plastidic ATP/ADP-transporter activity influences potato ( Solanum tuberosum L.) tuber morphology, yield and composition of tuber starch

The metabolic function of the plastidic ATP/ADP transporter (AATP) in heterotrophic plastids was examined in transgenic potato plants that exhibited increased or decreased amounts of the protein. Altered mRNA levels correlated with activities of the plastidic ATP/ADP transporter. Potato tubers with decreased plastidic ATP/ADP transporter activities exhibited reduced starch contents whereas sense lines accumulated increased amounts of tuber starch. Starch from wild-type tubers had an amylose content of 18.8%, starch from antisense plants contained 11.5–18.0% amylose, whereas starch from sense plants had levels of 22.7–27.0%. The differences in physiological parameters were accompanied with altered tuber morphology. These changes are discussed with respect to the stromal ATP supply during starch biosynthesis.     

Enhanced resistance to<Emphasis Type="Italic"> Phytophthora infestans</Emphasis> and<Emphasis Type="Italic"> Alternaria solani</Emphasis> in leaves and tubers,respectively, of potato plants with decreased activity of the plastidic ATP/ADP transporter

Recently, it has been reported that tubers of transgenic potato ( Solanum tuberosum L.) plants with decreased activity of the plastidic ATP/ADP transporter (AATP1) contain less starch, despite having an increased glucose level [P. Geigenberger et al. (2001) Plant Physiol 125:1667-1678]. The metabolic alterations correlated with enhanced resistance to the bacterium Erwinia carotovora. Here it is shown that transgenic potato tubers, possessing less starch yet increased glucose levels due to the expression of a cytoplasm-localized yeast invertase, exhibit drastic susceptibility to E. carotovora. In addition, it is demonstrated that AATP1 anti-sense tubers show an increased capacity to ward off the pathogenic fungus Alternaria solani. In contrast to AATP1 anti-sense tubers, the corresponding leaf tissue does not show changes in carbohydrate accumulation. However, upon elicitor treatment, AATP1 anti-sense leaves possess an increased capacity to release H(2)O(2) and activate various defence-related genes, reactions that are associated with substantially delayed appearance of disease symptoms caused by Phytophthora infestans. Grafting experiments between AATP1 anti-sense plants and wild-type plants indicate the presence of a signal that is generated in AATP1 rootstocks and primes wild-type scions for potentiated activation of cellular defence responses in leaves. Together, the results suggest that (i) the enhanced pathogen tolerance of AATP1 anti-sense tubers is not due to "high sugar resistance", (ii) the increased disease resistance of AATP1 anti-sense tubers is effective against different types of pathogen and (iii) a systemic signal induced by antisensing the plastidic ATP/ADP transporter in potato tubers confers increased resistance to pathogens.     

Isolation and characterization of a rice cDNA clone encoding ATP/ADP translocator

We isolated a rice cDNA clone which encodes an open reading frame of 382 amino acids. Its deduced amino acid sequence corresponds to an ATP/ADP translocator protein. Its homology with a maize ATP/ADP translocator was 83.9% in nucleotide sequence, and 90.2% of the amino acid level. Expression of this gene is regulated by such external stresses as salinity and low temperature.     

Increased fatty acid production in potato by engineering of acetyl-CoA carboxylase

In contrast to oil seeds, potato (Solanum tuberosum L.) is characterized by a high amount of starch stored in the tubers. To assess the capacity for oil synthesis in potato tubers, the changes in lipid content and flux into lipid synthesis were explored in transgenic potatoes altered in carbohydrate or lipid metabolism. A strong decrease in the amount of starch observed in antisense lines for ADP-glucose pyrophosphorylase or plastidic phosphoglucomutase had no effect on storage-lipid content. Similarly, potato lines over-expressing the Arabidopsis thaliana (L.) Heynh. plastidic ATP/ADP transporter that contained an increased amount of starch were not altered in oil content, indicating that the plastidic ATP level is not limiting fatty acid synthesis in potato tubers. However, over-expression of the acetyl-CoA carboxylase from Arabidopsis in the amyloplasts of potato tubers led to an increase in fatty acid synthesis and a more than 5-fold increase in the amount of triacylglycerol. Taken together, these data demonstrate that potato tubers have the capacity for storage-lipid synthesis and that malonyl-CoA, the substrate for elongation during fatty acid synthesis, represents one of the limiting factors for oil accumulation.Abbreviations AATP Plastidic ADP/ATP transporter - ACCase Acetyl-CoA:carboxylase - DGAT Acyl-CoA:diacylglycerol acyltransferase - FW Fresh weight - TLC Thin-layer chromatography - WT Wild typeSource for transgenic plant material. Upon request, transgenic potato lines altered in ACCase activity can be obtained from Peter Dörmann. For potato lines with alterations in AATP transporter activity, please refer to H. Ekkehard Neuhaus. Transgenic AGP and PGM lines are available from A. Fernie (Max-Planck-Institute of Molecular Plant Physiology, Golm, Germany).     

Sequences required for delivery and localization of the ADP/ATP translocator to the mitochondrial inner membrane.

The ADP/ATP translocator, a transmembrane protein of the mitochondrial inner membrane, is coded in Saccharomyces cerevisiae by the nuclear gene PET9. DNA sequence analysis of the PET9 gene showed that it encoded a protein of 309 amino acids which exhibited a high degree of homology with mitochondrial translocator proteins from other sources. This mitochondrial precursor, in contrast to many others, does not contain a transient presequence which has been shown to direct the posttranslational localization of proteins in the organelle. Gene fusions between the PET9 gene and the gene encoding beta-galactosidase (lacZ) were constructed to define the location of sequences necessary for the mitochondrial delivery of the ADP/ATP translocator protein in vivo. These studies reveal that the information to target the hybrid molecule to the mitochondria is present within the first 115 residues of the protein. In addition, these studies suggest that the "import information" of the amino-terminal region of the ADP/ATP translocator precursor is twofold. In addition to providing targeting function of the precursor to the organelle, these amino-terminal sequences act to prevent membrane-anchoring sequences located between residues 78 and 98 from stopping import at the outer mitochondrial membrane. These results are discussed in light of the function of distinct protein elements at the amino terminus of mitochondrially destined precursors in both organelle delivery and correct membrane localization.     

家蚕微孢子虫ADP/ATP转运蛋白的原核表达及间接免疫荧光定位分析

【目的】家蚕微孢子虫Nosema bombycis ADP/ATP转运蛋白可能参与搬运宿主细胞的能量。本研究克隆家蚕微孢子虫ADP/ATP转运蛋白基因,并进行原核表达、抗体制备及间接免疫荧光定位,为控制和防治家蚕微粒子病提供理论基础。【方法】通过同源序列比对鉴定家蚕微孢子虫N. bombycis ADP/ATP转运蛋白序列,采用生物合成的方法将编码3段面向膜内侧肽段的核酸序列拼接合成,在其两端引入BglⅡ和SalⅠ酶切位点,克隆至pUC57载体并测序,再亚克隆至含有二氢叶酸还原酶（dihydrofolate reductase,DHFR）标签的表达载体pQE40中,然后利用BamHⅠ和SalⅠ酶切获得含有DHFR标签的重组序列,并连接至pET30a（+）载体中进行诱导表达。通过SDS-PAGE、镍柱亲和层析和免疫印迹法鉴定表达蛋白,利用间接免疫荧光对ADP/ATP转运蛋白的分布进行检测。【结果】家蚕微孢子虫的ADP/ATP转运蛋白编码序列（GenBank登录号为EOB13854.1）全长1 524 bp,编码蛋白含有507个氨基酸残基,预测分子质量为59 kDa,等电点为9.35。具有12个跨膜结构域和TLC结构域,其中TLC结构域含有4个功能保守位点。与蜜蜂微孢子虫的ADP/ATP转运蛋白比较,氨基酸序列一致性达30%。系统进化分析表明微孢子虫ADP/ATP转运蛋白聚为一类,具有共同的起源。成功构建了NbADP/ATP-△TM-DHFR-pET30a原核表达重组质粒,目的基因获得表达,其融合蛋白分子量约为37 kDa,纯化重组蛋白并制备了多克隆抗体。免疫印迹分析表明,成熟微孢子虫中表达ADP/ATP转运蛋白;间接免疫荧光定位结果显示,家蚕微孢子虫孢子ADP/ATP转运蛋白定位于孢子质膜上。【结论】本研究将为阻断微孢子虫能量来源,达到控制和防治家蚕微粒子病提供新的思路。     

Non-mitochondrial ATP transport

Exchange of organelle ATP with cytosolic ADP through the ADP/ATP carrier is a well-characterized feature of mitochondrial metabolism. Obligate intracellular bacteria, such as Rickettsia prowazekii, and higher-plant plastids possess another type of adenylate transporter, which exchanges bacterial or plastidic ADP for ATP from the eukaryotic (host cell) cytoplasm. The bacterial and plastidic transporters are similar but do not share significant sequence similarities with the mitochondrial carrier. Recent molecular and biochemical studies are providing deeper insight into the functional and evolutionary relationships between the bacterial and the plant transport proteins.     

Identification, purification, and molecular cloning of a putative plastidic glucose translocator

During photosynthesis, part of the fixed carbon is directed into the synthesis of transitory starch, which serves as an intermediate carbon storage facility in chloroplasts. This transitory starch is mobilized during the night. Increasing evidence indicates that the main route of starch breakdown proceeds by way of hydrolytic enzymes and results in glucose formation. This pathway requires a glucose translocator to mediate the export of glucose from the chloroplasts. We have reexamined the kinetic properties of the plastidic glucose translocator and, using a differential labeling procedure, have identified the glucose translocator as a component of the inner envelope membrane. Peptide sequence information derived from this protein was used to isolate cDNA clones encoding a putative plastidic glucose translocator from spinach, potato, tobacco, Arabidopsis, and maize. We also present the molecular characterization of a candidate for a hexose transporter of the plastid envelope membrane. This transporter, initially characterized more than 20 years ago, is closely related to the mammalian glucose transporter GLUT family and differs from all other plant hexose transporters that have been characterized to date.     

Lawsonia intracellularis contains a gene encoding a functional rickettsia-like ATP/ADP translocase for host exploitation

ATP/ADP translocases are a hallmark of obligate intracellular pathogens related to chlamydiae and rickettsiae. These proteins catalyze the highly specific exchange of bacterial ADP against host ATP and thus allow bacteria to exploit their hosts' energy pool, a process also referred to as energy parasitism. The genome sequence of the obligate intracellular pathogen Lawsonia intracellularis (Deltaproteobacteria), responsible for one of the most economically important diseases in the swine industry worldwide, revealed the presence of a putative ATP/ADP translocase most similar to known ATP/ADP translocases of chlamydiae and rickettsiae (around 47% amino acid sequence identity). The gene coding for the putative ATP/ADP translocase of L. intracellularis (L. intracellularis nucleotide transporter 1 [NTT1(Li)]) was cloned and expressed in the heterologous host Escherichia coli. The transport properties of NTT1(Li) were determined by measuring the uptake of radioactively labeled substrates by E. coli. NTT1(Li) transported ATP in a counterexchange mode with ADP in a highly specific manner; the substrate affinities determined were 236.3 (+/- 36.5) microM for ATP and 275.2 (+/- 28.1) microM for ADP, identifying this protein as a functional ATP/ADP translocase. NTT1(Li) is the first ATP/ADP translocase from a bacterium not related to Chlamydiae or Rickettsiales, showing that energy parasitism by ATP/ADP translocases is more widespread than previously recognized. The occurrence of an ATP/ADP translocase in L. intracellularis is explained by a relatively recent horizontal gene transfer event with rickettsiae as donors.     

The plastidic pentose phosphate translocator represents a link between the cytosolic and the plastidic pentose phosphate pathways in plants

Plastids are the site of the reductive and the oxidative pentose phosphate pathways, which both generate pentose phosphates as intermediates. A plastidic transporter from Arabidopsis has been identified that is able to transport, in exchange with inorganic phosphate or triose phosphates, xylulose 5-phosphate (Xul-5-P) and, to a lesser extent, also ribulose 5-phosphate, but does not accept ribose 5-phosphate or hexose phosphates as substrates. Under physiological conditions, Xul-5-P would be the preferred substrate. Therefore, the translocator was named Xul-5-P/phosphate translocator (XPT). The XPT shares only approximately 35% to 40% sequence identity with members of both the triose phosphate translocator and the phosphoenolpyruvate/phosphate translocator classes, but a higher identity of approximately 50% to glucose 6-phosphate/phosphate translocators. Therefore, it represents a fourth group of plastidic phosphate translocators. Database analysis revealed that plant cells contain, in addition to enzymes of the oxidative branch of the oxidative pentose phosphate pathway, ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase in both the cytosol and the plastids, whereas the transketolase and transaldolase converting the produced pentose phosphates to triose phosphates and hexose phosphates are probably solely confined to plastids. It is assumed that the XPT function is to provide the plastidic pentose phosphate pathways with cytosolic carbon skeletons in the form of Xul-5-P, especially under conditions of a high demand for intermediates of the cycles.     

Effects of carbohydrate starvation on gene expression in citrus root

The roots of alternate-bearing citrus (Murcott, a Citrus reticulata hybrid) trees undergo extreme fluctuations of carbohydrate abundance and starvation. Using this system, we investigated the effect of root carbohydrate (total soluble sugar, sucrose and starch) depletion on carbohydrate-related gene expression. A series of genes, including those coding for starch phosphorylase ( STPH-L and STPH-H), ADP-glucose pyrophosphorylase, small subunit ( Agps), R1, plastidic ADP/ATP transporter ( AATP), phosphoglucomutase ( PGM-P and PGM-C), sucrose synthase ( CitSuS1 and CitSuSA), sucrose transporter ( SUT1 and SUT2), hexokinase ( HK) and alpha-amylase ( alpha-AMY), have been isolated and their expression analyzed. The genes were found to respond differentially to carbohydrate depletion. STPH-L, STPH-H, Agps, R1, AATP, PGM-P, PGM-C, CitSuS1 and HK were down-regulated while SUT1 and alpha-AMY were up-regulated during carbohydrate depletion. Two other genes, CitSuSA and SUT2, did not respond to carbohydrate depletion. Fruit removal, which interrupted the carbohydrate depletion induced by heavy fruiting, reversed these gene expression patterns. Trunk girdling and whole-plant darkening treatments, which brought about root carbohydrate depletion, induced the same changes in gene expression obtained in the alternate-bearing system. The possible roles of the up- and down-regulated genes in the metabolism of carbohydrate-depleted citrus roots are discussed. Although the specific signals involved have not been determined, the results support the feast/famine hypothesis of carbohydrate regulation proposed by Koch [K.E. Koch (1996) Annu Rev Plant Physiol Plant Mol Biol 47:509-540].     

Substrate specificity and ionic regulation of GlnPQ from Lactococcus lactis. An ATP-binding cassette transporter with four extracytoplasmic substrate-binding domains

We report on the functional characterization of GlnPQ, an ATP-binding cassette transporter with four extracytoplasmic substrate-binding domains. The first predicted transmembrane helix of GlnP was cleaved off in the mature protein and most likely serves as the signal sequence for the extracytoplasmic substrate-binding domains. Deletion analysis showed that the substrate-binding domain, in the primary sequence of GlnP nearest to the translocator domain, is used as the receptor that delivers the substrate to the translocator. Membrane reconstitution of the detergent-solubilized and purified GlnPQ complex yielded proteoliposomes that transported glutamine and glutamic acid at the expense of ATP. The transport activity of GlnPQ increased with lumenal salt concentration and internal pH, but the mechanism of ionic activation of the transporter is distinct from that of other osmoregulatory ATP-binding cassette transporters and does not depend on the presence of anionic lipids. The regulation of GlnPQ conforms to an electrostatic switch in which protein domain(s) and low molecular weight electrolytes participate.     

Isolation and sequence analysis of a cDNA encoding the ATP/ADP translocator of Zea mays L.

A cDNA complementary to the mRNA for the ATP/ADP translocator of maize (Zea mays L.) has been identified by virtue of hybridisation with the homologous gene from yeast. The cloned cDNA has been shown by DNA sequence analysis to contain an open reading frame of 954bp., which encodes a polypeptide of molecular weight 40,519. This polypeptide exhibits a high degree of homology to the translocator polypeptides of beef heart and Neurospora crassa mitochondria.     

Girdling affects carbohydrate-related gene expression in leaves,bark and roots of alternate-bearing citrus trees

Effects of girdling on carbohydrate status and carbohydrate-related gene expression in citrus trees were investigated. Alternate-bearing 'Murcott' (a Citrus reticulata hybrid of unknown origin) trees were girdled during autumn (25 Sep. 2001) and examined 10 weeks later. Girdling brought about carbohydrate (soluble sugar and starch) accumulation in leaves and shoot bark above the girdle, in trees during their fruitless, 'off' year. Trees during their heavy fruit load, 'on' year did not accumulate carbohydrates above the girdle due to the high demand for carbohydrates by the developing fruit. Girdling caused a strong decline in soluble sugar and starch concentrations in organs below the girdle (roots), in both 'on' and 'off' trees. Expression of STPH-L and STPH-H (two isoforms of starch phosphorylase), Agps (ADP-glucose pyrophosphorylase, small subunit), AATP (plastidic ADP/ATP transporter), PGM-C (phosphoglucomutase) and CitSuS1 (sucrose synthase), all of which are associated with starch accumulation, was studied. It was found that gene expression is related to starch accumulation in all 'off' tree organs. RNA levels of all the genes examined were high in leaves and bark that accumulated high concentrations of starch, and low in roots with declining starch concentrations. It may be hypothesized that changes in specific sugars signal the up- and down-regulation of genes involved in starch synthesis.     

Metabolite transporters in plastids

Communication between plastids and the surrounding cytosol occurs via the plastidic envelope membrane. Recent findings show that the outer membrane is not as freely permeable to low molecular weight solutes as previously thought, but contains different channel-like proteins that act as selectivity filters. The inner envelope membrane contains a variety of metabolite transporters that mediate the exchange of metabolites between both compartments. Two new classes of phosphate antiporters were recently described that are different in structure and function from the known triose phosphate/phosphate translocator from chloroplasts. In addition, a cDNA coding for an ATP/ADP antiporter from plastids was isolated that shows similarities to a bacterial adenylate translocator.