Molecular characterization of plastidic phosphoserine aminotransferase in serine biosynthesis from Arabidopsis

Serine biosynthesis in plants proceeds by two pathways; a photorespiratory pathway which is associated with photorespiration and a pathway from phosphoglycerate. A cDNA encoding plastidic phosphoserine aminotransferase (PSAT) which catalyzes the formation of phosphoserine from phosphohydroxypyruvate has been isolated from Arabidopsis thaliana . Genomic DNA blot analysis indicated that this enzyme is most probably encoded by a single gene and is mapped on the lower arm of chromosome 4. The deduced protein contains an N-terminal extension exhibiting the general features of a plastidic transit peptide, which was confirmed by subcellular organelle localization using GFP (green flourescence protein). Northern analysis indicated preferential expression of PSAT in roots of light-grown plants, supporting the idea that the phosphorylated pathway may play an important role in supplying the serine requirement of plants in non-green tissues. In situ hybridization analysis of PSAT revealed that the gene is generally expressed in all types of cells with a significantly higher amount in the meristem tissue of root tips.     

Chimers of two fused ADP/ATP carrier monomers indicate a single channel for ADP/ATP transport

The mitochondrial ADP/ATP carrier (AAC) is generally believed to function as a homodimer (Wt. Wt). It remains unknown whether the two monomers possess two independent but fully anticooperative channels or they form a single central channel for nucleotide transport. Here we generated fusion proteins consisting of two tandem covalent-linked AAC monomers and studied the kinetics of ADP/ATP transport in reconstituted proteoliposomes. Functional 64-kDa fusion proteins Wt-Wt and Wt-R294A (wild-type AAC linked to a mutant having low ATP transport activity) were expressed in mitochondria of yeast transformants. Compared to homodimer Wt. Wt, the fusion protein Wt-Wt retained the transport activity and selectivity of ADP versus ATP. The strongly divergent selectivities of Wt and R294A were partially propagated in the Wt-R294A fusion protein, suggesting a limited cooperativity during solute translocation. The rates of ADP or ATP transport were significantly higher than those predicted by the two-channel model. Fusion proteins for Wt-R204L (Wt linked to an inactive mutant) and R204L-Wt were not expressed in aerobically grown yeast cells, which contained plasmid rearrangements that regenerated the fully active 32-kDa homodimer Wt. Wt, suggesting that these fusion proteins are inactive in ADP/ATP transport. These results favor a single binding center gated pore model [Klingenberg, M. (1991) in A Study of Enzymes, Vol. 2: pp. 367-388] in which two AAC subunits cooperate for a coordinated ADP/ATP exchange through a single channel.     

The Molecular Mechanism of Transport by the Mitochondrial ADP/ATP Carrier

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Identification, expression, and functional analyses of a thylakoid ATP/ADP carrier from Arabidopsis

In plants the chloroplast thylakoid membrane is the site of light-dependent photosynthetic reactions coupled to ATP synthesis. The ability of the plant cell to build and alter this membrane system is essential for efficient photosynthesis. A nucleotide translocator homologous to the bovine mitochondrial ADP/ATP carrier (AAC) was previously found in spinach thylakoids. Here we have identified and characterized a thylakoid ATP/ADP carrier (TAAC) from Arabidopsis.(i) Sequence homology with the bovine AAC and the prediction of chloroplast transit peptides indicated a putative carrier encoded by the At5g01500 gene, as a TAAC. (ii) Transiently expressed TAAC-green fluorescent protein fusion construct was targeted to the chloroplast. Western blotting using a peptide-specific antibody together with immunogold electron microscopy revealed a major location of TAAC in the thylakoid membrane. Previous proteomic analyses identified this protein in chloroplast envelope preparations. (iii) Recombinant TAAC protein specifically imports ATP in exchange for ADP across the cytoplasmic membrane of Escherichia coli. Studies on isolated thylakoids from Arabidopsis confirmed these observations. (iv) The lack of TAAC in an Arabidopsis T-DNA insertion mutant caused a 30-40% reduction in the thylakoid ATP transport and metabolism. (v) TAAC is readily expressed in dark-grown Arabidopsis seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. We propose that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover in plants.     

Enlightening energy parasitism by analysis of an ATP/ADP transporter from chlamydiae

Energy parasitism by ATP/ADP transport proteins is an essential, common feature of intracellular bacteria such as chlamydiae and rickettsiae, which are major pathogens of humans. Although several ATP/ADP transport proteins have so far been characterized, some fundamental questions regarding their function remained unaddressed. In this study, we focused on the detailed biochemical analysis of a representative ATP/ADP transporter (PamNTT1), from the amoeba symbiont Protochlamydia amoebophila (UWE25) to further clarify the principle of energy exploitation. We succeeded in the purification of the first bacterial nucleotide transporter (NTT) and its functional reconstitution into artificial lipid vesicles. Reconstituted PamNTT1 revealed high import velocities for ATP and an unexpected and previously unobserved stimulating effect of the luminal ADP on nucleotide import affinities. Latter preference of the nucleotide hetero-exchange is independent of the membrane potential, and therefore, PamNTT1 not only structurally but also functionally differs from the well-characterized mitochondrial ADP/ATP carriers. Reconstituted PamNTT1 exhibits a bidirectional orientation in lipid vesicles, but interestingly, only carriers inserted with the N-terminus directed to the proteoliposomal interior are functional. The data presented here comprehensively explain the functional basis of how the intracellular P. amoebophila manages to exploit the energy pool of its host cell effectively by using the nucleotide transporter PamNTT1. This membrane protein mediates a preferred import of ATP, which is additionally stimulated by a high internal (bacterial) ADP/ATP ratio, and the orientation-dependent functionality of the transporter ensures that it is not working in a mode that is detrimental to P. amoebophila. Heterologous expression and purification of high amounts of PamNTT1 provides the basis for its crystallization and detailed structure/function analyses. Furthermore, functional reconstitution of this essential chlamydial protein paves the way for high-throughput uptake studies in order to screen for specific inhibitors potentially suitable as anti-chlamydial drugs.     

Molecular biology of the plastidic phosphorylated serine biosynthetic pathway in Arabidopsis thaliana

Summary. Serine biosynthesis in plants proceeds by two pathways; the glycolate pathway which is associated with photorespiration and the pathway from 3-phosphoglycerate which is presumed to take place in the plastids. The 3-phosphoglycerate pathway (phosphorylated pathway) involves three enzymes catalyzing three sequential reactions: 3-phosphoglycerate dehydrogenase (PGDH), 3-phosphoserine aminotransferase (PSAT) and 3-phosphoserine phosphatase (PSP). cDNA and genomic clones encoding these three enzymes from spinach and Arabidopsis thaliana were isolated by means of heterologous probe screening, homologous EST clones and genetic complementation in an Escherichia coli mutant. The identity of the isolated cDNAs was confirmed by functional complementation of serine auxotrophy in E. coli mutants and/or the detection of catalytic activity in the recombinant enzymes produced in E. coli. Northern blot analyses indicated the most preferential expression of these three genes in light-grown roots. In contrast, the mRNAs of two proteins involved in the glycolate pathway (H-protein of glycine decarboxylase multienzyme complex and serine hydroxymethyltransferase) accumulated to high levels in light-grown shoots. Environmental stresses, such as high salinity, flooding and low temperature, induced changes in mRNA levels of enzymes in the plastidic phosphorylated serine biosynthetic pathway but not in that of the glycolate pathway. These results indicate that the plastidic 3-phosphoglycerate pathway plays an important role in supplying serine in non-photosynthetic tissues in plants and under environmental stresses. Received December 9, 1999 Accepted February 2, 2000     

Identification and Characterization of ATP/ADP Isopentenyltransferases (ATP/ADP PpIPTs) Genes in Peach

ATP/ADP isopentenyltransferase (IPTs) genes encode key enzymes involved in cytokinin synthesis. In this study, the functions of ATP/ADP PpIPTs in peach were investigated. According to the genome sequence, we have found and verified that there are four members of this gene family in peach, namely, PpIPT1, PpIPT3, PpIPT5, and PpIPT7. Overexpression of each of these genes in Arabidopsis resulted in increased levels of cytokinins in the transgenic plants, confirming their roles in cytokinin synthesis. Numerous altered phenotypes were observed in the transgenic plants, including vigorous growth and enhanced salt resistance. ATP/ADP PpIPTs were expressed in tissues throughout the plant, but the expression patterns differed between the genes. Only PpIPT3 was upregulated within 2 h after the application of nitrate to N-deprived peach seedlings, and the increase was resistant to pre-treatment of a specific nitrate metabolism inhibitor. Results showed that ATP/ADP PpIPT expression levels decreased significantly in pulp within 2 weeks after flowering and remained low. However, pulp cytokinin levels were quite high during this time. Only PpIPT5 in seed increased significantly within 2 weeks after flowering, which was consistent with cytokinin levels during early fruit development, suggesting that PpIPT5 in seed is the key gene for cytokinin biosynthesis during early fruit development. ATP/ADP PpIPT expression also increased significantly during later fruit development in seed.

     

Activation of the ADP/ATP carrier from mitochondria by cationic effectors

The ADP/ATP carrier from the mitochondrial inner membrane was found to be influenced by cationic substances from the hydrophilic surroundings. Under low-ionic-strength conditions, addition of these cationic effectors fully activated the reconstituted adenine nucleotide translocator. The list of activators included divalent cations, polyamines, peptides and cationic proteins. The minimum requirement for an activator to be effective was the presence of at least two positive net charges, regardless of the size of the molecule. Cationic molecules were not activating when an intramolecular charge compensation was possible or when the two charges were too far apart from one another. The affinity of these activators varied from several hundred microM (diaminoalkanes, divalent cations) to 1 microM (cytochrome c, spermine) and even down to a few nM (polylysine). The activation by cations was fully reversible and was not due to fusion processes. It was not mediated by an interaction with the anionic substrates ADP and ATP, nor by interaction with the liposomes. The stimulation could directly and functionally be correlated to the reconstituted carrier protein. Activation was not observed in intact mitochondria, but could be demonstrated when the outer mitochondrial membrane had been removed by treatment with digitonin. These mitoplasts were stimulated by polycations similar to the ADP/ATP carrier in the reconstituted system.     

The molecular basis for relative physiological functionality of the ADP/ATP carrier isoforms in Saccharomyces cerevisiae

AAC2 is one of three paralogs encoding mitochondrial ADP/ATP carriers in the yeast Saccharomyces cerevisiae, and because it is required for respiratory growth it has been the most extensively studied. To comparatively examine the relative functionality of Aac1, Aac2, and Aac3 in vivo, the gene encoding each isoform was expressed from the native AAC2 locus in aac1Delta aac3Delta yeast. Compared to Aac2, Aac1 exhibited reduced capacity to support growth of yeast lacking mitochondrial DNA or of yeast lacking the ATP/Mg-P(i) carrier, both conditions requiring ATP import into the mitochondrial matrix through the ADP/ATP carrier. Sixteen AAC1/AAC2 chimeric genes were constructed and analyzed to determine the key differences between residues or sections of Aac1 and Aac2. On the basis of the growth rate differences of yeast expressing different chimeras, the C1 and M2 loops of the ADP/ATP carriers contain divergent residues that are responsible for the difference(s) between Aac1 and Aac2. One chimeric gene construct supported growth on nonfermentable carbon sources but failed to support growth of yeast lacking mitochondrial DNA. We identified nine independent intragenic mutations in this chimeric gene that suppressed the growth phenotype of yeast lacking mitochondrial DNA, identifying regions of the carrier important for nucleotide exchange activities.     

Role of Thylakoid ATP/ADP Carrier in Photoinhibition and Photoprotection of Photosystem II in Arabidopsis

The chloroplast thylakoid ATP/ADP carrier (TAAC) belongs to the mitochondrial carrier superfamily and supplies the thylakoid lumen with stromal ATP in exchange for ADP. Here, we investigate the physiological consequences of TAAC depletion in Arabidopsis (Arabidopsis thaliana). We show that the deficiency of TAAC in two T-DNA insertion lines does not modify the chloroplast ultrastructure, the relative amounts of photosynthetic proteins, the pigment composition, and the photosynthetic activity. Under growth light conditions, the mutants initially displayed similar shoot weight, but lower when reaching full development, and were less tolerant to high light conditions in comparison with the wild type. These observations prompted us to study in more detail the effects of TAAC depletion on photoinhibition and photoprotection of the photosystem II (PSII) complex. The steady-state phosphorylation levels of PSII proteins were not affected, but the degradation of the reaction center II D1 protein was blocked, and decreased amounts of CP43-less PSII monomers were detected in the mutants. Besides this, the mutant leaves displayed a transiently higher nonphotochemical quenching of chlorophyll fluorescence than the wild-type leaves, especially at low light. This may be attributed to the accumulation in the absence of TAAC of a higher electrochemical H⁺ gradient in the first minutes of illumination, which more efficiently activates photoprotective xanthophyll cycle-dependent and independent mechanisms. Based on these results, we propose that TAAC plays a critical role in the disassembly steps during PSII repair and in addition may balance the trans-thylakoid electrochemical H⁺ gradient storage.In plants, the chloroplast thylakoid membrane is the site of light-driven photosynthetic reactions coupled to ATP synthesis. There are four major protein complexes involved in these reactions, namely, PSI, PSII, the cytochrome b₆f, and the H⁺-translocating ATP synthase (for review, see Nelson and Ben-Shem, 2004). The photosystems and the cytochrome b₆f complex also contain redox components and pigments bound to protein subunits. Their synthesis, assembly, optimal function, and repair during normal development and stress require a number of transport and regulatory mechanisms. In this context, the water-oxidizing PSII complex composed of more than 25 integral and peripheral proteins attracts special attention since its reaction center D1 subunit is degraded and replaced much faster than the other subunits under excess and even growth light conditions (for review, see Aro et al., 2005). Thus, the D1 protein turnover is the major event in the repair cycle of the PSII complex and occurs subsequently to the inactivation of PSII electron transport. D1 degradation is most likely performed by thylakoid FtsH and Deg proteases, operating on both sides of the thylakoid membrane (Lindahl et al., 2000; Haussühl et al., 2001; Silva et al., 2003; Kapri-Pardes et al., 2007). The PSII repair cycle is regulated by reversible phosphorylation of several core subunits (Tikkanen et al., 2008).ATP is produced as a result of the light-driven photosynthetic reactions in the thylakoid membrane and mainly is utilized in the carbon fixation reactions occurring in the soluble stroma. Besides this, ATP also drives several energy-dependent processes occurring on the stromal side of the thylakoid membrane, including phosphorylation, folding, import, and degradation of proteins. Furthermore, experimental evidence for ATP transport across the thylakoid membrane and nucleotide metabolism inside the lumenal space has been reported (Spetea et al., 2004; for review, see Spetea and Thuswaldner, 2008; Spetea and Schoefs, 2010). The protein responsible for the thylakoid ATP transport activity has been identified in Arabidopsis (Arabidopsis thaliana) as the product of the At5g01500 gene and functionally characterized in Escherichia coli as an ATP/ADP exchanger (Thuswaldner et al., 2007). This protein is homologous to the extensively studied bovine mitochondrial ADP/ATP carrier and therefore has been named thylakoid ATP/ADP carrier (TAAC). In the same report, it has been demonstrated that TAAC transports ATP from stroma to lumen in exchange for ADP, as based on radioactive assays using thylakoids isolated from Arabidopsis wild-type plants and a T-DNA insertion knockout line (named taac). Furthermore, TAAC was shown to be mainly expressed in photosynthetic tissues with an up-regulation during greening, senescence, and stress (e.g. high light) conditions, implying a physiological role during thylakoid biogenesis and turnover.The ATP translocated by TAAC across the thylakoid membrane is converted to GTP by the lumenal nucleoside diphosphate kinase III; GTP can then be bound and hydrolyzed to GDP and inorganic phosphate by the PsbO protein, a lumenal extrinsic subunit of the PSII complex (Spetea et al., 2004; Lundin et al., 2007a). The anion transporter 1 from Arabidopsis has been proposed to export to the stroma the phosphate generated during nucleotide metabolism in the thylakoid lumen (Ruiz Pavón et al., 2008). Between the two PsbO isoforms in Arabidopsis, it has recently been reported that PsbO2 plays an essential role in D1 protein turnover during high light stress and that it has a higher GTPase activity than PsbO1 (Lundin et al., 2007b, 2008; Allahverdiyeva et al., 2009). The precise mechanism of PsbO2-mediated PSII repair is not known. Nevertheless, the requirement of GTP for efficient proteolytic removal of the D1 protein during repair of photoinactivated PSII was previously reported (Spetea et al., 1999). Furthermore, it has been proposed that the PsbO2 type of PSII complexes undergo more efficient repair. This has been attributed to the PsbO2-mediated GTPase activity that induces PsbO2 release from the complex, thus facilitating the next steps in the repair process, namely, dissociation of the CP43 subunit and proteolysis of the D1 subunit (Lundin et al., 2007b, 2008).TAAC may represent the missing link between ATP synthesis on the stromal side of the thylakoid membrane and nucleotide-dependent reactions in the lumenal space. The taac mutant provides an interesting tool to study whether there are any regulatory networks between the activity of TAAC and PSII repair. Based on phenotypic characterization of two different T-DNA insertion lines of the TAAC gene, we report in this article that the PSII repair cycle is malfunctioning in the absence of TAAC and that the thermal photoprotection is faster activated during light stress.     

Similar affinities of ADP and ATP for G-actin at physiological salt concentrations

The equilibrium constant for the exchange of ATP and ADP at G-actin was determined by fluorimetric titration of G-actin-bound ε-ATP by ATP or ADP. The affinity of ATP for G-actin was found to be only about 3-fold higher than the affinity of ADP for G-actin at 37°C, pH 7.5 and physiologically relevant salt concentrations (100 mmol K⁺/l, 0.8 mmol Mg²⁺/l, <0.01 mmol Ca²⁺/l).     

Import of ADP/ATP carrier into mitochondria: two receptors act in parallel

We have identified the yeast homologue of Neurospora crassa MOM72, the mitochondrial import receptor for the ADP/ATP carrier (AAC), by functional studies and by cDNA sequencing. Mitochondria of a yeast mutant in which the gene for MOM72 was disrupted were impaired in specific binding and import of AAC. Unexpectedly, we found a residual, yet significant import of AAC into mitochondria lacking MOM72 that occurred via the receptor MOM19. We conclude that both MOM72 and MOM19 can direct AAC into mitochondria, albeit with different efficiency. Moreover, the precursor of MOM72 apparently does not require a positively charged sequence at the extreme amino terminus for targeting to mitochondria.     

Molecular and functional characterization of a family of amino acid transporters from Arabidopsis

More than 50 distinct amino acid transporter genes have been identified in the genome of Arabidopsis, indicating that transport of amino acids across membranes is a highly complex feature in plants. Based on sequence similarity, these transporters can be divided into two major superfamilies: the amino acid transporter family and the amino acid polyamine choline transporter family. Currently, mainly transporters of the amino acid transporter family have been characterized. Here, a molecular and functional characterization of amino acid polyamine choline transporters is presented, namely the cationic amino acid transporter (CAT) subfamily. CAT5 functions as a high-affinity, basic amino acid transporter at the plasma membrane. Uptake of toxic amino acid analogs implies that neutral or acidic amino acids are preferentially transported by CAT3, CAT6, and CAT8. The expression profiles suggest that CAT5 may function in reuptake of leaking amino acids at the leaf margin, while CAT8 is expressed in young and rapidly dividing tissues such as young leaves and root apical meristem. CAT2 is localized to the tonoplast in transformed Arabidopsis protoplasts and thus may encode the long-sought vacuolar amino acid transporter.     

Identification and initial topological analysis of the Rickettsia prowazekii ATP/ADP translocase.

The Rickettsia prowazekii ATP/ADP translocase was identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis using antibodies raised against a synthetic peptide corresponding in sequence to the carboxyl-terminal 17 amino acids of the carrier. Both the translocase of R. prowazekii and that expressed by Escherichia coli transformants containing the rickettsial gene had an apparent molecular mass of 36,500 Da by SDS-PAGE analysis, a mass considerably less than that deduced from the sequence of the gene. The SDS-solubilized translocase aggregated upon heating at 100 degrees C in the presence of disulfide bond-reducing agents. Similar concentrations of disulfide bond-reducing agents inhibited the exchange transport of adenine nucleotides by both R. prowazekii and translocase-expressing E. coli. These data suggested that an intramolecular disulfide bond in the translocase was essential for transport activity. The antipeptide antibodies used for identification of the translocase bound preferentially to inside-out membrane vesicles of translocase-expressing E. coli relative to right-side-out spheroplasts, thus indicating that the carboxyl terminus of the carrier is located on the cytoplasmic side of the bacterial inner membrane. Protease studies were unable to localize the carboxyl terminus because of the resistance of this region of the native translocase to proteolytic cleavage. These data in conjunction with hydrophobicity analysis were used to construct an initial topological model of the translocase within the cell membrane.