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.     

The phosphoenolpyruvate/phosphate translocator is required for phenolic metabolism, palisade cell development, and plastid-dependent nuclear gene expression.

The Arabidopsis chlorophyll a/b binding protein (CAB) gene underexpressed 1 (cue1) mutant underexpresses light-regulated nuclear genes encoding chloroplast-localized proteins. cue1 also exhibits mesophyll-specific chloroplast and cellular defects, resulting in reticulate leaves. Both the gene underexpression and the leaf cell morphology phenotypes are dependent on light intensity. In this study, we determine that CUE1 encodes the plastid inner envelope phosphoenolpyruvate/phosphate translocator (PPT) and define amino acid residues that are critical for translocator function. The biosynthesis of aromatics is compromised in cue1, and the reticulate phenotype can be rescued by feeding aromatic amino acids. Determining that CUE1 encodes PPT indicates the in vivo role of the translocator in metabolic partitioning and reveals a mesophyll cell-specific requirement for the translocator in Arabidopsis leaves. The nuclear gene expression defects in cue1 suggest that a light intensity-dependent interorganellar signal is modulated through metabolites dependent on a plastid supply of phosphoenolpyruvate.     

Molecular cloning of two Arabidopsis UDP-galactose transporters by complementation of a deficient Chinese hamster ovary cell line

Nucleotide-sugar transporters (NSTs) form a family of structurally related transmembrane proteins that transport nucleotide-sugars from the cytoplasm to the endoplasmic reticulum and Golgi lumen. In these organelles, activated sugars are substrates for various glycosyltransferases involved in oligo- and polysaccharide biosynthesis. The Arabidopsis thaliana genome contains more than 40 members of this transporter gene family, of which only a few are functionally characterized. In this study, two Arabidopsis UDP-galactose transporter cDNAs (UDP-GalT1 and UDP-GalT2) are isolated by expression cloning using a Chinese hamster ovary cell line (CHO-Lec8) deficient in UDP-galactose transport. The isolated genes show only 21% identity to each other and very limited sequence identity with human and yeast UDP-galactose transporters and other NSTs. Despite this low overall identity, the two proteins clearly belong to the same gene family. Besides complementing Lec8 cells, the two NSTs are shown to transport exclusively UDP-galactose by an in vitro NST assay. The most homologous proteins with known function are plant transporters that locate in the inner chloroplast membrane and transport triose-phosphate, phosphoenolpyruvate, glucose-6-phosphate, and xylulose 5-phosphate. Also, the latter proteins are members of the same family, which therefore has been named the NST/triose-phosphate transporter family.     

Diversity of specificity and function of phosphate translocators in various plastids

This report gives a comparison of the specificity of phosphate translocators in various plastids. Whereas the phosphate translocator of the C₃ plant spinach mediates a counter exchange between inorganic phosphate, dihydroxyacetone phosphate, and 3-phosphoglycerate, the phosphate translocators in chloroplasts from C₄ and CAM plants transport phosphoenolpyruvate in addition to the above mentioned metabolites. In plastids from pea roots the phosphate translocator also transports glucose 6-phosphate. This diversity of phosphate translocators is discussed in view of the special functions of the various plastids.     

Comparison of the kinetic properties,inhibition and labelling of the phosphate translocators from maize and spinach mesophyll chloroplasts

The kinetic properties of the phosphate translocator from maize (Zea mays L.) mesophyll chloroplasts have been determined. We have used a double silicone-oil-layer centrifugation system in order to obtain true initial uptake rates in forward-reaction experiments. In addition, it was possible to perform back-exchange experiments and to study the effects of illumination and of preloading the chloroplasts with different substrates on transport. It is shown that the phosphate translocator from mesophyll chloroplasts of maize, a C₄ plant, transports inorganic phosphate and phosphorylated C₃ compounds in which the phosphate group is linked to the C₃ atom (e.g. 3-phosphoglycerate and triose phosphate). The affinities of the transported metabolites towards the translocator protein are about one order of magnitude higher than in mesophyll chloroplasts from the C₃ plant, spinach. In contrast to the phosphate translocator from C₃-mesophyll chloroplasts, that of C₄-mesophyll chloroplasts catalyzes in addition the transport of C₃ compounds where the phosphate group is attached to the C₂ atom (e.g. 2-phosphoglycerate, phosphoenolpyruvate). The phosphate translocator from both chloroplast types is strongly inhibited by pyridoxal-5-phosphate (PLP), 2,4,6-trinitrobenzenesulfonic acid and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS). In the case of the spinach translocator protein these inhibitors were shown to react with the same amino-acid residue at the substrate binding site, and one molecule of either DIDS or PLP is obviously required per substrate binding site for the inactivation of the translocation process. In the functionally active dimeric translocator protein only one substrate-binding site appears to be accessible at a particular time, indicating that the site might be exposed to each side of the membrane in turn. Using [³H]-H₂DIDS for the labelling of maize mesophyll envelopes the radioactivity was found to be associated with two polypeptides of about 29 and 30 kDa. Since Western-blot analysis showed that only the 30 kDa polypeptide reacted with an antiserum directed against the spinach phosphate translocator protein it is suggested that this polypeptide presumably represents the phosphate translocator from maize mesophyll chloroplasts.Abbreviations DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - PEP phosphoenolpyruvate - 2-,3-PGA 2-,3-phosphoglycerate - PLP pyridoxal-5-phosphate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TNBS 2,4,6-trinitrobenzenesulfonic acid - triose P triose phosphate This work was supported by the Deutsche Forschungsgemeinschaft     

Functional genomics of phosphate antiport systems of plastids

Plant cells require a co-ordination of metabolism between their major compartments, the plastids and the cytosol, in particular as certain metabolic pathways are confined to either compartments. The inner envelope membrane of the plastids forms the major barrier for metabolite exchange and is the site for numerous transport proteins, which selectively catalyse metabolite exchanges characteristic for green and/or non-green tissues. This report is focused on the molecular biology, evolution and physiological function of the family of phosphate translocators (PT) from plastids. Until now, four distinct subfamilies have been identified and characterized, which all share inorganic phosphate as common substrate, but have different spectra of counter exchange substrates to fulfil the metabolic needs of individual cells and tissues. The PTs are named after their main transported substrate, triose phosphate (TPT), phosphoenolpyruvate (PPT), glucose 6-phosphate (GPT) and xylulose 5-P (XPT). All PTs belong to the TPT/nucleotide sugar transporter (NST) superfamily, which includes yet uncharacterized PT homologues from plants and other eukaryotes. Transgenic plants or mutants with altered transport activity of some of the PTs have been generated or isolated. The analysis of these plant lines revealed new insights in the co-ordination and flexibility of plant metabolism.     

Cloning and in vivo expression of functional triose phosphate/phosphate translocators from C3- and C4-plants: evidence for the putative participation of specific amino acid residues in the recognition of phosphoenolpyruvate

The primary sequences of the chloroplast triose phosphate/phosphate translocator precursor proteins from C₄-plants (maize mesophyll cells and Flaveria trinervia ) and from the C₃-type Flaveria pringlei were determined. The mature parts of these translocators possess 83–94% identical amino acid residues. The C₄-translocator protein can be correctly targeted to C₃-type chloroplasts and inserted into the envelope membrane. Expression of the mature parts of these chloroplast translocators (cTPT) in transformed yeast cells and subsequent reconstitution of the functional proteins reveals the difference between the recombinant translocator proteins from the two cell types with respect to the transport of phosphoenolpyruvate. Comparison of the cTPT sequences from F. pringlei and F. trinervia in combination with computer-aided molecular modelling of the substrate translocation pore leads to the suggestion, that only minor exchanges of amino acid residues between the C₃- and C₄-translocator proteins are sufficient to extend their substrate specificities to recognize also phosphoenolpyruvate.     

Specific Transport of Inorganic Phosphate and C3- and C6-Sugar-Phosphates across the Envelope Membranes of Tomato (Lycopersicon esculentum) Leaf-Chloroplasts,Tomato Fruit-Chloroplasts and Fruit-Chromoplasts

Plastidial envelope membranes were isolated from tomato (Lycopersicon esculentum) leaves and green and red tomato fruits by isopycnic discontinuous sucrose density gradient centrifugation. Solubilized envelope membrane proteins were reconstituted into liposomes. Transport measurements revealed that the phosphate translocator from tomato leaves transports inorganic phosphate, 3-phosphoglycerate and triosephosphates. The phosphate translocators of green and red fruit plastids catalyze, in addition to the transport of these substrates, also the transport of glucose-6-phosphate, glucose-1-phosphate and phosphoenolpyruvate.     

Characterization of two functional phosphoenolpyruvate/phosphate translocator (PPT) genes in Arabidopsis--AtPPT1 may be involved in the provision of signals for correct mesophyll development

The Arabidopsis thaliana chlorophyll a/b-binding protein underexpressed 1 (cue1) mutant shows a reticulate leaf phenotype and is defective in a plastidic phosphoenolpyruvate (PEP)/phosphate translocator (AtPPT1). A functional AtPPT1 providing plastids with PEP for the shikimate pathway is therefore essential for correct leaf development. The Arabidopsis genome contains a second PPT gene, AtPPT2. Both transporters share similar substrate specificities and are therefore able to transport PEP into plastids. The cue1 phenotype could partially be complemented by ectopic expression of AtPPT2 but obviously not by the endogeneous AtPPT2. Both genes are differentially expressed in most tissues: AtPPT1 is mainly expressed in the vasculature of leaves and roots, especially in xylem parenchyma cells, but not in leaf mesophyll cells, whereas AtPPT2 is expressed ubiquitously in leaves, but not in roots. The expression profiles are corroborated by tissue-specific transport data. As AtPPT1 expression is absent in mesophyll cells that are severely affected in the cue1 mutant, we propose that the vasculature-located AtPPT1 is involved in the generation of phenylpropanoid metabolism-derived signal molecules that trigger development in interveinal leaf regions. This signal probably originates from the root vasculature where only AtPPT1, but not AtPPT2, is present.     

A cell-free translation and proteoliposome reconstitution system for functional analysis of plant solute transporters

We describe here a novel proteoliposome reconstitution system for functional analysis of plant membrane transporters that is based on a modified wheat germ cell-free translation system. We established optimized conditions for the reconstitution system with Arabidopsis thaliana phosphoenolpyruvate/phosphate translocator 1 (AtPPT1) as a model transporter. A high activity of AtPPT1 was achieved by synthesis of the protein in the presence of both a detergent such as Brij35 and liposomes. We also determined the substrate specificities of three putative rice PPT homologs with this system. The cell-free proteoliposome reconstitution system provides a valuable tool for functional analysis of transporter proteins.     

The phosphate translocator of the chloroplast envelope. Isolation of the carrier protein and reconstitution of transport

This report describes the solubilization and purification of the phosphate translocator of spinach chloroplasts and the reconstitution of its activity by incorporation into liposomes. (1) Prior to the isolation, the carrier is specifically labelled by treatment with 2,4,6-trinitrobenzenesulfonic acid and NaB[³H]H₄. (2) After preextraction of purified envelope membranes with Brij 58 for removing other loosely bound membrane proteins, the phosphate translocator is extracted with Triton X-100. After passing the resulting extract over a DEAE-Sepharose column followed by sucrose density gradient ultracentrifugation, the translocator protein is purified to apparent homogeneity. The 5–6-fold purification thus obtained concurs with earlier findings that the phosphate translocator protein represents 15–20% of the envelope membrane protein. This highly purified protein is suitable for studies of the hydrodynamic parameters of the translocator. (3) Since the exposure to detergents affects the activity of the translocator protein, alternatively, a rapid batch procedure for the purification of the translocator protein employing hydroxyapatite is used, yielding within 15 min the phosphate translocator protein of about 70% purity. (4) After incorporation of this protein fraction into liposomes, a specific transport of phosphate into these liposomes is observed, which van be terminated by inhibitor stop with pyridoxal 5′-phosphate. This uptake is only observed when the liposomes have been preloaded with phosphate or 3-phosphoglycerate, but not with 2-phosphoglycerate. Thus, like in intact chloroplasts, also the reconstituted transport facilitates an obligatory and specific counter exchange of anions. The apparent K_m for the transport of phosphate by this reconstituted system is about 0.8 mM, which is comparable to the corresponding value in intact chloroplasts. The calculated turnover of 150–300 min⁻¹ (20°C) accounts for 3–6% of the original activity.     

Interaction of acetyl phosphate and carbamyl phosphate with plant phosphoenolpyruvate carboxylase.

Acetyl phosphate produced an increase in the maximum velocity (Vmax. for the carboxylation of phosphoenolpyruvate catalysed by phosphoenolpyruvate carboxylase. The limiting Vmax. was 22.2 mumol X min-1 X mg-1 (185% of the value without acetyl phosphate). This compound also decreased the Km for phosphoenolpyruvate to 0.18 mM. The apparent activation constants for acetyl phosphate were 1.6 mM and 0.62 mM in the presence of 0.5 and 4 mM-phosphoenolpyruvate respectively. Carbamyl phosphate produced an increase in Vmax. and Km for phosphoenolpyruvate. The variation of Vmax./Km with carbamyl phosphate concentration could be described by a model in which this compound interacts with the carboxylase at two different types of sites: an allosteric activator site(s) and the substrate-binding site(s). Carbamyl phosphate was hydrolysed by the action of phosphoenolpyruvate carboxylase. The hydrolysis produced Pi and NH4+ in a 1:1 relationship. Values of Vmax. and Km were 0.11 +/- 0.01 mumol of Pi X min-1 X mg-1 and 1.4 +/- 0.1 mM, respectively, in the presence of 10 mM-NaHCO3. If HCO3- was not added, these values were 0.075 +/- 0.014 mumol of Pi X min-1 X mg-1 and 0.76 +/- 0.06 mM. Vmax./Km showed no variation between pH 6.5 and 8.5. The reaction required Mg2+; the activation constants were 0.77 and 0.31 mM at pH 6.5 and 8.5 respectively. Presumably, carbamyl phosphate is hydrolysed by phosphoenolpyruvate carboxylase by a reaction the mechanism of which is related to that of the carboxylation of phosphoenolpyruvate.     

Snapshots of catalysis: the structure of fructose-1,6-(bis)phosphate aldolase covalently bound to the substrate dihydroxyacetone phosphate.

Fructose-1,6-bis(phosphate) aldolase is an essential glycolytic enzyme found in all vertebrates and higher plants that catalyzes the cleavage of fructose 1,6-bis(phosphate) (Fru-1,6-P(2)) to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). Mutations in the aldolase genes in humans cause hemolytic anemia and hereditary fructose intolerance. The structure of the aldolase-DHAP Schiff base has been determined by X-ray crystallography to 2.6 A resolution (R(cryst) = 0.213, R(free) = 0.249) by trapping the catalytic intermediate with NaBH(4) in the presence of Fru-1,6-P(2). This is the first structure of a trapped covalent intermediate for this essential glycolytic enzyme. The structure allows the elucidation of a comprehensive catalytic mechanism and identification of a conserved chemical motif in Schiff-base aldolases. The position of the bound DHAP relative to Asp33 is consistent with a role for Asp33 in deprotonation of the C4-hydroxyl leading to C-C bond cleavage. The methyl side chain of Ala31 is positioned directly opposite the C3-hydroxyl, sterically favoring the S-configuration of the substrate at this carbon. The "trigger" residue Arg303, which binds the substrate C6-phosphate group, is a ligand to the phosphate group of DHAP. The observed movement of the ligand between substrate and product phosphates may provide a structural link between the substrate cleavage and the conformational change in the C-terminus associated with product release. The position of Glu187 in relation to the DHAP Schiff base is consistent with a role for the residue in protonation of the hydroxyl group of the carbinolamine in the dehydration step, catalyzing Schiff-base formation. The overlay of the aldolase-DHAP structure with that of the covalent enzyme-dihydroxyacetone structure of the mechanistically similar transaldolase and KDPG aldolase allows the identification of a conserved Lys-Glu dyad involved in Schiff-base formation and breakdown. The overlay highlights the fact that Lys146 in aldolase is replaced in transaldolase with Asn35. The substitution in transaldolase stabilizes the enamine intermediate required for the attack of the second aldose substrate, changing the chemistry from aldolase to transaldolase.     

Expression of genes encoding the tobacco chloroplast phosphate translocator is not light-regulated and is repressed by sucrose

A cDNA encoding the complete precursor of the phosphate translocator of the chloroplast inner envelope membrane has been isolated from a tobacco leaf (Nicotiana tabacum cv. Samsun) gt 11 library. The tobacco cDNA is 1546 by in length and encodes a precursor protein of 401 amino acid residues with a deduced molecular weight of 43705. A putative processing site between Ala-73 and Ala-74 of the precursor protein is suggested by comparison with the N-terminal sequences of the pea and spinach proteins. Removal of the transit peptide produces the mature protein of 328 amino acid residues with a molecular weight of 36038. Southern blot analysis suggests there is probably one copy of the phosphate translocator gene in the pea haploid genome and two copies in the tobacco haploid genome, one derived from each ancestral parental genome. Messenger RNAs essentially equivalent in size to the cDNAs (approx. 1.6 kb) were detected in extracts of all organs examined from tobacco and pea, including leaves, stems, sepals, petals, seed-pods, tendrils and roots. An immunochemically related protein of a similar size to the phosphate translocator was detected in the equivalent pea organs. The levels of both mRNA and protein in non-photosynthetic organs were lower than those in photosynthetic organs. Tobacco phosphate translocator mRNA was present at high levels in etiolated tissue and did not increase significantly after 24 h illumination. Germination and growth of tobacco seedlings in the presence of sucrose caused a 3.3-fold decrease in the level of the phoshate translocator mRNA.     

Phosphate Translocator of Isolated Guard-Cell Chloroplasts from Pisum sativum L. Transports Glucose-6-Phosphate

Chloroplasts were isolated from ruptured guard-cell protoplasts of the Argenteum mutant of Pisum sativum L. and purified by centrifugation through a Percoll layer. The combined volume of the intact plastids and the uptake of phosphate were determined by silicone oil-filtering centrifugation, using tritiated water and [14C]sorbitol as membrane-permeating and nonpermeating markers and [32P]phosphate as tracer for phosphate. The affinities of the phosphate translocator for organic phosphates were assessed by competition with inorganic phosphate. The affinities for dihydroxyacetone phosphate, 3-phosphoglycerate (PGA), and phosphoenolpyruvate were in the same order as those reported for mesophyll chloroplasts of several species. However, the guard-cell phosphate translocator had an affinity for glucose-6-phosphate that was as high as that for PGA. Guard-cell chloroplasts share this property with amyloplasts from the root of pea (H.W. Heldt, U.I. Flugge, S. Borchert [1991] Plant Physiol 95: 341-343). An ability to import glucose-6-phosphate enables guard-cell chloroplasts to synthesize starch despite the reported absence of a fructose-1,6-bisphosphatase activity in the plastids, which would be required if only C3 phosphates could enter through the translocator.     

Tyr‐Asp inhibition of glyceraldehyde 3‐phosphate dehydrogenase affects plant redox metabolism

How organisms integrate metabolism with the external environment is a central question in biology. Here, we describe a novel regulatory small molecule, a proteogenic dipeptide Tyr‐Asp, which improves plant tolerance to oxidative stress by directly interfering with glucose metabolism. Specifically, Tyr‐Asp inhibits the activity of a key glycolytic enzyme, glyceraldehyde 3‐phosphate dehydrogenase (GAPC), and redirects glucose toward pentose phosphate pathway (PPP) and NADPH production. In line with the metabolic data, Tyr‐Asp supplementation improved the growth performance of both Arabidopsis and tobacco seedlings subjected to oxidative stress conditions. Moreover, inhibition of Arabidopsis phosphoenolpyruvate carboxykinase (PEPCK) activity by a group of branched‐chain amino acid‐containing dipeptides, but not by Tyr‐Asp, points to a multisite regulation of glycolytic/gluconeogenic pathway by dipeptides. In summary, our results open the intriguing possibility that proteogenic dipeptides act as evolutionarily conserved small‐molecule regulators at the nexus of stress, protein degradation, and metabolism.