Lipid metabolism in chromoplast membranes from the daffodil: Glycosylation and acylation

The non-photosynthetic chromoplast membranes from the corona ofNarcissus pseudonarcissus L. were investigated for their lipid synthetic capabilities. The following activities were detected: galactosylation of diacylglycerol and galactosydiacylglycerols, glycosylation of sterols, acylation of monogalactosyldiacylglycerol and steryl glycosides from an unknown endogenous donor, acylation of phospholipids from acyl-CoA, and acylation of phosphatidyl inositol from phosphatidyl choline. Furthermore, activities of an acyl thioesterase, a sugar epimerase, and a phospholipase A₂ were measured.Abbreviations MGDG monogalactosyldiacylglycerol - DGDG digalactosyldiacylglycerol - TGDG tri-and tetragalactosyldiacylglycerol - SG steryl glycoside - SL sulfolipid - ACP acyl carrier protein     

Fatty acid synthesis by isolated chromoplasts from the daffodil. Energy sources and distribution patterns of the acids

1. Fatty acid synthesis in isolated intact chromoplasts from [1-¹⁴C]acetate was made possible by using ATP, ADP (via adenylate kinase), and, with decreasing efficiency, UTP, CTP, and GTP as energy sources. 2. The glycolytic path from dihydroxyacetone phosphate to acetyl-CoA operates within the chromoplasts. The glycolytic intermediates, especially 2-phosphoglycerate and phosphoenolpyruvate, served as very effective energy donors for fatty acid synthesis by phosphorylating the endogenous adenine nucleotide pool. 3. In the presence of exogenous ATP or ADP, appreciable amounts of in vitro formed fatty acids were found as acyl-CoA and subsequent products, mainly phosphatidylcholine. When other energy sources were used most of the acids formed were in the free form, and to a minor extent, in the phosphatidic acid and diacylglycerol fractions. Similar results have recently been reported for spinach chloroplasts (Kleinig and Liedvogel 1979, FEBS Lett.101, 339–342).Abbreviations ATP adenosine triphosphate - ADP adenosine diphosphate - UTP uridine triphosphate - CTP cytidine triphosphate - GTP gnanosine triphosphate     

Fatty acid and glycerolipid synthesis in abscised daffodil flowers after acetate feeding

The coronae of Narcissus pseudonarcissus flowers incorporated [1-¹⁴C]acetate almost exclusively into the fatty acid moieties of glycerolipids. After a 4 h incubation, the newly synthesized acids were: stearate plus palmitate (50%); oleate (17%); linoleate (32%); and linolenate (0.5%). Phosphatidylcholine and diacylglycerol were the principal labelled lipids. In pulse experiments these acids were further desaturated, with time, to an appreciable extent and, concurrently, transferred essentially from phosphatidylcholine to diacylglycerol, diacylgalactosylglycerol, and diacylgalabiosylglycerol. The labelling of diacylgalactosylglycerol and diacylgalabiosylglycerol paralleled the appearance of linolenate. The distribution of labelled acids in phosphatidylcholine, diacylgalactosylglycerol, and diacylgalabiosylglycerol was very different. The results were compared with those obtained in vitro with isolated coronae chromoplasts and discussed in relation to current schemes of fatty acid and glycerolipid synthesis in plant cells.     

Galactolipid synthesis in chromoplasts in vitro

Isolated chromoplasts from Narcissus pseudonarcissus flowers contain: a fatty acid synthesizing system; acyl-CoA synthetase (EC 6.2.1.3); glycero-phosphate acyltransferase (EC 2.3.1.15); acylglycero-phosphate acyltransferase; phosphatidate phosphatase (EC 3.1.3.4); diacylglycerol galactosyltransferase (EC 2.4.1.46); and diacylgalactosylglycerol galactosyltransferase, i.e. all enzymatic activities necessary for the synthesis of diacylgalactosylglycerol and diacylgalabiosylglycerol from acetate, HCO _- ³ , sn-glycerol 3-phosphate, and UDP-d-galactose. Diacylgalactosylglycerol and diacylgalabiosylglycerol, however, are synthesized from these precursors to only a very low extent in an in vitro system. This is attributed to a specificity of diacylglycerol galactosyltransferase for highly unsaturated diacylglycerols. Specificities of acyltransferase reactions were also found.     

The site of carotenogenic enzymes in chromoplasts from Narcissus pseudonarcissus L.

The membranes from the chromoplasts of Narcissus pseudonarcissus L. which are derived from the inner envelope membrane are the site of -carotene synthesis from [1-¹⁴C]isopentenyl diphosphate. The enzymes involved are partly peripheral membrane proteins (prenyltransferase, phytoene synthase) and partly integral membrane proteins (cis-trans isomerase, dehydrogenase(s), cyclase(s)). Metabolic channeling is suggested.Abbreviations IPP isopentenyl diphosphate - GGPP geranylgeranyl diphosphate     

β-Carotene synthesis in isolated chromoplasts from Narcissus pseudonarcissus

A system has been established from isolated intact chromoplasts of Narcissus pseudonarcissus flowers that synthesizes geranylgeraniol, an unknown polyprenoid alcohol, phytoene, and -carotene from [1-¹⁴C]isopentenyl pyrophosphate in a good yeild. Long chain pyrophosphates are not accumulated. San 6706 inhibits the dehydrogenation of phytoene, whereas nicotine does not lead to an accumulation of lycopene. Separation and identification of polyprenoid lipids was performed by HPLC. The properties and advantages of the chromoplast system are discussed.Abbreviations IPP isopentenyl pyrophosphate - TLC thin-layer chromatography - HPLC high pressure liquid chromatography - GC gas chromatography - Sau 6706 4-chloro-5-(dimethylamino)-2-,,-(trifluoro-m-tolyl)3(2H)-pyridazinone     

Origin and developmental changes of envelope proteins and translocator activities from plastids of Secale cereale L.

To determine the sites of synthesis of chloroplast-envelope proteins, we have analysed several enzyme and translocator functions ascribed to the envelope membranes, and investigated the envelope polypeptide composition of plastids isolated from 70S ribosome-deficient leaves of rye (Secale cereale L.) generated by growing the plants at a temperature of 32°C. Since the ribosomedeficient plastids are also achlorophyllous in light-grown leaves, not only were chloroplasts from mature, green leaves used for comparison, but also those from yellowing, aged leaves as well as etioplasts from dark-grown leaves raised at a temperature of 22° C. A majority of the plastidenvelope polypeptides appeared to be of cytoplasmic origin. The envelopes of ribosome-deficient plastids possessed ATPase (EC 3.6.1.3) activity; this was not, however, dependent on divalent cations, in contrast to the Mn²⁺- or Mg²⁺-dependent ATPase which is associated with chloroplast envelopes. Adenylate kinase (EC 2.7.4.3) was present in the stromal fraction of ribosome-deficient plastids and the stromal form of this enzyme is, therefore, of cytoplasmic origin. In contrast to previous findings, adenylate kinase was not, however, specifically associated with the chloroplast-envelope membranes, either in rye or in spinach. Measurements of the uptake of l-[¹⁴C]-malate into ribosome-deficient plastids indicated the presence and cytoplasmic origin of the dicarboxylate translocator. Malate uptake into rye etioplasts was, however, low. The phosphate translocator was assayed by the uptake of 3-phospho-[¹⁴C]glycerate. While rapid 3-phosphoglycerate uptake was observed for rye chloroplasts and etioplasts, it was hardly detectable for ribosome-deficient, plastids and rather low for chloroplasts from aged leaves. A polypeptide of M _r approx. 30000 ascribed to the phosphate translocator was greatly reduced in the envelope patterns of ribosome-deficient plastids and of chloroplasts from aged leaves.     

Changes in the adenylate energy charge and the induction of sporulation in Saccharomyces diastaticus

The induction of sporulation in yeast is generally accompanied by a sharp increase in energy metabolism which is evidenced by a rise of the adenylate energy charge by that time. The energy charge can be held at a low level by limitation of the phosphate supply in the growth medium. Ascus formation remains unaffected by this treatment. This suggests that the rise in ATP production normally encountered during early sporulation is not essential for the initiation of sporulation.     

On the compartmentation of isopentenyl diphosphate synthesis and utilization in plant cells

Purified spinach chloroplast and daffodil chromoplast preparations do not use mevalonate, phosphomevalonate, and diphosphomevalonate for the synthesis of isopentenyl diphosphate. Isopentenyl diphosphate, on the other hand, is incorporated into plastidal polyprenoids in large amounts. In the presence of a cytoplasmic supernatant, however, mevalonate and the phosphomevalonates were incorporated into the plastidal polyprenoids in equally large amounts, which demonstrates that the enzymes mevalonate kinase (EC 2.7.1.36), phosphomevalonate kinase (EC 2.7.4.2), and diphosphomevalonate decarboxylase (EC 4.1.1.33) are soluble cytoplasmic enzymes and that they apparently do not occur as isoenzymes within the plastids. The concept is developed that isopentenyl diphosphate is a central intermediate in plant polyprenoid formation which is channeled into several compartment for different biosynthetic pathways.Abbreviation IPP isopentenyl diphosphate - Chl_GG Chlorophyll a esterified with geranylgeraniol - HPLC high pressure liquid chromatography     

Effect of antisense repression of the chloroplast triose-phosphate translocator on photosynthetic metabolism in transgenic potato plants

The introduction of an antisense DNA into transgenic potato (Solanum tuberosum L.) plants decreased the expression of the chloroplast triose-phosphate translocator and lowered its activity by 20–30%. With plants propagated from tubers, the effect of the transformation on photosynthetic metabolism was analysed by measuring photosynthesis, the formation of leaf starch, and the total and subcellular metabolite contents in leaves. Although the transformants, in contrast to those propagated from cell cultures, did not differ from the wild-type plants in respect to rates of photosynthesis, plant appearance, growth and tuber production, their photosynthetic metabolism was found to be severely affected. The results show that the decrease in activity of the triose-phosphate translocator in the transformants caused a fourfold increase in the level of 3-phosphoglycerate and a corresponding decrease in inorganic phosphate in the stromal compartment, resulting in a large increase in the synthesis of starch. Whereas during a 12-h day period wild-type plants deposited 43% of their CO₂ assimilate into starch, this value rose to 61–89% in the transformants. In contrast to the wild-type plants, where the rate of assimilate export from the leaves during the night period was about 75% of that during the day, the export rate from leaves of transformants appeared to be much higher during the night than during the day. As the mobilisation of starch occurs in part hydrolytically, resulting in the formation of glucose, the triose-phosphate translocator loses its exclusive function in the export of carbohydrates from the chloroplasts when the photoassimilates are temporarily deposited as starch. It appears that by directing the CO₂ assimilates mainly into starch, the transformants compensate for the deficiency in triose-phosphate translocator activity in such a way that the productivity of the plants is not affected by the transformation.Abbreviations Chl chlorophyll - DHAP dihydroxyacetone phosphate - 3-PGA 3-phosphoglycerate - Rubisco ribulose,1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - trioseP triose phosphate - WT wild type The able technical assistance of Mrs. K. Wildenberger and Mrs. A. Großpietsch is gratefully acknowledged. This work has been supported by the Bundesminister für Forschung und Technologie.     

Molecular cloning and structural analysis of the phosphate translocator from pea chloroplasts and its comparison to the spinach phosphate translocator

Using an 5-AvaII fragment of the spinach (Spinacia oleracea L.) phosphate translocator cDNA as a probe for a hybridization screening of a pea (Pisum sativum L.) cDNA library we have cloned and sequenced a cDNA clone coding for the phosphate translocator precursor protein from pea chloroplasts. The full-length cDNA clone comprises 42 base pairs (bp) at the 5-non-coding region, a 1206-bp coding region corresponding to a polypeptide of 402 amino-acid residues (relative molecular mass 43 671) and 244 bp at the non-coding 3-region. Determination of the N-terminal sequence of the phosphate translocator from both pea and spinach chloroplasts revealed that the transit peptides consist of 72 and 80 amino-acid residues, respectively. These transit peptides are different from those of other chloroplastic transit peptides in that they both contain an amphiphilic -helix which is located either in close proximity to the processing site in pea or at the N-terminus in spinach. The mature proteins from pea and spinach both contain about 87% identical amino-acid residues and about seven putative membrane-spanning -helices. Some of these -helices have an amphiphilic character and might serve to form a hydrophilic translocation channel through the membrane. The in-vitro synthesized pea precursor protein is directed to the chloroplast and inserted into the chloroplast envelope membrane.Abbreviations bp base pairs - kDa kilodaltons - M_r relative moleculas mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We wish to thank Dr D. Pappin and R. Jakes (AFRC Sequencing Laboratory, Department of Biochemistry, University of Leeds, UK) for performing the N-terminal sequence determinations and are greatful to Dr J. S. Gantt (Botany Department, University of Georgia, Athens, USA) for a pea leaf cDNA library and to Professor J. C. Gray (University of Cambridge, Department of Botany, Cambridge, UK) for helpful discussions. This work was supported by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, the Science and Engineering Research Council and the Royal Society. D.L.W. was the recipient of the Royal Society Rosenheim research fellowship and K.F. was supported by a fellowship from the Studienstiftung des deutschen Volkes.     

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     

Isolation of adenylate cyclase mutants from Rhizobium meliloti deficient in nodulation

Six Rhizobium meliloti mutants were isolated after Tn5-mediated mutagenesis as resistant to inhibition by a mixture of amino acids (serine, methionine, glycine and leucine). All were defective in adenylate cyclase activity and failed to form nodules in infected roots of Medicago sativa. Furthermore, like other nodulation mutants, they showed altered motility and increased secretion of exopolysaccharides; addition of cAMP to the growth medium abolished some of these phenotypic defects. The possibility that adenylate cyclase participates in the transduction of signals inducing nodulation is discussed.     

Purification and properties of the adenylate kinases from Rhodopseudomonas palustris,Rhodopseudomonas sphaeroides and Rhodopseudomonas rubrum

The adenylate kinases (EC 2.7.4.3) from photosynthetically grown Rhodopseudomonas palustris, Rhodopseudomonas sphaeroides and Rhodospirillum rubrum were purified to homogeneity by the same procedure. The purified enzymes showed optimal rates of activity with MgCl₂ at 25° C and pH 8.0. They were found to be heat labile and were characterized by pI-values of 4.5. Apparent molecular weights of 33 500 for R. palustris, 34 400 for R. sphaeroides and 32 100 for R. rubrum were determined by high performance liquid chromatography. No separation into subunits was observed by use of sodium dodecylsulfate polyacrylamide gel electrophoresis. The apparent K _m -values for ADP corresponded to 0.26 mM for R. palustris, 0.27 mM for R. sphaeroides and 0.24 mM for R. rubrum. ADP in excess had a strong inhibitory effect. Competitive product inhibition was found for AMP, with K _i-values of 0.017 mM for R. palustris, 0.018 mM for R. sphaeroides and 0.014 mM for R. rubrum. A competitive inhibitor likewise was P¹,P⁵-di(adenosine-5)pentaphosphate with K _i-values of 0.020 M for R. palustris and R. sphaeroides, and 0.017 M for R. rubrum. Sulfhydryl-reacting reagents like p-chloromercuribenzoate and iodoacetic acid were found to be non-inhibitory. All measurements of adenylate kinase activity were carried out with the stabilized and most sensitive luciferin-luciferase system.     

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.     

Localization of adenylate cyclase activity in Populus: its relation to pollen-pistil recognition and incompatibility

Summary Adenylate cyclase has been localized cytochemically in female and male parents as well as during the pollen-stigma interaction with an original technique employing strontium as the capture ion and adenyl imidodiphosphate as the specific substrate. The specificity of the reaction was checked by using several controls. No final specific reaction product was detected in unpollinated P. deltoides stigmas or in the P. deltoides or P. alba pollen grains used for compatible and incompatible pollinations. In the compatible cross between P. deltoides × P. deltoides, fine dense precipitates were observed in the dictyosomes and the plasma membrane and exterior to the exine of hydrated pollen grains adhering to the stigma surface. Labeling of the stigmatic pellicle was also observed after pollen adhesion and hydration. This was accompanied by a strong reactivity of the cell wall and plasmalemma of the stigma papillae at the sites of pollen tube germination on the stigma surface and at the sites of penetration of pollen tubes between adjacent papillae. In the incompatible cross between P. deltoides x P. alba, adenylate cyclase activity was still present but reduced at the stigma surface following adhesion, hydration, and germination of P. alba pollen. This activity was completely abolished after the penetration of pollen tubes between stigma papillae. These findings suggest that in Populus, adenylate cyclase activity is correlated to pollen adhesion, hydration, and germination at the stigma surface, and that the abolition of this enzyme activity could be one of the cellular events governing the gametophytic phenotype of incompatibility in the cross between P. deltoides and P. alba.     

Fatty-acid synthesis in plastids from maturing safflower and linseed cotyledons

Plastids isolated from maturing, nongreen safflower (Carthamus tinctorius L.) cotyledons yielded unesterified fatty acids as the predominant product of fatty-acid synthesis from [1-¹⁴C]acetate. Exogenous reduced pyridine nucleotides were not required for this synthesis, but [1-¹⁴C]acetate incorporation was absolutely dependent on addition of ATP. Linseed (Linum usitatissimum L.) cotyledons are green during development and plastids isolated from them resembled leaf chloroplasts with developed grana. In contrast to the safflower plastids, those from linseed were able to carry out fatty-acid synthesis at low irradiances without the addition of either pyridine nucleotides or ATP. Intact linseed cotyledons were capable of net photosynthesis at rates up to 95 mol·mg^-1 chlorophyll·h^-1. However, the low-light environment inside the linseed capsule (approx. 15% of external) means that photosynthesis will not contribute appreciably to the carbon economy of the developing seed and its main role may be to supply cofactors for fatty-acid synthesis.Abbreviations ACP acyl carrier protein - DHAP dihydroxyacetone phosphate - PC phosphatidylcholine - PEP phosphoenolpyruvate - UFA unesterified fatty acids     

Structure, evolution and expression of the mitochondrial ADP/ATP translocator gene from Chlamydomonas reinhardtii

Summary The first AUG in the Chlamydomonas reinhardtii ADP/ATP translocator (CRANT) mRNA initiates an open reading frame (ORF) which is very similar (51–79% amino acid identity) to other ANT proteins. In contrast to higher plants, no evidence for a long amino-terminal extension was obtained. The 5 non-transcribed region of the single-copy CRANT gene contains sequence motifs present in other C. reinhardtii nuclear genes. Four introns, whose positions are not conserved in other ANT genes, interrupt the protein coding region. A short heat shock specifically reduces CRANT mRNA levels. CRANT mRNA levels were unaffected by a mutation in photosynthesis. In a dark/light regime CRANT mRNA levels are high in the dark phase and low in the early light phase. Data on translation initiation sites, splice junctions and the codon preferences of C. reinhardtii nuclear genes were compiled. With the exception of two rare codons, ACA and GGA, the CRANT gene exhibits the biased codon usage of C. reinhardtii nuclear genes that are highly expressed during normal vegetative growth.     

Effects of GTP,forskolin, sodium fluoride,serotonin, dopamine,and carbachol on adenylate cyclase inTeleost retina

Adenylate cyclase activity can be stimulated in goldfish retina by forskolin, GTP, NaF, dopamine and serotonin. Pharmacological characterisation of the dopamine and serotonin responses shows them to be mediated through specific receptors. A synergistic increase in the level of C-AMP is observed following application of forskolin together with NaF, GTP, dopamine, or serotonin. Dopamine and serotonin with or without GTP produced an additive response. When NaF and GTP are both together their combined effect in elevating C-AMP levels in the presence or absence of forskolin is less than additive. These results suggest that forskolin may be interacting with a G_s protein as well as directly stimulating adenylate cyclase. Increases in the level of C-AMP observed following application of forskolin or dopamine are decreased by carbachol in a dose-dependent manner. The carbachol response is blocked by pertussis toxin and is insensitive to the phosphodiesterase inhibitor, IBMX, suggesting an involvement of a G_i protein. Carbachol attenuation of elevated C-AMP levels is inhibited by atropine while pirenzapine has little effect suggesting the presence of a M2-type receptor.     

Transport of inorganic phosphate and C3- and C6-sugar phosphates across the envelope membranes of potato tuber amyloplasts

Amyloplasts have been isolated from tubers of potato plants (Solarium tuberosum. cv. Desirée). As it is difficult to isolate amyloplasts that have a high starch content, we used transformed plants in which the content of starch was reduced. This was achieved by decreasing the activity of ADP-glucose pyrophosphorylase by antisense techniques (Müller-Röber et al., 1992, EMBO. 11, 1229–1238). In the isolated plastids the activity of glutamine-oxoglutarate-aminotransferase (glutamate synthase, EC 2.6.1.53) was dependent upon the intactness of the plastids. For the supply of redox equivalents the addition of glucose-6-phosphate (Glc6P) was required. Glucose-1-phosphate (Glc1P) did not support glutamate synthesis. Plastids were treated with Triton X-100 and the solubilized proteins reconstituted into liposomes. Transport measurements with these liposomes revealed that inorganic phosphate (Pi), dihydroxyacetone phosphate (DHAP), 3-phosphoglycerate and Glc6P are transported in a counter-exchange mode. Transport of phosphoenolpyruvate was low and Glc1P was virtually not transported in exchange for Pi. Kinetic constants were determined for the Pi/Pi and Glc6P/Pi counter exchanges. For comparison, proteins of mitochondria from potato tubers and pea leaves were reconstituted into liposomes. As expected, the Pi/Pi exchange across the mitochondrial membrane was not affected by DHAP and Glc6P. Kinetic constants of the Pi/Pi counter exchange were determined for potato tuber mitochondria.Abbreviations DHAP dihydroxyacetone phosphate - Glc1P glucose-1-phosphate - Glc6P glucose-6-phosphate - PEP Phosphoenolpyruvate - 3-PGA 3-phosphoglycerate - Pi inorganic phosphate - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl] glycine This work was supported by Deutsche Forschungsgemeinschaft.