Adenine nucleotide translocase-dependent anion transport in pea chloroplasts

Pea chloroplasts were found to take up actively ATP and ADP and exchange the external nucleotides for internal ones. Using carrier-free [¹⁴C]ATP, the rate of nucleotide transport in chloroplasts prepared from 12–14-day-old plants was calculated to be 330 μmol ATP/g chlorophyll/min, and the transport was not affected by light or temperature between 4 and 22°C. Adenine nucleotide uptake was inhibited only slightly by carboxyatractylate, whereas bongkrekic acid was nearly as effective an inhibitor of the translocator in pea chloroplasts as it was in mammalian mitochondria. There was no counter-transport of adenine nucleotides with substrates carried on the phosphate translocator including inorganic phosphate, 3-phosphoglycerate and dihydroxyacetone phosphate. However, internal or external phosphoenolpyruvate, normally considered to be transported on the phosphate carrier in chloroplasts, was able to exchange readily with adenine nucleotides. Furthermore, inorganic pyrophosphate which is not transported by the phosphate carrier initiated efflux of phosphoenolpyruvate as well as ATP from the chloroplast. These findings illustrate some interesting similarities as well as differences between the various plant phosphate and nucleotide transport systems which may relate to their role in photosynthesis.     

Uptake of ATP analogs by isolated pea chloroplasts and their effect on CO2 fixation and electron transport.

1. The ATP analog, adenylyl-imidodiphosphate rapidly inhibited CO2-dependent oxygen evolution by isolated pea chloroplasts. Both alpha, beta- and beta, gamma-methylene adenosine triphosphate also inhibited oxygen evolution. The inhibition was relieved by ATP but only partially relieved by 3-phosphoglycerate. Oxygen evolution with 3-phosphoglycerate as substrate was inhibited by adenylyl-imidodiphosphate to a lesser extent than CO2-dependent oxygen evolution. The concentration of adenylylimidodiphosphate required for 50% inhibition of CO2-dependent oxygen evolution was 50 micronM. 2. Although non-cyclic photophosphorylation by broken chloroplasts was not significantly affected by adenylyl-imidodiphosphate, electron transport in the absence of ADP was inhibited by adenylyl-imidodiphosphate to the same extent as by ATP, suggesting binding of the ATP analog to the coupling factor of phosphorylation. 3. The endogenous adenine nucleotides of a chloroplast suspension were labelled by incubation with [14C]ATP and subsequent washing. Addition of adenylyl-imidodiphosphate to the labelled chloroplasts resulted in a rapid efflux of adenine nucleotides suggesting that the ATP analog was transported into the chloroplasts via the adenine nucleotide translocator. 4. It was concluded that uptake of ATP analogs in exchange for endogenous adenine nucleotides decreased the internal ATP concentration and thus inhibited CO2 fixation. Oxygen evolution was inhibited to a lesser extent in spinach chloroplasts which apparently have lower rates of adenine nucleotide transport than pea chloroplasts.     

Uptake of ATP analogs by isolated pea chloroplasts and their effect on CO2 fixation and electron transport

1. The ATP analog, adenylyl-imidodiphosphate rapidly inhibited CO₂-dependent oxygen evolution by isolated pea chloroplasts. Both α, β- and β, γ-methylene adenosine triphosphate also inhibited oxygen evolution. The inhibition was relieved by ATP but only partially relieved by 3-phosphoglycerate. Oxygen evolution with 3-phosphoglycerate as substrate was inhibited by adenylyl-imidodiphosphate to a lesser extent than CO₂-dependent oxygen evolution. The concentration of adenylyl-imidodiphosphate required for 50% inhibition of CO₂-dependent oxygen evolution was 50 μM.2. Although non-cyclic photophosphorylation by broken chloroplasts was not significantly affected by adenylyl-imidodiphosphate, electron transport in the absence of ADP was inhibited by adenylyl-imidodiphosphate to the same extent as by ATP, suggesting binding of the ATP analog to the coupling factor of phosphorylation.3. The endogenous adenine nucleotides of a chloroplast suspension were labelled by incubation with [¹⁴C]ATP and subsequent washing. Addition of adenylyl-imidodiphosphate to the labelled chloroplasts resulted in a rapid efflux of adenine nucleotides suggesting that the ATP analog was transported into the chloroplasts via the adenine nucleotide translocator.4. It was concluded that uptake of ATP analogs in exchange for endogenous adenine nucleotides decreased the internal ATP concentration and thus inhibited CO₂ fixation. Oxygen evolution was inhibited to a lesser extent in spinach chloroplasts which apparently have lower rates of adenine nucleotide transport than pea chloroplasts.     

Characterization of pyrophosphate exchange by the reconstituted adenine nucleotide translocator from mitochondria

The transport of inorganic pyrophosphate (PPi) by the adenine nucleotide translocator from beef heart mitochondria was studied in a reconstituted system. The transport of PPi is dependent on appropriate transmembrane substrates. The activity of PPi exchange is about one tenth as compared to the ADP/ATP exchange, whereas the transport affinity for PPi is very low (2-5 mM). The adenine nucleotide carrier catalyzes a strict counterexchange of PPi and nucleotides with an exchange stoichiometry close to 1. The inhibitor specificity of PPi exchange is comparable to that of ADP/ATP exchange.     

Transport of 3-phosphoglyceric acid,phosphoenolpyruvate, and inorganic phosphate in maize mesophyll chloroplasts,and the effect of 3-phosphoglyceric acid on malate and phosphoenolpyruvate production

Maize mesophyll chloroplasts loaded with radioactively labeled 3-phosphoglycerate or phosphoenolpyruvate exchange these compounds for externally provided inorganic phosphate, 3-phosphoglycerate, phosphoenolpyruvate, and dihydroxyacetone phosphate. These exchanges are inhibited by pyridoxal phosphate. 3-Phosphoglycerate uptake, which leads to accumulation of this substance in the stroma, is competitively inhibited by inorganic phosphate and phosphoenolpyruvate. These results are consistent with the transport of 3-phosphoglycerate, phosphoenolpyruvate, inorganic phosphate, and dihydroxyacetone phosphate being mediated by a common carrier (the phosphate translocator). The activation energy of 3-phosphoglycerate uptake as determined from its temperature dependence is 19.5 kcal (4–15 °C). In isolated chloroplasts malate and phosphoenolpyruvate production from oxalacetate and pyruvate, respectively, is inhibited by 3-phosphoglycerate, the extent of inhibition being dependent on the relative concentrations of inorganic phosphate and 3-phosphoglycerate. We propose that 3-phosphoglycerate from bundle-sheath cells may serve as a feedback regulator of mesophyll cell photosynthesis.     

Transport of AMP by Rickettsia prowazekii.

Rickettsia prowazekii possesses an exchange transport system for AMP. Chromatographic analysis of the rickettsiae demonstrated that transported AMP appeared intracellularly as AMP, ADP, and ATP, and no hydrolytic products appeared in either the intracellular or extracellular compartments. The phosphorylation of AMP to ADP and ATP was prevented by pretreatment of the cells with 1 mM N-ethylmaleimide without inhibiting the transport of AMP. Although no efflux was demonstrable in the absence of nucleotide in the medium, the intracellular adenine nucleotide pool could be exchanged with external unlabeled adenine nucleotides. Both ADP and ATP were as effective as AMP at inhibiting the uptake of [3H]AMP. Although this transport system was inhibited by low temperature (0 degrees C) and partially inhibited by the protonophore carbonyl cyanide-m-chlorophenyl hydrazone (1 mM), it was relatively insensitive to KCN (1 mM). The uptake of AMP at 34 degrees C had an apparent Kt for influx of 0.4 mM and a Vmax of 354 pmol min-1 per mg. At 0 degrees C there was a very rapid and unsaturable association of AMP with these organisms. Correction of the uptake data at 34 degrees C for the 0 degrees C component lowered the apparent Kt to 0.15 mM. Both magnesium and phosphate ions are required for optimal transport activity. Chemical measurements of the total intracellular nucleotide pools demonstrated that this system was not a net adenine nucleotide transport system, but that uptake of AMP was the result of an exchange with internal adenine nucleotides.     

Carboxyatractyloside-insensitive influx and efflux of adenine nucleotides in rat liver mitochondria

Unidirectional transport (influx and efflux) of adenine nucleotides in rat liver mitochondria was examined using carboxyatractyloside to inhibit rapid exchange of matrix and external adenine nucleotides via the adenine nucleotide translocase. Influx of adenine nucleotides was concentration-dependent. ATP was the preferred substrate with a Km of 2.67 mM and V of the preferred substrate with a Km of 2.67 mM and V of 8.33 nmol/min/mg of protein. For ADP, the Km was 14.7 mM and V was 10.8 nmol/min/mg of protein. Efflux of adenine nucleotides was also concentration-dependent, varying directly as a function of the matrix adenine nucleotide pool size. Any increase in the influx of adenine nucleotides was coupled to an increase in efflux. However, as the external ATP concentration was increased, influx was stimulated to a much greater extent than was efflux. This imbalance suggested that under certain conditions adenine nucleotide movement might be coupled to the movement of an alternate anion such as phosphate. Adenine nucleotide efflux increased as the external phosphate concentration was varied from 0.5 to 4 mM. Also, increasing the external phosphate concentration caused adenine nucleotide influx to decrease, suggesting competition. In the absence of external adenines and phosphate, no efflux occurred. Both adenine nucleotide influx and efflux were depressed if Mg2+ was omitted. Adenine nucleotide efflux in the presence of external phosphate was inhibited much less by lack of Mg2+ than was efflux in the presence of external ATP. This evidence supports a model in which either adenine nucleotides (probably with Mg2+) or phosphate can move across the mitochondrial membrane on a single carrier. Net adenine nucleotide movements can occur when adenine nucleotide movement is coupled to the movement of phosphate in the opposite direction.     

Pyrophosphate inhibition of carbon dioxide fixation in isolated pea chloroplasts by uptake in exchange for endogenous adenine nucleotides

Carbon dioxide-dependent O(2) evolution by isolated pea (Pisum sativum) chloroplasts was inhibited by inorganic pyrophosphate (PPi). Oxygen evolution was also inhibited by high concentrations of orthophosphate (Pi) and the inhibition was relieved by 3-phosphoglycerate. In contrast, the inhibition by PPi was not relieved by 3-phosphoglycerate, indicating that hydrolysis of PPi and accumulation of inhibitory concentrations of Pi were not occurring. In agreement with this suggestion, the percentage of (14)C-labeled products diffusing out of the chloroplasts was increased by Pi but not by PPi. The inhibition of O(2) evolution by PPi was reversed by ATP. The concentration of PPi required for 50% inhibition was 1.2 to 1.4 mm and the subsequent stimulation by ATP was half-maximal at 16 to 25 mum. Carbon dioxide-dependent O(2) evolution by spinach chloroplasts, or chloroplasts isolated from older pea plants, was not significantly inhibited by PPi.Chloroplasts were preloaded with (14)C-ATP and release of the labeled nucleotides was measured to assess the activity of adenine nucleotide transport across the inner chloroplast envelope membrane. A rapid exchange was promoted by the addition of exogenous ATP. Addition of PPi also resulted in a release of endogenous nucleotides. We suggest that PPi inhibits CO(2) fixation by entering the chloroplast in exchange for endogenous adenine nucleotides via the transporter on the inner envelope membrane. The subsequent depletion of the internal adenine nucleotide pool would result in decreased CO(2) fixation due to insufficient ATP. Addition of ATP to PPi-inhibited chloroplasts apparently results in uptake of catalytic amounts of ATP and restoration of the internal adenine nucleotide pool thus relieving the inhibition of CO(2) fixation.     

Relationship of phosphoenolpyruvate transport, acyl coenzyme A inhibition of adenine nucleotide translocase and calcium ion efflux in guinea pig heart mitochondria.

The transport of phosphoenolpyruvate by the adenine nucleotide translocase system of heart mitochondria may be directly involved in the mechanism of phosphoenolpyruvate-induced calcium ion efflux. In contrast to liver mitochondria, the transport of phosphoenolpyruvate via the tricarboxylate carrier system is low or absent in heart mitochondria. The translocation of phosphoenolpyruvate which catalyzed adenine nucleotide and calcium efflux from heart mitochondria was inhibited by palmitoyl-CoA as well as atractylate and ATP. These results suggest that phosphoenolpyruvate, which is preferentially transported on the tricarboxylate carrier of liver mitochondria, is transported primarily via the adenine nucleotide translocase system in heart mitochondria. As a result of its inward transport, phosphoenolpyruvate is able to catalyze calcium ion as well as adenine nucleotide efflux from the mitochondrial matrix. Although not yet proven, either or both phosphoenolpyruvate and long chain acyl-CoA esters may act as natural physiological effectors in the regulation and distribution of intracellular calcium.     

Transport in C4 mesophyll chloroplasts: evidence for an exchange of inorganic phosphate and phosphoenolpyruvate.

1. Mesophyll chloroplasts of the C4 plant Digitaria sanguinalis contain endogenous phosphoenolpyruvate which appears to distribute across the envelope according to the existing pH gradient. The phosphoenolpyruvate remaining in the stroma can be rapidly released by external inorganic phosphate or 3-phosphoglycerate while external pyruvate did not affect the distribution. 2. Phosphoenolpyruvate (PEP) was a competitive inhibitor (Ki (PEP) = 450 micrometer) of 32Pi uptake (Km(Pi)=200 micrometer) by chloroplasts in the dark and also reduced the steady-state internal concentration of 32Pi, which is consistent with phosphate and phosphoenolpyruvate sharing a common carrier. 3. Phosphoenolpyruvate formation by chloroplasts in the light in the presence of pyruvate but in the absence of inorganic phosphate was slow and the concentration ratio of phosphoenolpyruvate (internal/external) was high. Addition of 0.1 mM phosphate induced a high rate of phosphoenolpyruvate formation and the concentration ratio (internal/external) decreased 15-fold. It is proposed that external phosphate is required both for phosphoenolpyruvate formation and efflux from the chloroplast.     

Role of the intramitochondrial adenine nucleotides as intermediates in the uncoupler-induced hydrolysis of extramitochondrial ATP.

1. A formula is given that describes the appearance of [14C]ATPADP outside the mitochondria after the addition of [14C] 1atp during the steady-state uncoupler-induced hydrolysis of extramitochondrial ATP. If the transported adenine nucleotides equilibrate with the intramitochondrial pool, [14C]ADP0 would be expected to appear with a lag phase that corresponds with the time needed for the radioactive labelling of the intramitochondrial adenine nucleotide pool. 2. The rates of formation of [14C]ADP outside the mitochondria after addition of [14C]ATP during the steady-state uncoupler-induced ATP hydrolysis catalysed by rat-liver mitochondria at 0 degree C were measured. 3. In the presence of carbonyl cyanide m-chlorophenylhydrazone the time course of the [14]ADPo formation was the same as that predicted on the basis of the above assumption. 4. In the presence of the less effective uncoupler, 2,4-dinitrophenol, the time course of [14C]ADPo formation was not consistent with the theoretical predictions: no lag phase was present and the measured rate was higher than the maximal calculated rate. These results can be explained by assuming a functional interaction between the adenine nucleotide translocator and the mitochondrial ATPase (F1). 5. It is concluded that under phosphorylating as well as dephosphorylating conditions, the adenine nucleotide translocator and the mitochondrial ATPase can be functionally linked to catalyse phosphorylation or dephosphorylation of extramitochondrial ADP or ATP, without participation of the intramitochondrial adenine nucleotides.     

Stimulation of carbon dioxide fixation in isolated pea chloroplasts by catalytic amounts of adenine nucleotides

Carbon dioxide-dependent O(2) evolution by isolated pea (Pisum sativum var. Massey Gem) chloroplasts was increased two to 12 times by the addition of ATP. O(2) evolution was also stimulated by ADP and to a lesser extent by AMP. The ATP effects were not due to broken chloroplasts present in the preparations nor was ATP acting as a phosphate source. We concluded that the adenine nucleotides were acting catalytically. The concentration of ATP required for half-maximum rate of O(2) evolution was 16 to 25 mum. The degree to which ATP stimulated O(2) evolution depended on the age of pea plants from which the chloroplasts were isolated. Spinach (Spinacia oleracea var. True Hybrid 102) chloroplasts did not show a consistent stimulation of O(2) evolution by adenine nucleotides.The adenine nucleotide content of pea chloroplasts was not lower than that of spinach chloroplasts, but pea chloroplasts which showed a large stimulation of O(2) evolution by ATP contained an ATP-hydrolyzing reaction with rates of 10 to 50 mumol ATP hydrolyzed mg chlorophyll(-1) hour(-1). The rate of the ATP-consuming reaction was much lower in spinach chloroplasts and in chloroplasts from older pea plants which did not show large stimulation of O(2) evolution by ATP. We propose that the ATP-consuming reaction, with a high affinity for ATP, decreased the effective size of the ATP pool available for CO(2) fixation. Added adenine nucleotides could be transported into the chloroplasts increasing the concentration of internal nucleotides. Calculations showed that the adenine nucleotide transporter on the outer chloroplast membranes could operate at a sufficient rate to produce such an effect.     

An adenine nucleotide-phosphoenolpyruvate counter-transport system in C3 and C4 plant chloroplasts

A rapid counter-exchange between ATP and phosphoenolpyruvate (PEP) has been demonstrated in pea and maize mesophyll chloroplasts. Chloroplasts preloaded with either [14C] ATP or [14C] PEP readily exchange the radioactive compound with the externally added anions, ATP or PEP, whereas, cold external Pi counter-transports only with internal [14C] PEP. Flooding the system with cold Pi, however, will significantly reduce the counter-transport of external cold PEP with internal [14C] ATP. This ATP-PEP exchange is also markedly decreased by lowering the incubation temperature. The results indicate that the ATP-PEP counter-exchange could represent a key transport system in plant chloroplasts and may be particularly important in the photosynthesis of C4 plants. Furthermore, they provide information required to elucidate the mechanism of the ATP-PEP counter-transport system.     

Nucleotide transport in Rhodobacter capsulatus.

Cytoplasmic membrane vesicles isolated from the gram-negative photosynthetic bacterium Rhodobacter capsulatus catalyzed the transport of nucleotides. No transport occurred in the intact bacteria unless they were pretreated with EDTA. The transport rate was measured by incorporation of radioactive phosphate into externally added ADP or by incorporation of nonradioactive phosphate into added labeled ADP. The catalytic activities which utilized the added ADP were photosynthetic ATP synthesis, Pi-ADP exchange, and adenylate kinase. These activities were shown to occur on the cytoplasmic side of the internal membrane. The products were found in the outer medium. The rate of nucleotide transport across the membranes was comparable to the rate of photophosphorylation. These results indicated that nucleotides can be transported across the cytoplasmic membrane but not across the outer membrane of the native R. capsulatus cell. Therefore, by analogy to the mitochondrial ATP-ADP translocator, the exchange might function as an energy transfer system to the periplasm of these bacteria.     

ATP-Mg/Pi carrier activity in rat liver mitochondria.

The ATP-Mg/Pi carrier in liver mitochondria is activated by micromolar Ca2+ and mediates net adenine nucleotide transport into and out of the mitochondrial matrix. The purpose of this study was to characterize certain features of ATP-Mg/Pi carrier activity that are essential for understanding how the mitochondrial adenine nucleotide content is regulated. The relative importance of ATP and ADP as transport substrates was investigated using specific trap assays to measure their separate rates of carrier-mediated efflux with Pi as the external counterion. Under energized conditions ATP efflux accounted for 88% of total ATP+ADP efflux. With oligomycin present to lower the matrix ATP/ADP ratio, ATP efflux was eliminated and ADP efflux was relatively unaffected. Mg2+ was stoichiometrically required for ATP influx and is probably transported simultaneously with ATP. Ca2+ and Mn2+ could substitute for the stoichiometric Mg2+ requirement. ADP influx and Pi-induced adenine nucleotide efflux were unaffected by external Mg2+. Experiments with Pi analogues suggested that Pi is transported as the divalent anion, HPO4(2-). The results show that ATP-Mg and divalent Pi are the major transport substrates; the most probable transport mechanism for the ATP-Mg/Pi carrier is an electroneutral exchange. The results are consistent with the hypothesis that the direction and magnitude of net adenine nucleotide movements are determined mainly by the (ATP-Mg)2- and HPO4(2-) concentration gradients across the inner mitochondrial membrane.     

Phosphate translocator and adenylate translocator in chromoplast membranes

It is shown by the criteria of saturation kinetics, specificity, and inhibition experiments that chromoplast membranes from the daffodil flower contain a phosphate translocator for the counter-exchange of phosphate, and 3-phosphoglycerate, as well as phosphoenolpyruvate; they also contain an adenylate translocator. This is the first report on the occurrence of these translocators in non-green plastids. Both translocators exhibit certain dissimilar properties when compared to the corresponding systems of chloroplasts. The transport rates of both translocators are sufficient to allow a prominent fatty acid synthesis in isolated chromoplasts when C₃ intermediates of the glycolytic pathway or adenine nucleotides are used as energy sources.     

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.     

Transport in C4 mesophyll chloroplasts. Evidence for an exchange of inorganic phosphate and phosphoenolpyruvate

1. Mesophyll chloroplasts of the C₄ plant Digitaria sanguinalis contain endogenous phosphoenolpyruvate which appears to distribute across the envelope according to the existing pH gradient. The phosphoenolpyruvate remaining in the stroma can be rapidly released by external inorganic phosphate or 3-phosphoglycerate while external pyruvate did not affect the distribution.

2. Phosphoenolpyruvate (PEP) was a competitive inhibitor (K_i(PEP) = 450 μM) of ³²P_i uptake (K_m(P_i) = 200 μM) by chloroplasts in the dark and also reduced the steady-state internal concentration of ³²P_i, which is consistent with phosphate and phosphoenolpyruvate sharing a common carrier.

3. Phosphoenolpyruvate formation by chloroplasts in the light in the presence of pyruvate but in the absence of inorganic phosphate was slow and the concentration ratio of phosphoenolpyruvate (internal/external) was high. Addition of 0.1 mM phosphate induced a high rate of phosphoenolpyruvate formation and the concentration ratio (internal/external) decreased 15-fold. It is proposed that external phosphate is required both for phosphoenolpyruvate formation and efflux from the chloroplast.     

Inhibition of phosphoenolpyruvate transport via the tricarboxylate and adenine nucleotide carrier systems of rat liver mitochondria

The translocation of phosphoenolpyruvate by the tricarboxylate carrier system in rat liver mitochondria was shown to be inhibited by atractyloside and long chain fatty acyl CoA esters as well as benzene, 1, 2, 3 tricarboxylate. By contrast benzene 1, 2, 3 tricarboxylate did not inhibit atractyloside sensitive adenine nucleotide translocation catalyzed by phosphoenolpyruvate. These results indicate that although phosphoenoppyruvate is preferentially transported by the tricarboxylate carrier system, it may also be transported by the adenine nucleotide translocase. The inhibition of the adenine nucleotide and tricarboxylate carrier systems by atractyloside and long chain acyl CoA esters indicates a close functional interrelation-ship of these transport carriers in the inner mitochondrial membrane. Moreover, the potent inhibition of phosphoenolpyruvate, citrate, and adenine nucleotide transport by long chain acyl CoA's provides further evidence that these esters are natural effectors which participate in the regulation of gluconeogenesis, lipogenesis, and energy-linked respiration.     

Stimulation of adenine nucleotide translocation in reconstituted vesicles by phosphate and the phosphate transporter

Highly purified adenine nucleotide transporter from bovine heart mitochondria was reconstituted with phospholipids to form vesicles which catalyzed atractyloside-sensitive adenine nucleotide translocation. When internal ATP was exchanged with external ADP, this reaction was enhanced by agents capable of collapsing a membrane potential, but not by inorganic phosphate. When the purified nucleotide transporter was reconstituted together with a second protein fraction, nucleotide transport was stimulated by inorganic phosphate. The stimulated rate was eliminated by mersalyl or other SH reagents. The second protein fraction could be replaced by preparations of purified phosphate transporter.