Mitochondrial transport in proline catabolism in plants: the existence of two separate translocators in mitochondria isolated from durum wheat seedlings

Abiotic stresses, such as high salinity or drought, can cause proline accumulation in plants. Such an accumulation involves proline transport into mitochondria where proline catabolism occurs. By using durum wheat seedlings as a plant model system, we investigated how proline enters isolated coupled mitochondria. The occurrence of two separate translocators for proline, namely a carrier solely for proline and a proline/glutamate antiporter, is shown in a functional study in which we found the following: (1) Mitochondria undergo passive swelling in isotonic proline solutions in a stereospecific manner. (2) Externally added l-proline (Pro) generates a mitochondrial membrane potential (ΔΨ) with a rate depending on the transport of Pro across the mitochondrial inner membrane. (3) The dependence of the rate of generation of ΔΨ on increasing Pro concentrations exhibits hyperbolic kinetics. Proline transport is inhibited in a competitive manner by the non-penetrant thiol reagent mersalyl, but it is insensitive to the penetrant thiol reagent N-ethylmaleimide (NEM). (4) No accumulation of proline occurs inside the mitochondria as a result of the addition of proline externally, whereas the content of glutamate increases both in mitochondria and in the extramitochondrial phase. (5) Glutamate efflux from mitochondria occurs at a rate which depends on the mitochondrial transport, and it is inhibited in a non-competitive manner by NEM. The dependence of the rate of glutamate efflux on increasing proline concentration shows saturation kinetics. The physiological role of carrier-mediated transport in the regulation of proline catabolism, as well as the possible occurrence of a proline/glutamate shuttle in durum wheat seedlings mitochondria, are discussed.Catello Di Martino, Roberto Pizzuto these authors contributed equally to the paper     

Proline transport by tsetse fly Glossina morsitans flight muscle mitochondria.

1. Proline accumulation by tsetse fly Glossina morsitans flight muscle mitochondria was studied in vitro by the swelling technique and direct measurement of (U-14C) proline. 2. Proline transport was inhibited by the uncharged liposoluble -SH reagent, N-ethylmaleimide but not by ionic reagent, mersalyl, suggesting that the -SH groups involved in the transport of proline are located in a hydrophobic part of the membrane or on the matrix side of the membrane. 3. The kinetic study of proline accumulation revealed saturation kinetics and a high temperature dependence. It gave a Km of 85 microM and a Vmax of 962 pmol/min/mg protein and an activation energy (Ea) of 11 kcal/mol. 4. Certain other amino acids (L-valine, L-alanine, L-methionine, L-phenylalanine, L-tryptophan and L-hydroxyproline) significantly stimulated proline uptake. 5. These observations indicate that tsetse fly Glossina morsitans flight muscle mitochondria contain a proline transport mechanism.     

Glycine transport in rat brain and liver mitochondria

Transport of glycine by rat brain and liver mitochondria has been investigated by both [¹⁴C]glycine uptake and swelling experiments. Glycine enters mitochondria passively down its concentration gradient by a respiratory-independent carrier-mediated process. This view is supported by the following observations: (a) glycine inside the mitochondria reaches the incubation medium concentration; (b) mitochondria swell in the presence of isoosmotic solutions of glycine in a concentration-dependent fashion; (c) the uptake of glycine is not influenced by respiratory inhibitors such as KCN or by uncouplers such as carbonylcyanide p-trifluoromethoxyphenylhydrazone; (d) initial rates of uptake approach saturation kinetics, the apparent K_m of the rat brain mitochondria for glycine being 1.7 mM and that of the liver mitochondria being 5.7 mM; (e) the rate of swelling is inhibited by methylmalonate, propionate and, at pH 6.5, by mersalyl, and (f) uptake is inhibited by phosphoserine, methylmalonate and propionate, but not by alanine or proline.     

Special features of Pi transport in pig heart and rat liver mitochondria as revealed by 6,6'-dithiodinicotinic acid (CPDS).

CPDS (6,6'-dithiodinicotinic acid), a non permeant thiol agent which affects several mitochondrial functions in a way different to that of mersalyl [18-19] revealed striking differences between the phosphate translocating systems of pig heart and rat liver mitochondria. Pi entry was measured either by swelling in 0.12 M ammonium phosphate or by rapid centrifugation in 32Pi medium. Pi efflux was measured after preloading of mitochondria with 32Pi, by exchange against Pi or malate; the "ATP-FCCP" system has been tested previously [19]. In pig heart mitochondria, Pi entry seems to proceed exclusively via the Pi/OH- carrier; CPDS completely inhibits this transport and the energy-linked functions. In contrast n-butyl-malonate does not affect the Pi-entry and the energy-linked functions. The Pi efflux is not affected either by CPDS or mersalyl, which do not produce a swelling in the "ATP-uncoupler system". In rat liver mitochondria, CPDS inhibits only the Pi/OH- carrier; both CPDS and n-butylmalonate are necessary to inhibit completely Pi entry. CPDS as well as mersalyl provokes a swelling in the presence of the "APT-uncoupler system". The results suggest two distinct functions of phosphate transport in both types of mitochondria.     

Nonspecific lipid transfer protein (sterol carrier protein 2) is bound to rat liver mitochondria: its role in spontaneous intermembrane phospholipid transfer

In the present study we have investigated the transfer of phospholipids between vesicles and rat liver mitochondria. Transfer was measured by electron paramagnetic resonance spectroscopy using vesicles that contained spin-labeled phospholipids. A spontaneous transfer was observed which could be strongly inhibited by treating the mitochondria with the thiol reagent mersalyl. Transfer was also greatly reduced after a saline wash of the mitochondria; the transfer activity was then recovered in the wash. This activity was inhibited by tryptic digestion and mersalyl. By gel chromatography, enzyme immunoassay and immunoblotting it was demonstrated that the activity in the wash was due to the nonspecific lipid transfer protein (sterol carrier protein 2). We could estimate that up to 85% of the spontaneous phospholipid transfer between vesicles and rat liver mitochondria was mediated by this transfer protein.     

Phosphate transport in mitochondria and submitochondrial particles: The influence of thiol oxidation

Diamide, a thiol oxidizing agent, partially inhibited P _i uptake by rat liver mitochondria. The inhibition was temperature dependent; at 20°C, the optimal temperature for maximum inhibitory effect, diamide also reduced the minimal amount of mersalyl required for the inhibition of P _i transport. Under the same conditions no inhibitory effect on P _i efflux was observed. The amount of mitochondrial thiol groups titrated by the amounts of diamide needed for the inhibition of P _i uptake was on the order of 5 nmole/mg protein. Unlike liver mitochondria, the P _i transport system of heart mitochondria was insensitive to diamide. On the contrary, accumulation of P _i into submitochondrial heart vesicles, previously loaded with MnCl₂, was inhibited by diamide. These results outline the different positional character of membrane thiol groups of mitochondria from various sources, and provide further evidence of an asymmetric orientation of the P _i transport system in mitochondrial membranes.     

Parallel efflux of Ca2+ and Pi in energized rat liver mitochondria.

Addition of Ruthenium Red to energized rat liver mitochondria that have previously accumulated Ca2+ and phosphate from the external medium induces a parallel efflux of both these ions. Mersalyl or dithioerythritol, which decrease Ruthenium Red-insensitive Ca2+ efflux, also decrease phosphate efflux to the same extent. Conversely diazenedicarboxylic acid bis(NN-dimethylamide) (DDBA), which increases the Ruthenium Red-induced Ca2+ efflux concurrently increases phosphate release. Dithioerythritol and DDBA, reducing and oxidizing agents of thiol groups respectively, modify Ca2+ and Pi efflux without penetrating the mitochondrial inner membrane. Under all the adopted conditions the membrane potential is preserved. The release of resting respiration and the parallel efflux of Mg2+ and adenine nucleotides, events closely correlated to Ca2+ cycling, are equally prevented either by mersalyl, which inhibits phosphate transport, or dithioerythritol; DDBA has the opposite effect. These findings and the observation that suggest that Ca2+ and phosphate transport in energized liver mitochondria are closely related and dependent on the redox state of membrane-bound thiol groups.     

Ca2+ Induces a Cyclosporin A-Insensitive Permeability Transition Pore in Isolated Potato Tuber Mitochondria Mediated by Reactive Oxygen Species

Oxidative damage of mammalian mitochondria induced by Ca²⁺ and prooxidants is mediated by the attack of mitochondria-generated reactive oxygen species on membrane protein thiols promoting oxidation and cross-linkage that leads to the opening of the mitochondrial permeability transition pore (Castilho et al., 1995). In this study, we present evidence that deenergized potato tuber (Solanum tuberosum) mitochondria, which do not possess a Ca²⁺ uniport, undergo inner membrane permeabilization when treated with Ca²⁺ (>0.2 mM), as indicated by mitochondrial swelling. Similar to rat liver mitochondria, this permeabilization is enhanced by diamide, a thiol oxidant that creates a condition of oxidative stress by oxidizing pyridine nucleotides. This is inhibited by the antioxidants catalase and dithiothreitol. Potato mitochondrial membrane permeabilization is not inhibited by ADP, cyclosporin A, and ruthenium red, and is partially inhibited by Mg²⁺ and acidic pH, well known inhibitors of the mammalian mitochondrial permeability transition. The lack of inhibition of potato mitochondrial permeabilization by cyclosporin A is in contrast to the inhibition of the peptidylprolyl cis–trans isomerase activity, that is related to the cyclosporin A-binding protein cyclophilin. Interestingly, the monofunctional thiol reagent mersalyl induces an extensive cyclosporin A-insensitive potato mitochondrial swelling, even in the presence of lower Ca²⁺ concentrations (>0.01 mM). In conclusion, we have identified a cyclosporin A-insensitive permeability transition pore in isolated potato mitochondria that is induced by reactive oxygen species.     

The K+ conductance of the inner mitochondrial membrane. A study of the inducible uniport for monovalent cations

Addition of A23187 plus EDTA to rat liver mitochondria induces a common uniport pathway for monovalent cations. In this study, we have carried out a detailed characterization of the flow/force relationship for K+ transport along this pathway under steady state conditions. In the presence of EDTA, the K+ conductance is a linear function of external K+ in the range 0-20 mM K+, with a slope of 0.15 nmol of K+ x mg of protein-1 x min-1 x mV-1. The K+ conductance is inhibited by Mg2+ in the range 10(-9)-10(-6) M, while K+ flux is stimulated by the sulfhydryl group reagent mersalyl. Uniport activity can be detected in native mitochondria. These findings are compatible with the notion that electrophoretic K+ flux across the inner membrane takes place via a regulated K+ uniport with the potential of transporting K+ at rates in excess of 600 nmol x mg of protein-1 x min-1.     

Carrier Protein-mediated Transport of Neutral Amino Acids into Mung Bean Mitochondria

The transport of neutral amino acids into mitochondria isolated from the hypocotyl of mung bean (Roxb.) was studied by the swelling technique. Isolated mitochondria swelled when added to an isosmotic solution of proline, serine, methionine, threonine, alanine, and glycine. The swelling was stereospecific in that it was faster in the l-amino acid than in the corresponding d-amino acid. Preincubation of the mitochondria with the sulfhydryl modifying reagents, p-mercuribenzoate and mersalyl, resulted in an inhibition of the swelling caused by proline, serine, threonine, and glycine. The swelling induced by alanine was inhibited only by mersalyl, whereas that by methionine was inhibited only by p-mercuribenzoate. In all cases, the inhibition caused by the sulfhydryl modifying reagents was readily reversible by the subsequent treatment of the mitochondria with dithiothreitol. N-Ethylmaleimide, another sulfhydryl-modifying reagent, did not cause any inhibition of the swelling. The findings indicate the existence of a protein mediated mechanism for the transport of neutral amino acids into plant mitochondria.     

Effect of verapamil and sulfhydryl reagents on calcium transport in bovine spermatozoa

Calcium uptake by intact bovine epididymal spermatozoa is not affected by low concentrations (up to 0.75 mM) of the calcium transport blocker verapamil. Under these conditions, calcium transport into sperm mitochondria is highly inhibited. At higher verapamil concentrations (1.0, 1.5 mM), calcium transport into intact sperm is also inhibited, and this inhibition cannot be relieved by disrupting the plasma membrane with filipin. Calcium uptake into intact sperm is highly inhibited by mersalyl and this inhibitory effect can be completely relieved when the plasma membrane is disrupted by filipin. This effect of mersalyl is not dependent on the presence of phosphate in the incubation medium. Phosphate itself, up to 2 mM, enhances calcium uptake into the cells; this effect decreases at higher concentrations and is depressed 57% at 10 mM phosphate. This inhibitory effect of high phosphate concentration can be blocked by mersalyl. It is suggested that the calcium carrier itself and not a phosphate carrier of the plasma membrane is inhibited by mersalyl. It is possible that there is a symporter for calcium and phosphate in the plasma membrane of bovine spermatozoa.     

Carrier-mediated transport of metabolites in purified bean mitochondria

1. The mechanism of transport of Krebs cycle intermediates, phosphateand sulfurcontaining compounds across the membrane of purifiedbean mitochondria was investigated by directly measuring dieexchange between intramitochondrial labelled substrates andexternal anions and by testing die inhibitor sensitivity ofdiese transport processes.
2. The exchange between intramitochondrialphosphate and externalphosphate or sulfite is insensitive toN-ediylmaleimide or butylmalonatewhen either is added alone,but is completely inhibited by N-ethylmaleimideplus butylmalonateor by mersalyl. Internal phosphate is exchangedwith malate,succinate, oxaloacetate, sulfate and thiosulfate;these reactionsare inhibited by butylmalonate but not affectedby N-ethylmaleimide.
3. Internal sulfate is exchanged with malate, malonate, succinate,phosphate and sulfite in a butylmalonate- and mersalyl-sensitivereaction. Also the exchanges of malonate with phosphate, sulfateand sulfite are inhibited by butylmalonate and mersalyl. Onthe other hand, the exchange between intra- and extramitochondrialmalonate is completely inhibited only by the combination ofbutylmalonate and 1,2,3-benzenetricarboxylate.
4. Citrate isexchanged with some di- and tricarboxylates and phosphoenolpyruvate(but not with phosphate, sulfate, oxoglutarate, trans-aconitateand benzenetricarboxylates). These exchanges are inhibited by1,2,3-benzenetricarboxylate, but not by 1,2,4-benzenetricarboxylateor 1,3,5-pentanetricarboxylate.
5. Oxoglutarate is exchangedwith succinate, malate, malonate andoxaloacetate (but not withphosphate, citrate or phosphoenolpyruvate)in a mersalyl-insensitive,butylmalonate- and phenylsuccinate-sensitivereaction.
6. Weconcluded that bean mitochondria contain the following transportsystems: a phosphate carrier inhibited by N-ethylmaleimide ormersalyl, a dicarboxylate carrier inhibited by butylmalonateor mersalyl, a citrate carrier inhibited by 1,2,3-benzenetricarboxylateand an oxoglutarate carrier inhibited by phenylsuccinate orbutylmalonate but insensitive to mersalyl.

(Received June 23, 1976; )     

Importance of the flux of phosphate across the inner membrane of kidney mitochondria for the activation of glutaminase and the transport of glutamine

The effect of mersalyl, an inhibitor of phosphate transport across the inner mitochondrial membrane, was investigated on the uncoupled respiration of pig kidney mitochondria in the presence of glutamine as substrate and on the activity of the phosphate-dependent glutaminase in the intact organelles. In addition, the submitochondrial location of the enzyme was reinvestigated.

1. (1) It was found that mersalyl completely inhibits uncoupled respiration of the mitochondria in the presence of glutamine as substrate, whereas respiration with glutamate was not affected. The same amount of mersalyl which inhibits coupled oxidation of glutamine also inhibits coupled oxidation of glutamate and some other substrates.

2. (2) Mersalyl strongly inhibited the activation of glutaminase in intact mitochondria only in the presence of inhibitors of electron transport or of an uncoupler. The addition of a detergent prevented or fully released the inhibition. The effect of mersalyl was observed even when the mitochondria were pre-incubated with phosphate or incubated in the phosphate-free medium. If mersalyl and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were added 3 min after pre-incubation with phosphate the same intramitochondrial concentration of the anion as in control experiments was found, whereas the activity of glutaminase was severely inhibited. These findings suggest that the activation of the enzyme by phosphate in intact nonenergized mitochondria occurs only if the activator moves across the inner mitochondrial membrane.

3. (3) Mersalyl (plus CCCP) markedly decreased [¹⁴C]glutamine- and [³²P]-phosphate-permeable mitochondrial spaces. A close correlation between the decrease of phosphate and glutamine permeable spaces and the inhibition of glutaminase activity was found.

4. (4) If the activation energy of the enzyme was determined with frozen mitochondrial preparations, a discontinuity or break in the Arrhenius plot was observed, whereas the presence of a detergent completely abolished the break. Digitonin or ultrasonic treatment of the mitochondria followed by separation of the membrane and the soluble fraction revealed that glutaminase is a membrane-bound enzyme.

On the basis of these findings it is concluded that there is an association between the transport of phosphate on one side and the transport of glutamine and glutaminase activity on the other. It is possible that the movement of phosphate across the membrane activates the enzyme which facilitates diffusion of glutamine down a concentration gradient. However, the existence of a specific glutamine-phosphate carrier is not ruled out.     

A cyanine dye tri-S-C7(5). Phosphate-dependent cationic uncoupler of oxidative phosphorylation in mitochondria

The trinuclear cyanine dye, tri-S-C7(5), at about 10 microM stimulated State 4 respiration of rat liver mitochondria more than 6-fold and released oligomycin-inhibited respiration completely. Thus, the dye is concluded to be a very effective cationic uncoupler of oxidative phosphorylation in mitochondria. However, for exhibition of its uncoupling action, the presence of Pi (or arsenate) was necessary, and a phosphate-transport inhibitor, N-ethylmaleimide or mersalyl, inhibited its action. The stimulation of phosphate transport via the Pi carrier by the dye is suggested to be directly related to the uncoupling action.     

The transport of sulphate and sulphite in rat liver mitochondria

1. The mechanism of sulphite and sulphate permeation into rat liver mitochondria was investigated. 2. Extramitochondrial sulphite and sulphate elicit efflux of intramitochondrial phosphate, malate, succinate and malonate. The sulphate-dependent effluxes and the sulphite-dependent efflux of dicarboxylate anions are inhibited by butylmalonate, phenylsuccinate and mersalyl. Inhibition of the phosphate efflux produced by sulphite is caused by mersalyl alone and by N-ethylmaleimide and butylmalonate when present together. 3. External sulphite and sulphate cause efflux of intramitochondrial sulphate, and this is inhibited by butylmalonate, phenylsuccinate and mersalyl. 4. External sulphite and sulphate do not cause efflux of oxoglutarate or citrate. 5. Mitochondria swell when suspended in an iso-osmotic solution of ammonium sulphite; this is not inhibited by N-ethylmaleimide or mersalyl. 6. Low concentrations of sulphite, but not sulphate, produce mitochondrial swelling in iso-osmotic solutions of ammonium malate, succinate, malonate, sulphate, or phosphate in the presence of N-ethylmaleimide. 7. It is concluded that both sulphite and sulphate may be transported by the dicarboxylate carrier of rat liver mitochondria and also that sulphite may permeate by an additional mechanism; the latter may involve the permeation of sulphurous acid or SO(2) or an exchange of the sulphite anion for hydroxyl ion(s).     

Comparison of the effects of certain thiol reagents on alanine transport in plasma membrane vesicles from rat liver and their use in identifying the alanine carrier.

The Na+-dependent uptake of alanine into plasma membrane vesicles from rat liver was inhibited by N-ethylmaleimide (NEM) and by mersalyl. NEM did not inhibit alanine-independent Na+ uptake and the inhibition of alanine transport by NEM was protected by pre-incubation with an excess of substrate. It was therefore concluded that NEM acted by binding to the alanine carrier. A protein of Mr 20 000 was found to bind NEM with a concentration dependence parallel to the NEM inhibition of alanine transport. The inhibition of binding of [3H]NEM to this protein by mersalyl had a concentration dependence similar to that of the inhibition of transport by mersalyl. Preincubation with L-alanine, but not with D-alanine, led to protection of the Mr 20 000 protein from binding NEM. It is concluded that this protein is an essential component of the alanine transport system.     

A cyanine dye tri-S-C7(5). Phosphate-dependent cationic uncoupler of oxidative phosphorylation in mitochondria

The trinuclear cyanine dye, tri-S-C₇(5), at about 10 μM stimulated State 4 respiration of rat liver mitochondria more than 6-fold and released oligomycin-inhibited respiration completely. Thus, the dye is concluded to be a very effective cationic uncoupler of oxidative phosphorylation in mitochondria. However, for exhibition of its uncoupling action, the presence of P_i (or arsenate) was necessary, and a phosphate-transport inhibitor, N-ethylmaleimide or mersalyl, inhibited its action. The stimulation of phosphate transport via the P_i carrier by the dye is suggested to be directly related to the uncoupling action.     

Evidence of a phosphate-transporter system in the inner membrane of isolated mitochondria

1. The organic mercurial sodium mersalyl, formaldehyde, dicyclohexylcarbodiimide and tributyltin each blocked respiratory-chain-linked ATP synthesis in rat liver mitochondria. 2. Mersalyl and formaldehyde also blocked a number of other processes dependent on the entry of inorganic phosphate into mitochondria, including mitochondrial respiration and swelling stimulated by cations and phosphate, the substrate-level phosphorylation reaction of the citric acid cycle, and swelling in ammonium phosphate. 3. Dicyclohexylcarbodi-imide and tributyltin did not inhibit the entry of phosphate into mitochondria. 4. Mersalyl and formaldehyde had a relatively slight effect on succinate oxidation and swelling stimulated by cations when phosphate was replaced by acetate, on succinate oxidation stimulated by uncoupling agents, and on swelling in solutions of ammonium salts other than phosphate or arsenate. 5. Formaldehyde blocked the oxidation of NAD-linked substrates in mitochondria treated with 2,4-dinitrophenol and the ATP-dependent reduction of NAD by succinate catalysed by ox heart submitochondrial particles. Both these effects appear to be due to an inhibition by formaldehyde of the NAD-flavin region of the respiratory chain. 6. Concentrations of dicyclohexylcarbodiimide or tributyltin sufficient to abolish ADP-stimulated respiration blocked the dinitrophenol-stimulated adenosine triphosphatase activity, whereas mersalyl and formaldehyde caused only partial inhibition of ATP hydrolysis. 7. When mitochondria were incubated with dinitrophenol and ATP, less than 10% of the total inorganic phosphate liberated was recovered in the mitochondria and no swelling occurred. In the presence of mersalyl or formaldehyde at least 80% of the total inorganic phosphate liberated was retained in the mitochondria and extensive swelling was observed. This swelling was inhibited by oligomycin but not by antimycin or rotenone. 8. The addition of mersalyl to mitochondria swollen by treatment with valinomycin, K(+) and phosphate blocked the contraction induced by dinitrophenol and caused an increase in the phosphate content of the mitochondria, but had no effect on the contraction of mitochondria when phosphate was replaced by acetate. 9. It is concluded that mitochondria contain a phosphate-transporter system, which catalyses the movement of phosphate in either direction across the mitochondrial membrane, and that this system is inactivated by organic mercurials and by formaldehyde. Evidence is presented that the phosphate-transporter system is situated in the inner membrane of rat liver mitochondria and is also present in other types of mammalian mitochondria.     

Characterization of the inhibitor sensitivity of the coenzyme a transport system in isolated rat heart mitochondria

The effect of protein labeling agents on coenzyme A (CoA) transport into isolated rat heart mitochondria was studied. CoA transport was substantially inhibited by sulfhydryl reagents (mersalyl, pCMB) as well as by the tyrosine-selective reagent N-acetylimidazole. The effect of pCMB was reversed by DTT. Moreover, CoA uptake was completely abolished by agents selective for lysine and amino terminal residues (pyridoxal 5-phosphate, dansyl chloride). In contrast arginine-selective reagents (2, 3-butanedione, phenylglyoxal) caused considerably less inhibiton of CoA uptake. Moreover, partial inhibition of transport was observed with the stilbene disulfonic acid derivatives DIDS and SITS. Finally, measurement of the effects of the labeling agents on the mitochondrial membrane potential indicated that the inhibition of CoA transport into mitochondria is not a secondary effect that arises from an alteration in the electric potential gradient across the inner mitochondrial membrane. These results provide the first information on the types of amino acid residues that may be essential to the CoA transport mechanism and provide additional support for the existence of a CoA transport protein within the mitochondrial inner membrane. Furthermore, the identification of effective inhibitors of the CoA transport system will greatly facilitate the functional reconstitution of this transporter in a proteoliposomal system following its solubilization and purification.