Reactivity of sulphydryl groups of cytosolic and mitochondrial bovine aspartate aminotransferases

Summary Reactivity of sulphydryl groups of cytosolic and mitochondrial aspartate aminotransferases from ox heart has been studied. A total of 5 and 7 cysteine residues per monomer are present in cAATo and mAATo, respectively. In native conditions only a single sulphydryl group can be titrated by Nbs₂ while the catalytic activity remains unchanged, however in the mitochondrial isozyme the reactivity depends on the functional state of the enzyme. Reactivity toward NEM reveals the existence of a syncatalytic sulphydryl group in the cytosolic isozyme. Titration of cAATo with pMB at pH 8 and pH 5 confirms the existence of two exposed sulphydryl groups with a different reactivity. The results compared with those reported on the corresponding isozymes from pig and chicken heart show that syncatalytic sulphydryl groups are of general occurrence in these enzymes.     

Reactivity of the -SH groups of the mitochondrial phosphate carrier under native, solubilized and reconstituted conditions

The isolated and liposome-reconstituted mitochondrial phosphate carrier exhibits a sigmoidal inhibition curve by mersalyl, similar to that found with intact mitochondria. In contrast a hyperbolic inhibition curve is found (a) by titration of the soluble carrier with mersalyl before reconstitution in liposomes and (b) by titration of the reconstituted carrier with mersalyl after successively pretreatment of the mitochondria with low, non-inhibitory concentrations of mersalyl, excess N-ethylmaleimide and dithiothreitol. The inhibition of the reconstituted, but not of the soluble, phosphate carrier by mersalyl can be reversed by dithiothreitol. Cupric di(1,10-phenanthroline) inhibits the soluble but not the reconstituted phosphate carrier. The inhibited phosphate carrier can be reactivated by dithiothreitol in the soluble state but not after reconstitution in liposomes. The data support the previously suggested model of the phosphate carrier, assuming a dimer of two identical subunits for the active unit.     

Characterization of sulphydryl groups of the mitochondrial phosphate translocator by a maleimide spin label

A maleimide spin label strongly inhibits the phosphate/H+ symporter of rat liver mitochondria. While inducing half-maximal inhibition of transport, the spin label reacts preferentially with the SH groups of the carrier, which are at least of two types. One type of SH group is localized close to the surface of the membrane and its environment does not significantly influence the mobility of the probe. The second type of SH group is buried in the membrane, is not accessible to ascorbate or chromium oxalate and its environment greatly restricts the motion of the probe.     

Photoaffinity labeling of the mitochondrial phosphate carrier by 4-azido-2-nitrophenyl phosphate

The effect of 4-azido-2-nitrophenyl phosphate (ANPP), a photoreactive analogue of phosphate, on the phosphate carrier of pig-heart mitochondria has been investigated. In the dark, ANPP inhibits the transport of phosphate in a competitive manner with a Ki of 3.2 mM. Upon photoirradiation with visible light, [32P]ANPP binds covalently to the phosphate carrier and the inhibition becomes irreversible. Both the inhibition of phosphate transport and the incorporation of [32P]ANPP into the phosphate carrier depend on the concentration of the inhibitor and the pH of the medium. Incubation of the mitochondria with phosphate during illumination in the presence of ANPP protects the carrier against inactivation and decreases the amount of radioactivity which is found to be associated with the purified protein. By extrapolation it is calculated that at 100% inactivation of the phosphate carrier 0.35 mol of reagent are bound per mol of 33 kDa carrier protein. It is concluded that ANPP can be used for photoaffinity labeling of the mitochondrial phosphate carrier at the substrate-binding site.     

The roles of phosphate and the phosphate carrier in the mitochondrial permeability transition pore

Phosphate activation of the mitochondrial permeability transition pore (MPTP) opening is well-documented and could involve the phosphate carrier (PiC) that we have proposed is the pore's cyclophilin-D binding component. However, others have reported that following CyP-D ablation Pi inhibits MPTP opening while cyclosporine-A (CsA) inhibits MPTP opening only when Pi is present. Here we demonstrate that Pi activates MPTP opening under all energised and de-energised conditions tested while CsA inhibits pore opening whether or not Pi is present. Using siRNA in HeLa cells we could reduce PiC expression by 65-80% but this inhibited neither mitochondrial calcium accumulation nor MPTP opening.     

The transport of inorganic phosphate by the mitochondrial dicarboxylate carrier

1. N-Ethylmaleimide inhibited the influx and efflux of P(i) in rat liver mitochondria. 2. The efflux was stimulated by either succinate or malate in the presence of N-ethylmaleimide, and this stimulation was reversed by 2-n-butylmalonate. 2-Oxoglutarate and citrate, even in the presence of low concentrations of malate, were relatively ineffective in stimulating efflux of P(i) under these conditions, as was glutamate. 3. By using radioactively labelled P(i) and dicarboxylate ions an exchange was demonstrated, the stoicheiometry of which was 1.3+/-0.5 dicarboxylate ions:1 P(i) (n=10). 4. An exchange between unlabelled and labelled P(i) in the presence of N-ethylmaleimide was found which was sensitive to 2-n-butylmalonate. 5. It is concluded that the mitochondrial dicarboxylate carrier can transport phosphate by an exchange diffusion with certain penetrant dicarboxylic acids or with phosphate itself. The exchange mechanism is sensitive to 2-n-butylmalonate but is unaffected by N-ethylmaleimide; the action of mersalyl in this context is commented on.     

Interaction of mitochondrial phosphate carrier with fatty acids and hydrophobic phosphate analogs

Mitochondrial transporters, in particular uncoupling proteins and the ADP/ATP carrier, are known to mediate uniport of anionic fatty acids (FAs), allowing FA cycling which is completed by the passive movement of FAs across the membrane in their protonated form. This study investigated the ability of the mitochondrial phosphate carrier to catalyze such a mechanism and, furthermore, how this putative activity is related to the previously observed HgCl(2)-induced uniport mode. The yeast mitochondrial phosphate carrier was expressed in Escherichia coli and then reconstituted into lipid vesicles. The FA-induced H(+) uniport or Cl(-) uniport were monitored fluorometrically after HgCl(2) addition. These transport activities were further characterized by testing various inhibitors of the two different transport modes. The phosphate carrier was found to mediate FA cycling, which led to H(+) efflux in proteoliposomes. This activity was insensitive to ATP, mersalyl or N-ethylmaleimide and was inhibited by methylenediphosphonate and iminodi(methylenephosphonate), which are new inhibitors of mitochondrial phosphate transport. Also, the HgCl(2) induced Cl(-) uniport mediated by the reconstituted yeast PIC, was found to be inhibited by these reagents. Both methylenediphosphonate and iminodi(methylenephosphonate) blocked unidirectional Cl(-) uptake, whereas Cl(-) efflux was inhibited by iminodi(methylenephosphonate) and phosphonoformic acid only. These results suggest that a hydrophobic domain, interacting with FAs, exists in the mitochondrial phosphate carrier, which is distinct from the phosphate transport pathway. This domain allows for FA anion uniport via the phosphate carrier and consequently, FA cycling that should lead to uncoupling in mitochondria. This might be considered as a side function of this carrier.     

Specific elution from hydroxylapatite of the mitochondrial phosphate carrier by cardiolipin

The role of cardiolipin in the purification of the mitochondrial phosphate carrier by hydroxylapatite has been investigated. Without added cardiolipin, the reconstituted phosphate-transport activity in the hydroxylapatite eluate is small and only confined to the first fraction. With cardiolipin added to the extract, the eluted activity is much higher and present until fraction 6. The activity retained by hydroxylapatite in the absence of cardiolipin is eluted after addition of this phospholipid to the column. The requirement of added cardiolipin diminishes on increasing the concentration of solubilized mitochondria. The hydroxylapatite eluate contains five protein bands in the Mr-region of 30 000-35 000, which are differently distributed in the various fractions. Among these, only the presence and the relative amount of band 3 of Mr 33 000 corresponds to the phosphate transport activity. Cardiolipin is the only phospholipid tested which causes elution of band 3 from hydroxylapatite; on the other hand, it prevents the elution of band 2 and retards that of band 5 (the ADP/ATP carrier). Band 1 starts to appear in the second fraction even without cardiolipin. On increasing the concentration of cardiolipin, in the first fraction of the hydroxylapatite eluate band 3 increases and the contamination of band 4 decreases. Under optimal conditions a preparation of band 3 about 90% pure and with high reconstituted phosphate transport activity is obtained. It is concluded that the elution of the phosphate carrier from hydroxylapatite requires cardiolipin and that the phosphate carrier is identical with (or with part of) band 3 of the hydroxylapatite eluate.     

Genetic deletion of the mitochondrial phosphate carrier desensitizes the mitochondrial permeability transition pore and causes cardiomyopathy

The mitochondrial phosphate carrier (PiC) is critical for ATP synthesis by serving as the primary means for mitochondrial phosphate import across the inner membrane. In addition to its role in energy production, PiC is hypothesized to have a role in cell death as either a component or a regulator of the mitochondrial permeability transition pore (MPTP) complex. Here, we have generated a mouse model with inducible and cardiac-specific deletion of the Slc25a3 gene (PiC protein). Loss of PiC protein did not prevent MPTP opening, suggesting it is not a direct pore-forming component of this complex. However, Slc25a3 deletion in the heart blunted MPTP opening in response to Ca²⁺ challenge and led to a greater Ca²⁺ uptake capacity. This desensitization of MPTP opening due to loss or reduction in PiC protein attenuated cardiac ischemic-reperfusion injury, as well as partially protected cells in culture from Ca²⁺ overload induced death. Intriguingly, deletion of the Slc25a3 gene from the heart long-term resulted in profound hypertrophy with ventricular dilation and depressed cardiac function, all features that reflect the cardiomyopathy observed in humans with mutations in SLC25A3. Together, these results demonstrate that although the PiC is not a direct component of the MPTP, it can regulate its activity, suggesting a novel therapeutic target for reducing necrotic cell death. In addition, mice lacking Slc25a3 in the heart serve as a novel model of metabolic, mitochondrial-driven cardiomyopathy.The mitochondrial oxidative phosphorylation (OXPHOS) system is the primary source of cellular energy production. Defects in OXPHOS occur with a frequency of 1 in 5000 live births¹ and underlie a wide range of mitochondrial disorders that often affect multiple organ systems and tissues with high oxidative energy demands, such as brain, skeletal muscle, and heart.² Cardiac phenotypes associated with mitochondrial disease are diverse, and can range from cardiomyopathies to cardiac conduction defects.^{3, 4, 5}The mitochondrial phosphate carrier (PiC) is a member of the solute carrier 25A family that has a critical role in OXPHOS, serving as the primary route for inorganic phosphate (Pi) import into the mitochondrial matrix.^{6, 7} PiC, together with the adenine nucleotide translocator (ANT) and the ATP synthase, forms the ATP synthasome whereby all of the metabolites needed to generate ATP are within one immediate microdomain.^{8, 9} The importance of PiC in facilitating energy production is highlighted by the profound disease phenotype observed in patients presenting with mutations in the skeletal muscle-specific isoform of this gene.^{10, 11} Such patients present with a multisystemic disorder characterized by muscle hypotonia, lactic acidosis, severe hypertrophic cardiomyopathy, and shortened lifespan.^{10, 11} Similarly, patients with SLC25A4 (ANT1 protein) deficiency present with cardiomyopathy,¹² as do mice lacking the Slc25a4 gene,¹³ likely due to a similar molecular defect in the efficiency of ATP production within the mitochondria.In addition to its role in mitochondrial energy metabolism, PiC has been implicated in regulating cell death by serving either as a modulator or a direct component of the mitochondrial permeability transition pore (MPTP).^{14, 15, 16} The MPTP is a non-selective channel that forms in response to Ca²⁺ overload and oxidant stress that allows inner-membrane permeability to solutes up to 1500 Da in size, leading to loss of mitochondrial membrane potential, mitochondrial swelling and rupture, and eventually cell death through necrosis.¹⁵ Structurally, the MPTP complex has been proposed to be comprised of the ATP synthase^{17, 18} and to be regulated by cyclophilin D (CypD),^{19, 20} ANT,²¹ and the pro-apoptotic proteins Bax and Bak in the outer mitochondrial membrane.^{22, 23} PiC has also been suggested to be an inner-membrane component of the MPTP because it can form nonspecific channels in lipid membranes and because the MPTP is known to be activated by Pi.^{24, 25, 26, 27} Finally, PiC directly interacts with CypD in the mitochondrial matrix, which is a verified regulator and component of the MPTP.¹⁶ Saccharomyces cerevisiae lacking PiC have altered MPTP characteristics with a smaller pore size, suggesting it might directly participate in the mitochondrial permeability pore.²⁸ However, partial reduction of PiC by siRNAs in cultured cells had no effect on mitochondrial permeability activity, suggesting that PiC is not required for MPTP function.²⁷ Definitive genetic proof of PiC''s involvement in MPTP formation/function is currently lacking.In the present study, we tested the role of PiC in MPTP regulation and cell death in vivo using a mouse model with inducible cardiomyocyte-specific deletion of the Slc25a3 gene (encodes PiC). We found that cardiac mitochondria depleted of PiC were able to undergo permeability transition, suggesting that PiC is not a requisite component of the MPTP. However, the extent of Ca²⁺-induced MPTP opening was blunted, suggesting that PiC serves to regulate this activity. Furthermore, Slc25a3 deletion produced a unique mouse model of mitochondrial-driven hypertrophic cardiomyopathy that recapitulates features observed in human patients with phosphate carrier deficiency and metabolic cardiomyopathy.     

The location of sulphydryl groups in alpha-crystallin

The microenvironments of the sulphydryl groups in the multimeric protein, alpha-crystallin, were studied by examining: the rate of the reaction of the groups with DTNB; the effect of increasing urea concentrations on their accessibilities; and the quenching of a fluorescent probe. In foetal bovine alpha-crystallin (1 SH/alpha A subunit) both kinetic and quenching studies indicated that over 90% of the sulphydryl groups fell into a single buried class; the remainder was exposed. In the human protein (2 SH/alpha A subunit), half of the groups were buried and the other half exposed. Accessible sulphydryl groups increased gradually as the urea concentration was increased, with complete exposure at about 4.0 M. Sedimentation velocity analyses revealed that no significant dissociation of the aggregates into subunits occurred below 3.5 M urea, at which point over 80% of the sulphydryl groups were exposed. An age-dependent increase (3-35%) was found in the proportion of exposed sulphydryl groups in bovine alpha-crystallin and a decrease in the urea concentration required to expose the remainder. It was concluded that the single cysteine is buried in the newly synthesized protein, but becomes solvent-exposed as a result of age-related conformational changes. Our observations are consistent with a quaternary structure in which all alpha A subunits occupy equivalent sites.     

Nucleotide sequence of a human heart cDNA encoding the mitochondrial phosphate carrier.

We have isolated and characterized a full length cDNA clone encoding the precursor of the human heart mitochondrial phosphate carrier protein. The entire clone is 1330 bp in length with 5'- and 3'-untranslated regions of 48 and 184 bp, respectively. The open reading frame encodes the mature protein consisting of 312 amino acids, preceded by a presequence of 49 amino acids. The amino acid sequence of the mature human phosphate carrier is 93.6, 94.2 and 33.6% identical to that of the phosphate carrier from beef, rat and yeast, respectively. Like other mitochondrial transport proteins, the human phosphate carrier has a tripartite structure. Each of the three repeats contains two hydrophobic regions which presumably span the membrane in the form of alpha-helices.     

The mitochondrial phosphate carrier reconstituted in liposomes is inhibited by doxorubicin

The phosphate carrier has been isolated from beef heart mitochondria in the presence of cardiolipin and reconstituted in asolectin vesicles. It has been found that 100 microM doxorubicin and 100 microM Br-daunomycin inhibit the unidirectional phosphate uptake in the reconstituted liposomes to the same extent as N-ethylmaleimide. The inhibition by Br-daunomycin is not due to covalent interaction with the carrier. The specific interaction between doxorubicin and cardiolipin is responsible for the inhibition of the phosphate carrier. Br-daunomycin interacts with 3 mitochondrial proteins of apparent Mr approximately 45 000, approximately 35 000 and approximately 30 000.     

Interaction of phenylisothiocyanates with the mitochondrial phosphate carrier. I. Covalent modification and inhibition of phosphate transport

The effects of phenylisothiocyanate (PITC) and of the polar analogue p-sulfophenylisothiocyanate (p-sulfoPITC) on the phosphate carrier of bovine heart mitochondria have been investigated. Incubation of mitochondria with the two phenylisothiocyanates leads to inhibition of the phosphate carrier protein. The inhibition of phosphate transport by PITC is unaffected by the addition of dithioerythritol (DTE) or by variation of the pH. The inhibition by p-sulfoPITC is in part removed by DTE; the remaining inactivation of the phosphate carrier, which can be attributed to the reaction with NH2 groups, is temperature and pH-dependent. Inhibition of phosphate transport by both p-sulfoPITC and PITC depends on the time of incubation and the concentration of the inhibitor. Preincubation with mersalyl protects the carrier protein against the inactivation by p-sulfoPITC but not against PITC. Other SH reagents tested do not show any protective effect. It can thus be concluded that two types of lysine residues are essential for the activity of the phosphate carrier. Lysine(s) of the former type are located at the surface of the membrane and are topologically related to the functional SH groups of the protein. Lysine residue(s) of the latter type are buried in the hydrophobic phase of the membrane.     

Interaction of phenylisothiocyanates with the mitochondrial phosphate carrier. I. Covalent modification and inhibition of phosphate transport

The effects of phenylisothiocyanate (PITC) and of the polar analogue p-sulfophenylisothiocyanate (p-sulfoPITC) on the phosphate carrier of bovine heart mitochondria have been investigated. Incubation of mitochondria with the two phenylisothiocyanates leads to inhibition of the phosphate carrier protein. The inhibition of phosphate transport by PITC is unaffected by the addition of dithioerythritol (DTE) or by variation of the pH. The inhibition by p-sulfoPITC is in part removed by DTE; the remaining inactivation of the phosphate carrier, which can be attributed to the reaction with NH₂ groups, is temperature and pH-dependent. Inhibition of phosphate transport by both p-sulfoPITC and PITC depends on the time of incubation and the concentration of the inhibitor. Preincubation with mersalyl protects the carrier protein against the inactivation by p-sulfoPITC but not against PITC. Other SH reagents tested do not show any protective effect. It can thus be concluded that two types of lysine residues are essential for the activity of the phosphate carrier. Lysine(s) of the former type are located at the surface of the membrane and are topologically related to the functional SH groups of the protein. Lysine residue(s) of the latter type are buried in the hydrophobic phase of the membrane.