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.     

Reactivity of the sulphydryl groups of the mitochondrial phosphate carrier

The rate of reaction of - SH groups of the mitochondrial phosphate carrier with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) and N-ethylmaleimide (MalNEt) was followed by measuring the inhibition of phosphate transport. The changes in the rate of reaction caused by alterations of the ionic composition of the matrix were compared with changes of the total intramitochondrial phosphate content, the intramitochondrial K+ content and the value of intramitochondrial pH. The ionic composition was manipulated by addition of valinomycin to non-respiring or to respiring mitochondria and by addition of inorganic phosphate to respiring and non-respiring mitochondria. From all these variables it was the changes of the intramitochondrial pH which correlated with the - SH group reactivity. Internal acidification decreased and internal alkalinization increased the rate of reaction of mitochondrial phosphate carrier with both Nbs2 and MalNEt. Nbs2 did not penetrate the inner mitochondrial membrane as assayed by determination of the acid-soluble thiol content of the matrix. From this fact it follows that the Nbs2-reactive SH groups of the carrier were accessible from the outer surface of the inner membrane in our experiments. It is concluded that intramitochondrial pH modifies the reactivity of the externally oriented - SH groups indirectly. A hypothesis is presented according to which protonation and deprotonation of the carrier molecule on the inner side could induce a conformational change of the whole protein altering also the microenvironment of the - SH groups near the opposite surface.     

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 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.     

The mitochondrial phosphate carrier interacts with cyclophilin D and may play a key role in the permeability transition

The mitochondrial permeability transition pore (MPTP) plays a key role in cell death, yet its molecular identity remains uncertain. Although knock-out studies have confirmed critical roles for both cyclophilin-D (CyP-D) and the adenine nucleotide translocase (ANT), given a strong enough stimulus MPTP opening can occur in the absence of either. Here we provide evidence that the mitochondrial phosphate carrier (PiC) may also be a critical component of the MPTP. Phenylarsine oxide (PAO) was found to activate MPTP opening in the presence of carboxyatractyloside (CAT) that prevents ANT binding to immobilized PAO. Only four proteins from solubilized CAT-treated beef heart inner mitochondrial membranes bound to immobilized PAO, one of which was the PiC. GST-CyP-D pull-down and co-immunoprecipitation studies revealed CsA-sensitive binding of PiC to CyP-D; this increased following diamide treatment. Co-immunoprecipitation of the ANT with the PiC was also observed but was insensitive to CsA treatment. N-ethylmaleimide and ubiquinone analogues (UQ(0) and Ro 68-3400) inhibited phosphate transport into rat liver mitochondria with the same concentration dependence as their inhibition of MPTP opening. UQ(0) and Ro 68-3400 also induced the "m" conformation of the ANT, as does NEM, and reduced the binding of both the PiC and ANT to the PAO column. We propose a model for the MPTP in which a calcium-triggered conformational change of the PiC, facilitated by CyP-D, induces pore opening. An interaction of the PiC with the ANT may enable agents that bind to either transporter to modulate pore opening.     

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 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.     

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.     

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.     

Inhibition of the mitochondrial phosphate carrier by a reaction with a carboxyl group reagent

The effect of N-ethyl-5-phenylisoxazolium 3"-sulfonate (Woodward's reagent K, WRK), a reagent forming covalent bonds with protein carboxyl groups, on the activity of the mitochondrial phosphate carrier was investigated. Treatment with WRK of mitochondria or of extracted carrier incorporated into liposomes, inhibited phosphate transport in a reconstituted liposomal system. Increasing the binding of WRK resulted in increased inhibition: the modified carrier protein showed a reduced affinity for phosphate, but binding of WRK had no effect on the Vmax of phosphate transport. It was concluded that WRK caused a conformational change in the carrier protein not involving the phosphate or H+ carrier sites such that its affinity for phosphate was lowered.     

Transmembrane topography of the mitochondrial phosphate carrier explored by peptide-specific antibodies and enzymatic digestion

Two peptides corresponding to the amino acid sequences 1-10 (N-terminal peptide) and 303-313 (C-terminal peptide) of the bovine heart mitochondrial phosphate carrier have been synthesized. After being coupled to ovalbumin, they were injected into rabbits to raise polyclonal antibodies. The specificity of the generated antibodies was tested by enzyme-linked immunosorbent assay (ELISA) and/or Western blot. Anti-N-terminal antibodies and anti-C-terminal antibodies exclusively reacted with the corresponding terminal peptide, they also reacted with the isolated phosphate carrier as well as with the phosphate carrier protein in mitochondrial lysates. Both anti-N-terminal and anti-C-terminal antibodies bound to freeze-thawed mitochondria, indicating that both termini of the membrane-bound phosphate carrier are exposed to the cytoplasmic side of the inner mitochondrial membrane. These immunological data were complemented with results concerning enzymatic cleavage of the membrane-bound phosphate carrier by carboxypeptidase A and by an arginine-specific endoprotease. Carboxypeptidase A markedly decreased the binding of anti-C-terminal antibodies to phosphate carrier in freeze-thawed mitochondria. Arg-endoprotease cleaved the phosphate carrier in inside-out submitochondrial particles, but not in right-side-out particles, yielding two fragments of similar apparent molecular weight (Mr approximately equal to 14.5K), which were immunodetected only by the anti-N-terminal antiserum, and a fragment of Mr approximately equal to 17K which was detected only by the anti-C-terminal antiserum. It appears, therefore, that Arg-endoprotease cleavage sites of the phosphate carrier are present only at the matrix side of the inner mitochondrial membrane, at Arg-140 and/or Arg-152.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of mitochondrial phosphate carrier in metabolism-secretion coupling in rat insulinoma cell line INS-1

In pancreatic β-cells, glucose-induced mitochondrial ATP production plays an important role in insulin secretion. The mitochondrial phosphate carrier PiC is a member of the SLC25 (solute carrier family 25) family and transports Pi from the cytosol into the mitochondrial matrix. Since intramitochondrial Pi is an essential substrate for mitochondrial ATP production by complex V (ATP synthase) and affects the activity of the respiratory chain, Pi transport via PiC may be a rate-limiting step for ATP production. We evaluated the role of PiC in metabolism-secretion coupling in pancreatic β-cells using INS-1 cells manipulated to reduce PiC expression by siRNA (small interfering RNA). Consequent reduction of the PiC protein level decreased glucose (10 mM)-stimulated insulin secretion, the ATP:ADP ratio in the presence of 10 mM glucose and elevation of intracellular calcium concentration in response to 10 mM glucose without affecting the mitochondrial membrane potential (Δψm) in INS-1 cells. In experiments using the mitochondrial fraction of INS-1 cells in the presence of 1 mM succinate, PiC down-regulation decreased ATP production at various Pi concentrations ranging from 0.001 to 10 mM, but did not affect Δψm at 3 mM Pi. In conclusion, the Pi supply to mitochondria via PiC plays a critical role in ATP production and metabolism-secretion coupling in INS-1 cells.     

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.     

Natural and azido fatty acids inhibit phosphate transport and activate fatty acid anion uniport mediated by the mitochondrial phosphate carrier

The electroneutral P(i) uptake via the phosphate carrier (PIC) in rat liver and heart mitochondria is inhibited by fatty acids (FAs), by 12-(4-azido-2-nitrophenylamino)dodecanoic acid (AzDA) and heptylbenzoic acid ( approximately 1 microm doses) and by lauric, palmitic, or 12-azidododecanoic acids ( approximately 0.1 mm doses). In turn, reconstituted E. coli-expressed yeast PIC mediated anionic FA uniport with a similar pattern leading to FA cycling and H(+) uniport. The kinetics of P(i)/P(i) exchange on recombinant PIC in the presence of AzDA better corresponded to a competitive inhibition mechanism. Methanephosphonate was identified as a new PIC substrate. Decanephosphonate, butanephosphonate, 4-nitrophenylphosphate, and other P(i) analogs were not translocated and did not inhibit P(i) transport. However, methylenediphosphonate and iminodi(methylenephosphonate) inhibited both electroneutral P(i) uptake and FA cycling via PIC. AzDA analog 16-(4-azido-2-nitrophenylamino)-[(3)H(4)]-hexadecanoic acid ((3)H-AzHA) bound upon photoactivation to several mitochondrial proteins, including the 30- and 34-kDa bands. The latter was ascribed to PIC due to its specific elution pattern on Blue Sepharose and Affi-Gel. (3)H-AzHA photolabeling of recombinant PIC was prevented by methanephosphonate and diphosphonates and after premodification with 4-azido-2-nitrophenylphosphate. Hence, the demonstrated PIC interaction with monovalent long-chain FA anions, but with divalent phosphonates of short chain only, indicates a pattern distinct from that valid for the mitochondrial uncoupling protein-1.     

Steroidogenic activity of StAR requires contact with mitochondrial VDAC1 and phosphate carrier protein

The steroidogenic acute regulatory protein (StAR) is required for adrenal and gonadal steroidogenesis and for male sexual differentiation. StAR acts on the outer mitochondrial membrane (OMM) to facilitate movement of cholesterol from the OMM to the inner mitochondrial membrane to be converted to pregnenolone, the precursor of all steroid hormones. The mechanisms of the action of StAR remain unclear; the peripheral benzodiazepine receptor, an OMM protein, appears to be involved, but the identity of OMM proteins that interact with StAR remain unknown. Here we demonstrate that phosphorylated StAR interacts with voltage-dependent anion channel 1 (VDAC1) on the OMM, which then facilitates processing of the 37-kDa phospho-StAR to the 32-kDa intermediate. In the absence of VDAC1, phospho-StAR is degraded by cysteine proteases prior to mitochondrial import. Phosphorylation of StAR by protein kinase A requires phosphate carrier protein on the OMM, which appears to interact with StAR before it interacts with VDAC1. VDAC1 and phosphate carrier protein are the first OMM proteins shown to contact StAR.     

In Saccharomyces cerevisiae, the phosphate carrier is a component of the mitochondrial unselective channel

The mitochondrial permeability transition (PT) involves the opening of a mitochondrial unselective channel (MUC) resulting in membrane depolarization and increased permeability to ions. PT has been observed in many, but not all eukaryotic species. In some species, PT has been linked to cell death, although other functions, such as matrix ion detoxification or regulation of the rate of oxygen consumption have been considered. The identification of the proteins constituting MUC would help understand the biochemistry and physiology of this channel. It has been suggested that the mitochondrial phosphate carrier is a structural component of MUC and we decided to test this in yeast mitochondria. Mersalyl inhibits the phosphate carrier and it has been reported that it also triggers PT. Mersalyl induced opening of the decavanadate-sensitive Yeast Mitochondrial Unselective Channel (YMUC). In isolated yeast mitochondria from a phosphate carrier-null strain the sensitivity to both phosphate and mersalyl was lost, although the permeability transition was still evoked by ATP in a decavanadate-sensitive fashion. Polyethylene glycol (PEG)-induced mitochondrial contraction results indicated that in mitochondria lacking the phosphate carrier the YMUC is smaller: complete contraction for mitochondria from the wild type and the mutant strains was achieved with 1.45 and 1.1 kDa PEGs, respectively. Also, as expected for a smaller channel titration with 1.1 kDa PEG evidenced a higher sensitivity in mitochondria from the mutant strain. The above data suggest that the phosphate carrier is the phosphate sensor in YMUC and contributes to the structure of this channel.