Peptide-specific antibodies and proteases as probes of the transmembrane topology of the bovine heart mitochondrial porin.

We have investigated the transmembrane topology of the bovine heart mitochondrial porin by means of proteases and antibodies raised against the amino-terminal region of the protein. The antisera against the human N-terminus reacted with porin in Western blots of NaDodSO4-solubilized bovine heart mitochondria and with the membrane-bound porin in enzyme-linked immunosorbent assay (ELISA). The immunoreaction with mitochondria coated on microtiter wells showed that the amino-terminal region of the protein is not embedded in the lipid bilayer but is exposed to the cytosol. Back-titration of unreacted anti-N-terminal antibodies after their incubation with intact mitochondria demonstrated that the porin N-terminus is also exposed in "noncoated" mitochondria. No difference in antisera reactivity was observed between intact and broken mitochondria. Intact and broken mitochondria were subjected to proteolysis by specific proteases. The membrane-bound bovine heart porin was strongly resistant to proteolysis, but a few specific cleavage sites were observed. Staphylococcus aureus V8 protease gave a large 24K N-terminal peptide, trypsin produced a 12K N-terminal and an 18K C-terminal peptide, and chymotrypsin gave two peptides of Mr 19.5K and 12.5K, which were both recognized by the antiserum against the human N-terminus. Carboxypeptidase A was ineffective in cleaving the membrane-bound porin in both intact and broken mitochondria. Thus, the carboxy-terminal part of the protein is probably not exposed to the water phase. The cleavage patterns of membrane-bound porin, obtained with S. aureus V8 protease, trypsin, and chymotrypsin, showed no difference between intact and broken mitochondria, thus indicating that all porin molecules have the same orientation in the membrane. The computer analysis of the sequence of human B-lymphocyte porin suggested that 16 beta-strands can span the phospholipid bilayer. This result, together with the overall information presented, allowed us to draw a possible scheme of the transmembrane arrangement of mammalian mitochondrial porin.     

Peptide-specific antibodies as probes of the orientation of the glucose transporter in the human erythrocyte membrane

Antibodies were raised in rabbits against synthetic peptides corresponding to the N-terminal (residues 1-15) and the C-terminal (residues 477-492) regions of the human erythrocyte glucose transporter. The antisera recognized the intact transporter in enzyme-linked immunosorbent assays (ELISA) and Western blots. In addition, the anti-C-terminal peptide antibodies were demonstrated, by competitive ELISA and by immunoadsorption experiments, to bind to the native transporter. Competitive ELISA, using intact erythrocytes, unsealed erythrocyte membranes, or membrane vesicles of known sidedness as competing antigen, showed that these antibodies bound only to the cytoplasmic surface of the membrane, indicating that the C terminus of the protein is exposed to the cytoplasm. On Western blots, the anti-N-terminal peptide antiserum labeled the glycosylated tryptic fragment of the transporter, of apparent Mr = 23,000-42,000, showing that this originates from the N-terminal half of the protein. The anti-C-terminal peptide antiserum labeled higher Mr precursors of the Mr = 18,000 tryptic fragment, although not the fragment itself, indicating that the latter, with its associated cytochalasin B binding site, is derived from the C-terminal half of the protein. Antiserum against the intact transporter recognized the C-terminal peptide on ELISA, and the Mr = 18,000 fragment but not the glycosylated tryptic fragment on Western blots.     

Topography of the membrane-bound ADP/ATP carrier assessed by enzymatic proteolysis.

The folding of the peptide chain of the beef heart ADP/ATP carrier in the inner mitochondrial membrane was investigated by enzymatic and immunochemical approaches, using specific proteases and polyclonal antibodies directed against the whole protein and specific regions of the carrier. The accessibility of the membrane-bound ADP/ATP carrier to proteases was followed by immunodetection of the cleavage products, using mitochondria devoid of outer membrane (mitoplasts) and inside-out submitochondrial particles (SMP) in the presence of either carboxyatractyloside (CATR) or bongkrekic acid (BA), two specific inhibitors which are able to bind to the outer face or the inner face of the carrier, respectively. Four types of particles were investigated, namely, mitoplasts-CATR, mitoplasts-BA, SMP-CATR, and SMP-BA. Only the ADP/ATP carrier in SMP-BA was cleaved by two specific proteases, namely, trypsin and lysine C endoprotease, at low doses for short periods of time. Two initial cleavage sites were found between Lys-42 and Glu-43, and between Lys-244 and Gly-245. After a longer period of incubation, an additional cleavage site between Lys-146 and Gly-147 could be demonstrated. Despite cleavage of the membrane-embedded carrier, the binding capacity and affinity of SMP for BA were not altered. A number of other proteases tested, including V8 protease, proline C endoprotease, thrombin, alpha-chymotrypsin, and thermolysin had virtually no effect. These results are explained by a dynamic model of the arrangement of the peptide chain of the ADP/ATP carrier.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro synthesis and assembly of a 68 kDa outer mitochondrial membrane protein from rat liver

Outer mitochondrial membrane was purified from rat liver. Its constituent proteins were analyzed by SDS-polyacrylamide gel electrophoresis and by electrophoretic immunoblotting employing antibodies raised against total outer mitochondrial membrane. Anti-outer mitochondrial membrane antiserum reacted with only one polypeptide (15 kDa) in rough microsomes, whereas no immunological cross-reactivity was observed with other mitochondrial compartments (intermembrane space, inner membrane, or matrix) or with lysosomes or total cytosol. The antiserum was employed to characterize precursors of outer mitochondrial membrane proteins synthesized in vitro in a rabbit reticulocyte cell-free system. One product (a 68 kDa polypeptide designated OMM-68) bound efficiently to mitochondria in vitro but did not interact with either dog pancreas or rat liver microsomes, either co-translationally or post-translationally. OMM-68 was synthesized exclusively by the membrane-free class of polyribosomes. Attachment of precursor OMM-68 to mitochondria was not accompanied by processing of the polypeptide to a different size.     

Mitochondrial phosphate transport. Import of the H+/Pi symporter and role of the presequence

The rat liver mitochondrial phosphate transporter contains a 44-amino acid presequence. The role of this presequence is not clear since the ADP/ATP carrier and the brown fat uncoupling protein, related members of a family of inner membrane anion transporters, lack a presequence and contain targeting information within the mature protein. Here, we present evidence that the rat liver mitochondrial phosphate transporter can be synthesized in vitro, imported into mitochondria, and processed to a protein of Mr 33,000. Import requires the membrane potential and external nucleotide triphosphate. The presequence inserts into the outer mitochondrial membrane, and import proceeds via a process similar to other proteins destined for the inner membrane or matrix. A mutant phosphate transporter lacking 35 amino acids at the NH2 terminus of the presequence has little capacity for mitochondrial import. The rat liver phosphate transporter is also imported and processed by rat kidney mitochondria and by mitochondria from the yeast Saccharomyces cerevisiae. A site-directed mutation of the N-ethyl-maleimide reactive cysteine 41 does not affect import or processing. The results presented show that optimal import of the mitochondrial phosphate transporter, unlike the ADP/ATP carrier and the brown fat uncoupling protein, is dependent on a presequence. As these carriers are believed to have evolved from a single gene, it seems likely that the H+/Pi carrier, known to be present in prokaryotes, appeared first and that subsequent evolutionary events leading to the other anion carriers eliminated the presequence.     

Mitochondrial proton/phosphate transporter. An antibody directed against the COOH terminus and proteolytic cleavage experiments provides new insights about its membrane topology

Molecular cloning and sequencing of a full-length cDNA encoding the rat liver mitochondrial phosphate transporter (H+/Pi symporter) has revealed its primary structure (Ferreira, G. C. Pratt, R. D., and Pedersen, P. L. (1989) J. Biol. Chem. 264, 15628-15633). To date, no experimental data pertinent to the membrane topology of this transporter are available. For this reason, four different peptides which represent different regions of the H+/Pi symporter were synthesized and used to raise polyclonal antibodies. Each of the antipeptide antibodies exhibits immunoreactivity with its synthetic peptide antigen, but only antiserum against a COOH-terminal peptide reacts with the native transporter, suggesting that the other peptides are either conformally restricted or located in the interior of the protein. Competitive radioimmunoassays, using intact "mitoplasts" (outer membrane-free mitochondria) and inverted inner membrane vesicles, show that the COOH-terminal antibodies bind only to the cytoplasmic surface of the inner membrane, indicating that the COOH terminus of the protein is normally exposed to the mitochondrial intermembrane space. In support of this conclusion, tryptic digestion of mitoplasts but not of the inside-out vesicles, cleaves the antigenic site for the COOH-terminal antibodies. In other experiments, it was shown that N-ethylmalemide, a sulfhydryl alkylating agent known to inhibit the mitochondrial phosphate transporter, markedly reduces the accessibility of the COOH terminus to trypsin. These studies provide the first direct experimental data relevant to the membrane topology of the mitochondrial H+/Pi symporter. In addition, they support the view that alkylation of a reactive cysteine residue induces a significant conformational change in the transporter.     

THE DISTRIBUTION OF SOLUBLE AND MEMBRANE-BOUND FORMS OF GLUTAMINASE IN PIG BRAIN

Abstract— About 10% of the glutaminase activity associated with pig brain mitochondria was readily extractable by a variety of techniques but the remainder was very resistant to extraction. These two forms, which have been termed the soluble and membrane-bound forms respectively, have been shown to differ in their responses to activation by phosphate and phosphate-borate containing buffers. Submitochondrial fractionation studies indicated that the soluble form was located in the mitochondrial inner matrix whereas the membrane-bound form was associated with the inner membrane. The mitochondria associated with the synaptosomes were found to contain only the membrane-bound form of the enzyme whereas both forms were present in the free brain mitochondria.     

The mitochondrial tricarboxylate carrier of silver eel: dimeric structure and cytosolic exposure of both N- and C-termini

The mitochondrial tricarboxylate carrier plays a fundamental role in the hepatic fatty acid synthesis. In this study, we investigated the transmembrane organization of this protein in the inner membrane of eel liver mitochondria using anti–N-terminal and anti–C-terminal antibodies. These antibodies recognized the N- and C-termini of the tricarboxylate carrier in intact mitoplasts, thus suggesting a cytosolic exposure of these regions in the membrane-bound protein. This structural arrangement of the tricarboxylate carrier was further confirmed by protease treatment of intact mitoplasts. Moreover, the oligomeric state of the native tricarboxylate carrier was investigated by blue native electrophoresis. A dimeric form of the carrier protein was found when eel liver mitochondria were solubilized with the mild detergent digitonin. These findings suggest an arrangement of the dimeric tricarboxylate carrier into an even number of membrane-spanning domains, with the N-terminal and C-terminal regions oriented toward the intermembrane space of fish mitochondria.     

Conserved structural domains among species and tissues-specific differences in the mitochondrial phosphate-transport protein and the ADP/ATP carrier

Peptide maps were generated of the CNBr-digested mitochondrial phosphate-transport protein and ADP/ATP carrier from bovine and rat heart, rat liver and blowfly flight muscle. Total mitochondrial proteins from the same sources plus pig heart were separated by SDS-polyacrylamide gel electrophoresis. The peptide maps and the total mitochondrial proteins were electroblotted onto nitrocellulose membranes and reacted with rabbit antisera raised against the purified bovine heart phosphate-transport protein and the ADP/ATP carrier. On the basis of antibody specificity, mobility in SDS-polyacrylamide gel electrophoresis, and peptide maps the following was concluded. Phosphate-transport protein alpha and phosphate-transport protein beta (pig and bovine heart) react equally with the first and also with the second of two independent phosphate-transport protein-antisera. Tissue-specific structural domains exist for both the phosphate-transport protein and the ADP/ATP carrier, i.e., one phosphate-transport protein-antiserum reacts with the phosphate-transport protein from all assayed sources, the other only with the cardiac phosphate-transport protein. These differences may reflect tissue-specific regulation of phosphate and adenine nucleotide transport. Homologies among the different species are found for the phosphate transport protein and the ADP/ATP carrier, except for the flight muscle ADP/ATP carrier. These conserved structural domains of the phosphate-transport protein may relate directly to catalytic activity. Alkylation of the purified phosphate-transport proteins and the ADP/ATP carriers by the transport inhibitor N-ethylmaleimide affects electrophoretic mobilities but not the antibody binding. Neither of the two phosphate-transport protein-antisera nor the ADP/ATP-carrier antiserum react with both phosphate transport protein and ADP/ATP carrier, even though these two proteins possess similarities in primary structure and function. Possible mechanisms for generating tissue-specific structural differences in the proteins are discussed.     

Yeast mitochondria lacking the phosphate carrier/p32 are blocked in phosphate transport but can import preproteins after regeneration of a membrane potential.

Two different functions have been proposed for the phosphate carrier protein/p32 of Saccharomyces cerevisiae mitochondria: transport of phosphate and requirement for import of precursor proteins into mitochondria. We characterized a yeast mutant lacking the gene for the phosphate carrier/p32 and found both a block in the import of phosphate and a strong reduction in the import of preproteins transported to the mitochondrial inner membrane and matrix. Binding of preproteins to the surface of mutant mitochondria and import of outer membrane proteins were not inhibited, indicating that the inhibition of protein import occurred after the recognition step at the outer membrane. The membrane potential across the inner membrane of the mutant mitochondria was strongly reduced. Restoration of the membrane potential restored preprotein import but did not affect the block of phosphate transport of the mutant mitochondria. We conclude that the inhibition of protein import into mitochondria lacking the phosphate carrier/p32 is indirectly caused by a reduction of the mitochondrial membrane potential (delta(gamma)), and we propose a model that the reduction of delta(psi) is due to the defective phosphate import, suggesting that phosphate transport is the primary function of the phosphate carrier/p32.     

Membrane topology and quaternary structure of cardiac gap junction ion channels.

The membrane topology and quaternary structure of rat cardiac gap junction ion channels containing alpha 1 connexin (i.e. Cx43) have been examined using anti-peptide antibodies directed to seven different sites in the protein sequence, cleavage by an endogenous protease in heart tissue and electron microscopic image analysis of native and protease-cleaved two-dimensional membrane crystals of isolated cardiac gap junctions. Specificity of the peptide antibodies was established using dot immunoblotting, Western immunoblotting, immunofluorescence and immunoelectron microscopy. Based on the folding predicted by hydropathy analysis, five antibodies were directed to sites in cytoplasmic domains and two antibodies were directed to the two extracellular loop domains. Isolated gap junctions could not be labeled by the two extracellular loop antibodies using thin-section immunogold electron microscopy. This is consistent with the known narrowness of the extracellular gap region that presumably precludes penetration of antibody probes. However, cryo-sectioning rendered the extracellular domains accessible for immunolabeling. A cytoplasmic "loop" domain of at least Mr = 5100 (residues (101 to 142) is readily accessible to peptide antibody labeling. The native Mr = 43,000 protein can be protease-cleaved on the cytoplasmic side of the membrane, resulting in an Mr approximately 30,000 membrane-bound fragment. Western immunoblots showed that protease cleavage occurs at the carboxy tail of the protein, and the cleavage site resides between amino acid residues 252-271. Immunoelectron microscopy demonstrated that the Mr approximately 13,000 carboxy-terminal peptide(s) is released after protease cleavage and does not remain attached to the Mr approximately 30,000 membrane-bound fragment via non-covalent interactions. Electron microscopic image analysis of two-dimensional membrane crystals of cardiac gap junctions revealed that the ion channels are formed by a hexagonal arrangement of protein subunits. This quaternary arrangement is not detectably altered by protease cleavage of the alpha 1 polypeptide. Therefore, the Mr approximately 13,000 carboxyterminal domain is not involved in forming the transmembrane ion channel. The similar hexameric architecture of cardiac and liver gap junction connexins indicates conservation in the molecular design of the gap junction channels formed by alpha or beta connexins.     

MRS3 and MRS4, two suppressors of mtRNA splicing defects in yeast, are new members of the mitochondrial carrier family.

When present in high copy number plasmids, the nuclear genes MRS3 and MRS4 from Saccharomyces cerevisiae can suppress the mitochondrial RNA splicing defects of several mit- intron mutations. Both genes code for closely related proteins of about Mr 32,000; they are 73% identical. Sequence comparisons indicate that MRS3 and MRS4 may be related to the family of mitochondrial carrier proteins. Support for this notion comes from a structural analysis of these proteins. Like the ADP/ATP carrier protein (AAC), the mitochondrial phosphate carrier protein (PiC) and the uncoupling protein (UCP), the two MRS proteins have a tripartite structure; each of the three repeats consists of two hydrophobic domains that are flanked by specific amino acid residues. The spacing of these specific residues is identical in all domains of all proteins of the family, whereas spacing between the hydrophobic domains is variable. Like the AAC protein, the MRS3 and MRS4 proteins are imported into mitochondria in vitro and without proteolytic cleavage of a presequence and they are located in the inner mitochondrial membrane. In vivo studies support this mitochondrial localization of the MRS proteins. Overexpression of the MRS3 and MRS4 proteins causes a temperature-dependent petite phenotype; this is consistent with a mitochondrial function of these proteins. Disruption of these genes affected neither mitochondrial functions nor cellular viability. Their products thus have no essential function for mitochondrial biogenesis or for whole yeast cells that could not be taken over by other gene products. The findings are discussed in relation to possible functions of the MRS proteins in mitochondrial solute translocation and RNA splicing.     

Conserved structural domains among species and tissues-specific differences in the mitochondrial phosphate-transport protein and the ADP/ATP carrier

Peptide maps were generated of the CNBr-digested mitochondrial phosphate-transport protein and ADP/ATP carrier from bovine and rat heart, rat liver and blowfly flight muscle. Total mitochondrial proteins from the same sources plus pig heart were separated by SDS-polyacrylamide gel electrophoresis. The peptide maps and the total mitochondrial proteins were electroblotted onto nitrocellulose membranes and reacted with rabbit antisera raised against the purified bovine heart phosphate-transport protein and the ADP/ATP carrier. On the basis of antibody specificity, mobility in SDS-polyacrylamide gel electrophoresis, and peptide maps the following was concluded. (1) Phosphate-transport protein and phosphate-transport protein β (pig and bovine heart) react equally with the first and also with the second of two independent phosphate-transport protein-antisera. (2) Tissue-specific structural domains exist for both the phosphate-transport protein and the ADP/ATP carrier, i.e., one phosphate-transport protein-antiserum reacts with the phosphate-transport protein from all assayed sources, the other only with the cardiac phosphate-transport protein. These differences may reflect tissue-specific regulation of phosphate and adenine nucleotide transport. (3) Homologies among the different species are found for the phosphate transport protein and the ADP/ATP carrier, except for the flight muscle ADP/ATP carrier. These conserved structural domains of the phosphate-transport protein may relate directly to catalytic activity. (4) Alkylation of the purified phosphate-transport proteins and the ADP/ATP carriers by the transport inhibitor N-ethylmaleimide affects electrophoretic mobilities but not the antibody binding. (5) Neither of the two phosphate-transport protein-antisera nor the ADP/ATP-carrier antiserum react with both phosphate transport protein and ADP/ATP carrier, even though these two proteins possess similarities in primary structure and function. Possible mechanisms for generating tissue-specific structural differences in the proteins are discussed.     

Purification and characterization of the phosphate/hydroxyl ion antiport protein from rat liver mitochondria.

The phosphate transport protein was purified from rat liver mitochondria by extraction in an 8% (v/v) Triton X-100 buffer followed by adsorption chromatography on hydroxyapatite and Celite. SDS/polyacrylamide-gel electrophoresis (10%, w/v) demonstrated that the purified polypeptide was apparently homogeneous when stained with Coomassie Blue and had a subunit Mr of 34,000. However, lectin overlay analysis of this gel with 125I-labelled concanavalin A demonstrated the presence of several low- and high-Mr glycoprotein contaminants. To overcome this problem, mitochondria were pre-extracted with a 0.5% (v/v) Triton X-100 buffer as an additional step in the purification of phosphate transport protein. SDS/polyacrylamide gradient gel electrophoresis (14-20%, w/v) of the hydroxyapatite and Celite eluates revealed one major band of Mr 34,000 when stained with Coomassie Blue. The known thiol group sensitivity of the phosphate transporter was employed to characterize the isolated polypeptide further. Labelling studies with N-[2-3H]ethylmaleimide showed that only the 34,000-Mr band was labelled in both the hydroxyapatite and Celite fractions, when purified from rat liver mitochondria. Further confirmation of its identity has been provided with an antiserum directed against the 34,000-Mr protein. Specific partial inhibition of phosphate uptake, as measured by iso-osmotic swelling in the presence of (NH4)2HPO4, was achieved when mitoplasts (mitochondria minus outer membrane) were incubated with this antiserum. Finally, amino acid analysis of the rat liver mitochondrial phosphate/hydroxyl ion antiport protein indicates that it is similar in composition to the equivalent protein isolated from ox heart.     

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.     

Release of the NILE and Other Glycoproteins from Cultured PC 12 Rat Pheochromocytoma Cells and Sympathetic Neurons

Studies were carried out on the glycoproteins (GPs) released by cultured rat sympathetic neurons and by cultured PC12 rat pheochromocytoma cells with and without nerve growth factor (NGF) treatment. Cultures were prelabeled with [3H]fucose and then incubated for 4-8 h in fresh unlabeled medium. The material released into the medium was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. The patterns of labeled material released by all three types of cultures were similar. One of the major components released was of apparent Mr less than or equal to 230,000. Another major component of apparent Mr = 55,000 as well as minor components of apparent Mr less than or equal to 180,000, 140,000, 118,000, and 105,000 were also detected. An additional peptide of apparent Mr less than or equal to 210,000 was released only by the sympathetic neurons. The soluble released Mr less than or equal to 230,000 component appeared to be derived from a previously characterized neuronal integral membrane GP referred to as the NILE (NGF-inducible large external) GP. Evidence for this included recognition of the released component by a monospecific antiserum prepared against membrane-derived NILE GP. At least several of the other released GPs appeared to be derived from membrane-bound components with which they share immuno-crossreactivity. Since the soluble NILE and other released GPs had somewhat faster mobilities on SDS-polyacrylamide gels than their apparent membrane-bound correspondents, release could either be due to, or accompanied by, minor changes in molecular structure.     

The nucleoside diphosphate kinase D (NM23-H4) binds the inner mitochondrial membrane with high affinity to cardiolipin and couples nucleotide transfer with respiration

Nucleoside diphosphate kinase (NDPK/Nm23), responsible for intracellular di- and triphosphonucleoside homeostasis, plays multiple roles in cellular energetics, signaling, proliferation, differentiation and tumor invasion. The only human NDPK with a mitochondrial targeting sequence is NDPK-D, the NME4 gene product, which is a peripheral protein of mitochondrial membranes. Subfractionation of rat liver and HEK 293 cell mitochondria revealed that NDPK-D is essentially bound to the inner membrane. Surface plasmon resonance analysis of the interaction using recombinant NDPK-D and model liposomes showed that NDPK-D interacts electrostatically with anionic phospholipids, with highest affinity observed for cardiolipin. Mutation of the central arginine (Arg-90) in a surface-exposed basic RRK motif unique to NDPK-D strongly reduced interaction with anionic phospholipids. Due to its symmetrical hexameric structure, NDPK-D was able to cross-link anionic phospholipid-containing liposomes, suggesting that NDPK-D could promote intermembrane contacts. Latency assays with isolated mitochondria and antibody binding to mitoplasts indicated a dual orientation for NDPK-D. In HeLa cells, stable expression of wild type but not of the R90D mutant led to membrane-bound enzyme in vivo. Respiration was significantly stimulated by the NDPK substrate TDP in mitochondria containing wild-type NDPK-D, but not in those expressing the R90D mutant, which is catalytically equally active. This indicates local ADP regeneration in the mitochondrial intermembrane space and a tight functional coupling of NDPK-D with oxidative phosphorylation that depends on its membrane-bound state.     

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.     

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.     

Transmembrane topology,genes, and biogenesis of the mitochondrial phosphate and oxoglutarate carriers

Phosphate and oxoglutarate carriers transport phosphate and oxoglutarate across the inner membranes of mitochondria in exchange for OH^– and malate, respectively. Both carriers belong to the mitochondrial carrier protein family, characterized by a tripartite structure made up of related sequences about 100 amino acids in length. The results obtained on the topology of the phosphate and oxoglutarate carriers are consistent with the six -helix model proposed by Saraste and Walker. In both carriers the N- and C-terminal regions are exposed toward the cytosol. In addition, the oxoglutarate carrier has been shown to be a dimer by means of cross-linking studies. The bovine and human genes coding for the oxoglutarate carrier are split into eight and six exons, respectively, and five introns are found in the same position in both genes. The bovine and human phosphate carrier genes have the same organization with nine exons separated by eight introns at exactly the same positions. The phosphate carrier of mammalian mitochondria is synthesized with a cleavable presequence, in contrast to the oxoglutarate carrier and the other members of the mitochondrial carrier family. The precursor of the phosphate carrier is efficiently imported, proteolytically processed, and correctly assembled in isolated mitochondria. The presequence-deficient phosphate carrier is imported with an efficiency of about 50% as compared with the precursor of the phosphate carrier and is correctly assembled, demonstrating that the mature portion of the phosphate carrier contains sufficient information for import and assembly into mitochondria.