Functional integration of mitochondrial and hydrogenosomal ADP/ATP carriers in the Escherichia coli membrane reveals different biochemical characteristics for plants, mammals and anaerobic chytrids.

The expression of mitochondrial and hydrogenosomal ADP/ATP carriers (AACs) from plants, rat and the anaerobic chytridiomycete fungus Neocallimastix spec. L2 in Escherichia coli allows a functional integration of the recombinant proteins into the bacterial cytoplasmic membrane. For AAC1 and AAC2 from rat, apparent Km values of about 40 microm for ADP, and 105 microm or 140 microm, respectively, for ATP have been determined, similar to the data reported for isolated rat mitochondria. The apparent Km for ATP decreased up to 10-fold in the presence of the protonophore m-chlorocarbonylcyanide phenylhydrazone (CCCP). The hydrogenosomal AAC isolated from the chytrid fungus Neocallimastix spec. L2 exhibited the same characteristics, but the affinities for ADP (165 microm) and ATP (2.33 mm) were significantly lower. Notably, AAC1-3 from Arabidopsis thaliana and AAC1 from Solanum tuberosum (potato) showed significantly higher external affinities for both nucleotides (10-22 microm); they were only slightly influenced by CCCP. Studies on intact plant mitochondria confirmed these observations. Back exchange experiments with preloaded E. coli cells expressing AACs indicate a preferential export of ATP for all AACs tested. This is the first report of a functional integration of proteins belonging to the mitochondrial carrier family (MCF) into a bacterial cytoplasmic membrane. The technique described here provides a relatively simple and highly reproducible method for functional studies of individual mitochondrial-type carrier proteins from organisms that do not allow the application of sophisticated genetic techniques.     

The human mitochondrial ADP/ATP carriers: kinetic properties and biogenesis of wild-type and mutant proteins in the yeast S. cerevisiae

The mitochondrial adenine nucleotide carrier, or Ancp, plays a key role in the maintenance of the energetic fluxes in eukaryotic cells. Human disorders have been found associated to unusual human ANC gene (HANC) expression but also to direct inactivation of the protein, either by autoantibody binding or by mutation. However, the individual biochemical properties of the three HAncp isoforms have not yet been deciphered. To do so, the three HANC ORF were expressed in yeast under the control of the regulatory sequences of ScANC2. Each of the three HANC was able to restore growth on a nonfermentable carbon source of a yeast mutant strain lacking its three endogenous ANC. Their ADP/ATP exchange properties could then be measured for the first time in isolated mitochondria. HANC3 was the most efficient to restore yeast growth, and HAnc3p presented the highest V(M) (80 nmol ADP min(-1) mg protein(-1)) and K(ADP)(M)(8.4 microM). HAnc1p and HAnc2p presented similar kinetic constants (V(M) approximately 30-40 nmol ADP min(-(1) mg protein(-1) and K(ADP)(M) approximately 2.5-3.7 microM), whose values were consistent with HANC1's and HANC2's lower capacity to restore yeast growth. However, the HANC genes restored growth at a lower level than ScANC2, indicating that HAncp amount may be limiting in vivo. To optimize the HAncp production, we investigated their biogenesis into mitochondria by mutagenesis of two charged amino acids in the N-terminus of HAnc1p. Severe effects were observed with the D3A and D3K mutations that precluded yeast growth. On the contrary, the K10A mutation increased yeast growth complementation and nucleotide exchange rate as compared to the wild type. These results point to the importance of the N-terminal region of HAnc1p for its biogenesis and transport activity in yeast mitochondria.     

Structural properties of mammalian mitochondrial ADP/ATP carriers: identification of possible amino acids that determine functional differences in its isoforms

The structural properties of isoforms of the mitochondrial ADP/ATP carrier expressed in mammals were characterized in order to understand their possible functional differences. To accomplish this, the cDNA clone of the bovine type 2 isoform was isolated and characterized. We also extensively explored the rat type 3 isoform, but it was not detected. We next compared the amino acid sequences of the ten ADP/ATP carriers, which are expressed in mammals. As a result, amino acids at positions 45, 147 and 164 were found to show strict isoform dependency regardless of species differences. Thus, they are expected to determine functional differences in the isoforms of the ADP/ATP carrier.     

Suggested mitochondrial ancestry of non-mitochondrial ATP/ADP

One of the major evolutionary events that transformed endosymbiotic bacterium into mitochondrion was an acquisition of ATP/ADP carrier in order to supply the host with respiration-derived ATP. Along with mitochondrial carrier, unrelated carrier is known which is characteristic of intracellular chlamydiae, plastids, parasitic intracellular eukaryote Encephalitozoon cuniculi, and the genus Rickettsia of obligate endosymbiotic alpha-Proteobacteria. This non-mitochondrial ATP/ADP carrier was recently described in rickettsia-like endosymbionts - a group of obligate intracellular bacteria, classified with the order Rickettsiales, which have diverged after free-living alpha-Proteobacteria but before sister groups of the Rickettsiaceae assemblage (true rickettsiae) and mitochondria. Published controversial phylogenetic data on the non-mitochondrial carrier were reanalysed in the present work using both DNA and protein sequences, and various methods including Bayesian analysis. The data presented are consistent with classic endosymbiont theory for the origin of mitochondria and also suggest that even last but one common ancestor of rickettsiae and organelles may have been an endosymbiotic bacterium in which ATP/ADP carrier has first originated.     

A divergent ADP/ATP carrier in the hydrogenosomes of Trichomonas gallinae argues for an independent origin of these organelles

The evolution of mitochondrial ADP and ATP exchanging proteins (AACs) highlights a key event in the evolution of the eukaryotic cell, as ATP exporting carriers were indispensable in establishing the role of mitochondria as ATP-generating cellular organelles. Hydrogenosomes, i.e. ATP- and hydrogen-generating organelles of certain anaerobic unicellular eukaryotes, are believed to have evolved from the same ancestral endosymbiont that gave rise to present day mitochondria. Notably, the hydrogenosomes of the parasitic anaerobic flagellate Trichomonas seemed to be deficient in mitochondrial-type AACs. Instead, HMP 31, a different member of the mitochondrial carrier family (MCF) with a hitherto unknown function, is abundant in the hydrogenosomal membranes of Trichomonas vaginalis. Here we show that the homologous HMP 31 of closely related Trichomonas gallinae specifically transports ADP and ATP with high efficiency, as do genuine mitochondrial AACs. However, phylogenetic analysis and its resistance against bongkrekic acid (BKA, an efficient inhibitor of mitochondrial-type AACs) identify HMP 31 as a member of the mitochondrial carrier family that is distinct from all mitochondrial and hydrogenosomal AACs studied so far. Thus, our data support the hypothesis that the various hydrogenosomes evolved repeatedly and independently.     

Valine 181 is critical for the nucleotide exchange activity of human mitochondrial ADP/ATP carriers in yeast

We isolated yeast Saccharomyces cerevisiae cells transformed with one of the three human adenine nucleotide carrier genes (HANC) that exhibited higher growth capacity than previously observed. The HANC genes were isolated from these clones, and we identified two independent mutations of HANC that led to replacement of valine 181 located in the fourth transmembrane segment by methionine or phenylalanine. Tolerance of this position toward substitution with various amino acids was systematically investigated, and since HANC/V181M was among the most efficient in growth complementation, it was more extensively studied. Here we show that increased growth capacities were associated with higher ADP/ATP exchange activities and not with higher human carrier amount in yeast mitochondria. These results are discussed in the light of the bovine Ancp structure, that shares more than 90% amino acid identity with Hancps, and its interaction with the lipid environment.     

Yeast mitochondrial ADP/ATP carriers are monomeric in detergents as demonstrated by differential affinity purification

Most mitochondrial carriers carry out equimolar exchange of substrates and they are believed widely to exist as homo-dimers. Here we show by differential tagging that the yeast mitochondrial ADP/ATP carrier AAC2 is a monomer in mild detergents. Carriers with and without six-histidine or hemagglutinin tags were co-expressed in defined molar ratios in yeast mitochondrial membranes. Their specific transport activity was unaffected by tagging or by co-expression. The co-expressed carriers were extracted from the membranes with mild detergents and purified rapidly by affinity chromatography. All of the untagged carriers were in the flow-through of the affinity column, whereas all of the tagged carriers bound to the column and were eluted subsequently, showing that stable dimers, consisting of associated tagged and untagged carriers, were not present. The specific inhibitors carboxyatractyloside and bongkrekic acid and the substrates ADP, ATP and ADP plus ATP were added during the experiments to determine whether lack of association might have been caused by carriers being prevented from cycling through the various states in the transport cycle where dimers might form. All of the protein was accounted for, but stable dimers were not detected in any of these conditions, showing that yeast ADP/ATP carriers are monomeric in detergents in agreement with their hydrodynamic properties and with their structure. Since strong interactions between monomers were not observed in any part of the transport cycle, it is highly unlikely that the carriers function cooperatively. Therefore, transport mechanisms need to be considered in which the carrier is operational as a monomer.     

Conformational dynamics of the mitochondrial ADP/ATP carrier: a simulation study

The mitochondrial ADP/ATP carrier is a six helix bundle membrane transport protein, which couples the exit of ATP from the mitochondrial matrix to the entry of ADP. Extended (4x20 ns) molecular dynamics simulations of the carrier, in the presence and absence of bound inhibitor (carboxyatractyloside), have been used to explore the conformational dynamics of the protein in a lipid bilayer environment, in the presence and absence of the carboxyatractyloside inhibitor. The dynamic flexibility (measured as conformational drift and fluctuations) of the protein is reduced in the presence of bound inhibitor. Proline residues in transmembrane helices H1, H3 and H5 appear to form dynamic hinges. Fluctuations in inter-helix salt bridges are also observed over the time course of the simulations. Inhibitor-protein and lipid-protein interactions have been characterised in some detail. Overall, the simulations support a transport mechanism in which flexibility about the proline hinges enables a transition between a 'closed' and an 'open' pore-like state of the carrier protein.     

Conformational dynamics of the mitochondrial ADP/ATP carrier: a simulation study

The mitochondrial ADP/ATP carrier is a six helix bundle membrane transport protein, which couples the exit of ATP from the mitochondrial matrix to the entry of ADP. Extended (4×20 ns) molecular dynamics simulations of the carrier, in the presence and absence of bound inhibitor (carboxyatractyloside), have been used to explore the conformational dynamics of the protein in a lipid bilayer environment, in the presence and absence of the carboxyatractyloside inhibitor. The dynamic flexibility (measured as conformational drift and fluctuations) of the protein is reduced in the presence of bound inhibitor. Proline residues in transmembrane helices H1, H3 and H5 appear to form dynamic hinges. Fluctuations in inter-helix salt bridges are also observed over the time course of the simulations. Inhibitor-protein and lipid-protein interactions have been characterised in some detail. Overall, the simulations support a transport mechanism in which flexibility about the proline hinges enables a transition between a ‘closed’ and an ‘open’ pore-like state of the carrier protein.     

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.     

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.     

Suggested mitochondrial ancestry of nonmitochondrial ATP/ADP carrier

One of the major evolutionary events that transformed an endosymbiotic bacterium into a mitochondrion was the acquisition of the ATP/ADP carrier (AAC) in order to supply the host with respiration-derived ATP. Along with the mitochondrial carrier, an unrelated carrier is known, which is characteristic of intracellular chlamydiae, plastids, parasitic intracellular eukaryote Encephalitozoon cuniculi, and the genus Rickettsia of obligate endosymbiotic α-proteobacteria. This nonmitochondrial carrier was recently described in rickettsia-like endosymbionts (RLE), a group of obligate intracellular bacteria classified with the order Rickettsiales, which have diverged after free-living α-proteobacteria but before sister groups of the Rickettsiaceae assemblage (true rickettsiae) and mitochondria. Published controversial phylogenetic data on nonmitochondrial AAC were re-analyzed in the present work, using both DNA and protein sequences and various methods including Bayesian analysis. The data presented are consistent with the classic endosymbiont theory for the origin of mitochondria and suggest that even the last but one common ancestor of rickettsiae and organelles was an endosymbiotic bacterium, in which AAC first originated.     

Structural basis for lipid-mediated interactions between mitochondrial ADP/ATP carrier monomers

The oligomerization state of the ADP/ATP carrier is an important issue in understanding the mechanism underlying nucleotide exchange across the inner mitochondrial membrane. The first high resolution structure obtained in the presence of carboxyatractyloside revealed a large cavity formed within a monomer in which the inhibitor is strongly bound. Whereas the protein-protein interactions implicated in the first crystal form are not biologically relevant, the new crystal form described herein, highlights favorable protein-protein interactions. The interactions are mediated by endogenous cardiolipins, which are tightly bound to the protein, two cardiolipins being sandwiched between the monomers on the matrix side. The putative dimerization interface evidenced here is consistent with other structural, biochemical or functional data published so far.     

Mitochondrial ATP synthasome: three-dimensional structure by electron microscopy of the ATP synthase in complex formation with carriers for Pi and ADP/ATP

The terminal steps involved in making ATP in mitochondria require an ATP synthase (F(0)F(1)) comprised of two motors, a phosphate carrier (PIC), and an adenine nucleotide carrier (ANC). Under mild conditions, these entities sub-fractionate as an ATP synthase/PIC/ANC complex or "ATP synthasome" (Ko, Y.H., Delannoy, M, Hullihen, J., Chiu, W., and Pedersen, P.L. (2003) J. Biol. Chem. 278, 12305-12309). As a first step toward obtaining three-dimensional information about this large complex or "metabolon" and the locations of PIC and ANC therein, we dispersed ATP synthasomes into single complexes and visualized negatively stained images by electron microscopy (EM) that showed clearly the classical headpiece, central stalk, and basepiece. Parallel immuno-EM studies revealed the presence of PIC and ANC located non-centrally in the basepiece, and other studies implicated an ATP synthase/PIC/ANC stoichiometry near 1:1:1. Single ATP synthasome images (7506) were boxed, and, using EMAN software, a three-dimensional model was obtained at a resolution of 23 A. Significantly, the basepiece is oblong and contains two domains, the larger of which connects to the central stalk, whereas the smaller appears as an extension. Docking studies with known structures together with the immuno-EM studies suggest that PIC or ANC may be located in the smaller domain, whereas the other transporter resides nearby in the larger domain. Collectively, these finding support a mechanism in which the entry of the substrates ADP and P(i) into mitochondria, the synthesis of ATP on F(1), and the release and exit of ATP are very localized and highly coordinated events.     

Chimers of two fused ADP/ATP carrier monomers indicate a single channel for ADP/ATP transport

The mitochondrial ADP/ATP carrier (AAC) is generally believed to function as a homodimer (Wt. Wt). It remains unknown whether the two monomers possess two independent but fully anticooperative channels or they form a single central channel for nucleotide transport. Here we generated fusion proteins consisting of two tandem covalent-linked AAC monomers and studied the kinetics of ADP/ATP transport in reconstituted proteoliposomes. Functional 64-kDa fusion proteins Wt-Wt and Wt-R294A (wild-type AAC linked to a mutant having low ATP transport activity) were expressed in mitochondria of yeast transformants. Compared to homodimer Wt. Wt, the fusion protein Wt-Wt retained the transport activity and selectivity of ADP versus ATP. The strongly divergent selectivities of Wt and R294A were partially propagated in the Wt-R294A fusion protein, suggesting a limited cooperativity during solute translocation. The rates of ADP or ATP transport were significantly higher than those predicted by the two-channel model. Fusion proteins for Wt-R204L (Wt linked to an inactive mutant) and R204L-Wt were not expressed in aerobically grown yeast cells, which contained plasmid rearrangements that regenerated the fully active 32-kDa homodimer Wt. Wt, suggesting that these fusion proteins are inactive in ADP/ATP transport. These results favor a single binding center gated pore model [Klingenberg, M. (1991) in A Study of Enzymes, Vol. 2: pp. 367-388] in which two AAC subunits cooperate for a coordinated ADP/ATP exchange through a single channel.     

Subunits of the yeast mitochondrial ADP/ATP carrier: cooperation within the dimer

The mitochondrial ADP/ATP carrier, or Ancp, is a member of the mitochondrial carrier family (MCF). It exchanges ADP and ATP between matrix and intermembrane space. It is postulated from numerous experiments that the inactive Ancp bound to one of its inhibitors (CATR or BA) is a dimer, and it is inferred that the active unit is a dimer, too. However, the structure of beef Ancp bound to CATR obtained at high resolution is that of a monomer. To ascertain the dimeric organization of Ancp, we have constructed covalent tandem dimers of which one "subunit" (protomer) is the wild type and the other is inactive for ADP/ATP exchange. We have chosen either the op1 mutant or another member of the MCF, the phosphate carrier (Picp). Activities of the chimeras were first evaluated in vivo. The Ancp/op1 constructs exchange the adenine nucleotides. The Anc/Pic chimeras are considered as bifunctional forms since they exchange ADP and ATP and transport P(i) within the same cells. We have then controlled the fact that the chimeras are stable in vivo and in vitro. Proteinase K digestion showed that both protomers of Ancp/op1 have similar organization in the membrane. Analyses of kinetic properties indicated that protomers of Ancp/op1 chimeras crosstalk during the nucleotide exchange unlike those of Anc/Pic. However, full inhibition of phosphate uptake by CATR, a very specific inhibitor of Ancp, strongly suggests that the native functional unit of Ancp, and thus of Picp, is a dimer.     

Roles of adjoining Asp and Cys residues of first matrix-facing loop in transport activity of yeast and bovine mitochondrial ADP/ATP carriers

The mitochondrial ADP/ATP carrier (AAC) transports substrate by interconversion of its conformation between m- and c-states. The 1st loop facing the matrix (LM1) is extruded into the matrix in the m-state and is suggested to intrude into the mitochondrial membrane on conversion to the c-state conformation [Hashimoto, M., Majima, E., Goto, S., Shinohara, Y., and Terada, H. (1999) Biochemistry 38, 1050-1056]. To elucidate the mechanism of the translocation of LM1, we examined the effects of site-directed mutagenesis of two adjoining residues, Cys56 and Asp55 in the bovine type 1 AAC and Cys73 and Asp72 in the yeast type 2 AAC, on the substrate transport activity. We found that (i) replacement of the Cys by bulky and hydrophilic residues was unfavorable for efficient transport activity, (ii) the carboxyl groups of the Asp residues of the bovine and yeast AACs were essential and strictly position-specific, and (iii) hence, the mutation to Glu showed transport activity comparable to that of the native AACs. Based on these results, we discussed the functional role of LM1 in the transport activity of AAC.