Structural and functional implications of the instability of the ADP/ATP transporter purified from mitochondria as revealed by FTIR spectroscopy

The ADP/ATP transporter shows a high instability when solubilized, making it difficult to obtain functional protein with sufficient purity for long-term spectroscopic studies. When solubilized in the detergent dodecyl maltoside the protein is in equilibrium between the so-called CATR and BA conformations and in a few hours it becomes nonfunctional, unable to bind either its inhibitors or its substrates. By Fourier transform infrared spectroscopy, we studied the structural changes involved in this denaturation process. To do so, the carboxyatractyloside-inhibited protein was used as a structural model for the protein in the CATR conformation and its spectrum was compared with that of the unliganded time-inactivated protein. From the difference spectra of the amide I, amide II, and amide A bands combined with dichroism spectra of the carboxyatractyloside-inhibited protein, we concluded that few structural differences exist between both states, affecting as few as 11 amino acids (3.5% of the protein); the structural changes consisted in the disappearance of large loop structure and the appearance of aggregated strands. We hypothesize that some mitochondrial loop (tentatively loop M1) shows a high tendency to aggregate, being responsible for the observed features. The functional consequences of this hypothesis are discussed.     

Conformation-dependent swinging of the matrix loop m2 of the mitochondrial Saccharomyces cerevisiae ADP/ATP carrier

Structure-function relationships of the membrane-embedded Saccharomyces cerevisiae mitochondrial ADP/ATP carrier were investigated through two independent approaches, namely, limited proteolysis and cysteine labeling. Experiments were carried out in the presence of either carboxyatractyloside (CATR) or bongkrekic acid (BA), two specific inhibitors of the ADP/ATP transport that bind to two distinct conformers involved in the translocation process. The proteolysis approach allowed us to demonstrate (i) that N- and C-terminal extremities of ADP/ATP carrier are facing the intermembrane space and (ii) that the central region of the carrier corresponding to the matrix loop m2 is accessible to externally added trypsin in a conformation-sensitive manner, being cleaved at the Lys163-Gly164 and Lys178-Thr179 bonds in the carrier-CATR and the carrier-BA complexes, respectively. The cysteine labeling approach was carried out on the S161C mutant of the ADP/ATP carrier. This variant of the carrier is fully active, displaying nucleotide transport kinetic parameters and inhibitor binding properties similar to that of wild-type carrier. Alkylation experiments, carried out on mitochondria with the nonpermeable reagents eosin-5-maleimide and iodoacetamidyl-3,6-dioxaoctanediamine-biotin, showed that Cys 161 is accessible from the outside in the carrier-CATR complex, whereas it is masked in the carrier-BA complex. Taken together, our results indicate that the matrix loop m2 connecting the transmembrane helices H3 to H4 intrudes to some extent into the inner mitochondrial membrane. Its participation in the translocation of ADP/ATP is strongly suggested, based on the finding that its accessibility to reagents added outside mitochondria is modified according to the conformational state of the carrier.     

Effect of habitat and landscape structure on waterbird abundance in wetlands of central Argentina

Determining the response of birds to local habitat characteristics and landscape structure is essential to understanding habitat selection and its consequences for the distribution of species. This study identified the influence of environmental factors as determinants of the waterbird assemblage composition in 39 wetlands in the Pampas of central Argentina. Multivariate analysis allowed the identification of environmental factors affecting the use of habitat by waterbird species, whose variable numbers were explained by local and landscape environmental factors. Interspecific variation in responses to changes in environmental factors shows that habitat selection occurs at a species-specific level, although species with similar ecological requirements tend to respond similarly to environmental heterogeneity. Plovers and flamingos were mostly associated with high salinity and the presence of a muddy shoreline and temporary ponds; ducks, herons and ibises were associated with vegetation abundance and decrease in salinity; piscivores and carnivorous species were associated with water depth. The small degree of overlap of habitat requirements between some species suggests a high level of specialization within waterbird assemblages. This knowledge can be used for the design of appropriate conservation and management strategies in central Argentina, where the alarming loss of wetlands requires management strategies that ensure the permanence of the greatest diversity of waterbirds.     

Control of oxidative phosphorylation in rat heart mitochondria: The role of the adenine nucleotide carrier

1. Inhibitor titration experiments carried out with carboxyatractyloside, oligomycin and rotenone show that in the case of heart mitochondria the membrane-bound ATPase and the respiratory chain are the major factors controlling the rate of oxidative phosphorylation whereas the adenine nucleotide carrier exhibits no control strength. 2. As shown by carboxyatractyloside titration curves under different conditions, the relative importance of the adenine nucleotide carrier depends on the mode of regeneration (F₁-ATPase or glucose plus hexokinase) of ADP from ATP exported outside mitochondria, on the total concentration of adenine nucleotides present in the medium and on the mode of limitation of the rate of respiration (cyanide, rotenone, oligomycin or mersalyl). 3. Concomitantly with the inhibition of O₂ consumption, carboxyatractyloside brings about a rise in membrane potential. The inverse relationship between the two processes is observed for carboxyatractyloside concentrations ranging between 0.7 and 1.5 nmol per mg protein. Carboxyatractyloside concentrations below and above this range increase the membrane potential without affecting significantly the rate of respiration. 4. Titration experiments aimed at comparing the effects of ADP, carboxyatractyloside and the uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, corroborate the conclusion that in heart mitochondria a major limiting factor in oxidative phosphorylation is the capacity of the respiratory chain.     

Topological analysis of ATAD3A insertion in purified human mitochondria

ATAD3 is a mitochondrial inner membrane-associated protein that has been predicted to be an ATPase but from which no associated function is known. The topology of ATAD3 in mitochondrial membranes is not clear and subject to controversy. A direct interaction of the N-terminal domain (amino-acids 44–247) with the mtDNA has been described, but the same domain has been reported to be sensitive to limited proteolysis in purified mitochondria. Furthermore, ATAD3 has been found in a large purified nucleoid complex but could not be cross-linked to the nucleoid. To resolve these discrepancies we used two immunological approaches to test whether the N-terminal (amino-acids 40–53) and the C-terminal (amino-acids 572–586) regions of ATAD3 are accessible from the cytosol. Using N-terminal and C-terminal specific anti-peptide antibodies, we carried out back-titration ELISA measurements and immuno-fluorescence analysis on freshly purified human mitochondria. Both approaches showed that the N-terminal region of ATAD3A is accessible to antibodies in purified mitochondria. The N-terminal region of ATAD3A is thus probably in the cytoplasm or in an accessible intermembrane space. On the contrary, the C-terminal region is not accessible to the antibody and is probably located within the matrix. These results demonstrate both that the N-terminal part of ATAD3A is outside the inner membrane and that the C-terminal part is inside the matrix.     

Conformational Dynamics of the Bovine Mitochondrial ADP/ATP Carrier Isoform 1 Revealed by Hydrogen/Deuterium Exchange Coupled to Mass Spectrometry

The mitochondrial adenine nucleotide carrier (Ancp) catalyzes the transport of ADP and ATP across the mitochondrial inner membrane, thus playing an essential role in cellular energy metabolism. During the transport mechanism the carrier switches between two different conformations that can be blocked by two toxins: carboxyatractyloside (CATR) and bongkrekic acid. Therefore, our understanding of the nucleotide transport mechanism can be improved by analyzing structural differences of the individual inhibited states. We have solved the three-dimensional structure of bovine carrier isoform 1 (bAnc1p) in a complex with CATR, but the structure of the carrier-bongkrekic acid complex, and thus, the detailed mechanism of transport remains unknown. Improvements in sample processing in the hydrogen/deuterium exchange technique coupled to mass spectrometry (HDX-MS) have allowed us to gain novel insights into the conformational changes undergone by bAnc1p. This paper describes the first study of bAnc1p using HDX-MS. Results obtained with the CATR-bAnc1p complex were fully in agreement with published results, thus, validating our approach. On the other hand, the HDX kinetics of the two complexes displays marked differences. The bongkrekic acid-bAnc1p complex exhibits greater accessibility to the solvent on the matrix side, whereas the CATR-bAnc1p complex is more accessible on the intermembrane side. These results are discussed with respect to the structural and biochemical data available on Ancp.     

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.     

Chemical,immunological, enzymatic,and genetic approaches to studying the arrangement of the peptide chain of the ADP/ATP carrier in the mitochondrial membrane

In the process of oxidative phosphorylation, the exchange of cytosolic ADP^3– against mitochondrial ATP^4– across the inner mitochondrial membrane is mediated by a specific carrier protein. Two different conformations for this carrier have been demonstrated on the basis of interactions with specific inhibitors, namely carboxyatractyloside (CATR) and bongkrekic acid (BA). The two conformations, referred to as CATR and BA conformations, are interconvertible, provided that ADP or ATP are present. The functional ADP/ATP carrier is probably organized as a tetramer. In the presence of CATR or BA the tetramer is split into two dimers combined with either of the two inhibitors. The amino acid sequence of the beef heart carrier monomer (297 residues) contains three repeats of about 100 residues each. Experimental results obtained through different approaches, including photolabeling, immunochemistry, and limited proteolysis, can be interpreted on the basis of a model with five or six transmembrane helices per carrier monomer. Two mobile regions involved in the binding of nucleotides and accessible to proteolytic enzymes have been identified. Each of them may be visualized as consisting of two pairs of short amphipathic helices, which can be juxtaposed to form hydrophilic channels facilitating the nucleotide transport. Mutagenesis in yeast is currently being used to detect strategic amino acids in ADP/ATP transport.     

Nucleotide exchange in mitochondria: insight at a molecular level

Mitochondrial carrier proteins are embedded in the inner mitochondrial membrane and ensure the transport of many important metabolites. The ADP/ATP carrier imports ADP into the mitochondrial matrix in exchange for ATP after synthesis. It is the most studied mitochondrial carrier and its structure was the first to be unraveled at high resolution. The structure reveals six transmembrane helices forming a tightly closed bundle toward the matrix and a funnel-shaped cavity opening toward the intermembrane space. The cavity ends in a narrow pit 10A from the matrix. The analysis of residues located in the cavity hints at the mechanism of binding of adenine nucleotides. Additionally, the presence of conserved proline residues in three sharply kinked helices suggests a translocation mechanism.