Gérard Brandolin

The mitochondrial ADP/ATP carrier: structural, physiological and pathological aspects.

Under the conditions of oxidative phosphorylation, the mitochondrial ADP/ATP carrier catalyses the one to one exchange of cytosolic ADP against matrix ATP across the inner mitochondrial membrane. The ADP/ATP transport system can be blocked very specifically by two families of inhibitors: atractyloside (ATR) and carboxyatractyloside (CATR) on one hand, and bongkrekic acid (BA) and isobongkrekic acid (isoBA) on the other hand. It is well established that these inhibitors recognise two different conformations of the carrier protein, the CATR- and BA-conformations, which exhibit different chemical, immunochemical and enzymatic reactivities. The reversible transition of the ADP/ATP carrier between the two conformations was studied by fluorometric techniques. This transconversion, which is only triggered by transportable nucleotides, is probably the same as that which occurs during the functioning of ADP/ATP transport system. The fluorometric approach, using the tryptophanyl residues of the yeast carrier as intrinsic fluorescence probes, was combined to a mutagenesis approach to elucidate the ADP/ATP transport mechanism at the molecular level. Finally, recent reports that myopathies might result from defect in ADP/ATP transport led us to develop a method to quantify the carrier protein in muscular biopsies.

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 ADP3− against mitochondrial ATP4− 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 α 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 α 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.

Effects of p47phox C Terminus Phosphorylations on Binding Interactions with p40phox and p67phox STRUCTURAL AND FUNCTIONAL COMPARISON OF p40phox and p67phox SH3 DOMAINS

The neutrophil NADPH oxidase produces superoxide anions in response to infection. This reaction is activated by association of cytosolic factors, p47phox and p67phox, and a small G protein Rac with the membranous flavocytochrome b558. Another cytosolic factor, p40phox, is associated to the complex and is reported to play regulatory roles. Initiation of the NADPH oxidase activation cascade has been reported as consecutive to phosphorylation on serines 359/370 and 379 of the p47phox C terminus. These serines surround a polyproline motif that can interact with the Src homology 3 (SH3) module of p40phox (SH3p40) or the C-terminal SH3 of p67phox (C-SH3p67). The latter one presents a higher affinity in the resting state for p47phox. A change in SH3 binding preference following phosphorylation has been postulated earlier. Here we report the crystal structures of SH3p40 alone or in complex with a 12-residue proline-rich region of p47phox at 1.46 Å resolution. Using intrinsic tryptophan fluorescence measurements, we compared the affinity of the strict polyproline motif and the whole C terminus peptide with both SH3p40 and C-SH3p67. These data reveal that SH3p40 can interact with a consensus polyproline motif but also with a noncanonical motif of the p47phox C terminus. The electrostatic surfaces of both SH3 are very different, and therefore the binding preference for C-SH3p67 can be attributed to the polyproline motif recognition and particularly to the Arg-368p47 binding mode. The noncanonical motif contributes equally to interaction with both SH3. The influence of serine phosphorylation on residues 359/370 and 379 on the affinity for both SH3 domains has been checked. We conclude that contrarily to previous suggestions, phosphorylation of Ser-359/370 does not modify the SH3 binding affinity for both SH3, whereas phosphorylation of Ser-379 has a destabilizing effect on both interactions. Other mechanisms than a phosphorylation induced switch between the two SH3 must therefore take place for NADPH oxidase activation cascade to start.

Effective removal of nonionic detergents in protein mass spectrometry, hydrogen/deuterium exchange, and proteomics.

Detergents are frequently used for protein isolation and solubilization. Their presence is crucial in membrane protein protocols or in lipid raft proteomics. However, they are usually poorly compatible with mass spectrometry. Several different sample preparation protocols are routinely used, but they are either laborious or suffer from sample losses. Here, we describe our alternative method for nonionic detergent removal. It is based on selective detergent extraction after capture of the sample on a reversed phase cartridge. The extraction is performed by chlorinated solvents and works well for polyoxyethylene based nonionic detergents, but also for polymers like polyethylene and propylene glycol. Detergent removal can be also carried out on the protein level but a special care must be taken with hydrophobic proteins. In such cases, it is preferable to perform detergent removal after proteolysis which digests the protein to peptides and reduces the hydrophobicity. The method can easily be automated and is compatible with hydrogen/deuterium exchange coupled to mass spectrometry.

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.

A covalent tandem dimer of the mitochondrial ADP/ATP carrier is functional in vivo.

The adenine nucleotide carrier, or Ancp, is an integral protein of the inner mitochondrial membrane. It is established that the inactive Ancp bound to one of its inhibitors (CATR or BA) is a dimer, but different contradictory models were proposed over the past years to describe the organization of the active Ancp. In order to decide in favor of a single model, it is necessary to establish the orientations of the N- and C-termini and thus the parity of the Ancp transmembrane segments (TMS). According to this, we have constructed a gene encoding a covalent tandem dimer of the Saccharomyces cerevisiae Anc2p and we demonstrate that it is stable and active in vivo as well as in vitro. The properties of the isolated dimer are strongly similar to those of the native Anc2p, as seen from nucleotide exchange and inhibitor binding experiments. We can therefore conclude that the native Anc2p has an even number of TMS and that the N- and C-terminal regions are exposed to the same cellular compartment. Furthermore, our results support the idea of a minimal dimeric functional organization of the Ancp in the mitochondrial membrane and we can suggest that TMS 1 of one monomer and TMS 6 of the other monomer in the native dimer are very close to each other.

Photoaffinity labeling of the adenine nucleotide carrier in heart and yeast mitochondria by an arylazido ADP analog

Abstract 1. 1. Arylazido analogs of ADP and ATP ( N -4-azido-2-nitrophenyl-aminobutyryl-ADP and N -4-azido-2-nitrophenylaminobutyryl-ATP have been prepared in radioactive form and used in photolabeling experiments to identify the adenine nucleotide carrier in mitochondria and sonic submitochondrial particles. 2. 2. When added in the dark to beef heart mitochondria, azidonitrophenyl-aminobutyryl-ADP binds to the adenine-nucleotide carrier. It is not transported across the membrane to the matrix space, but it inhibits ADP transport in mito-chondria. The inhibition is of a mixed type with a K i value of about 10 μM. 3. 3. The nitrene derivative formed upon photoirradiation of tritiated azidonitrophenylaminobutyryl-ADP or -ATP binds to a polypeptide of apparent molecular weight 30 000 in beef heart mitochondria and 37 000 in Saccharomyces cerevisiae mitochondria. The photolabeling is prevented by preincubation of the mitochondria with atractyloside or carboxyatractyloside. 4. 4. Photoirradiation of sonic submitochondrial particles from beef heart (inside-out particles) with tritiated azidonitrophenylaminobutyryl-ADP or -ATP results in the labeling of the 30 000-dalton polypeptide and also in the labeling of higher molecular weight peptides (50 000–55 000) probably belonging to F 1 -ATPase. Addition of bongkrekic acid specifically decreases the photolabeling of the 30 000-dalton polypeptide. 5. 5. An arylazido derivative of atractyloside ( N -4-azido-2-nitrophenylaminobutyryl atractyloside) binds upon photoirradiation to the 30 000-dalton polypeptide in beef heart mitochondria and to the 37 000-dalton polypeptide in S. cerevisiae mitochondria. 6. 6. Since the adenine nucleotide carrier is readily damaged by ultraviolet light, nitro-arylazido analogs of ADP and ATP or of atractyloside, which are photoactivated in visible light, were used in preference to other azido analogs, which require ultraviolet light for photoactivation. 7. 7. Data presented in this paper support the view that the same mitochondrial protein belonging to the adenine nucleotide transport system is able to bind ADP (or ATP) and atractyloside.

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.

Aryl-azido atractylosides as photoaffinity labels for the mitochondrial adenine nucleotide carrier

It is well known that atractyloside (ATR) and carboxyatractyloside (CATR) are potent and specific inhibitors of ADP transport in mitochondria [1,2]. Labeled ATR or CATR have been used for the purification of an ATRor CATR-binding protein [3-5 ]. Isolation of the ATRor CATR-protein complex was made possible by mild techniques of purification which do not dissociate the inhibitor from its binding site in the protein. These techniques include affinity chromatography [3] and hydroxyapatite chromatography [4]. In contrast strong detergents, such as sodium dodecylsulfate (SDS) resulted in the displacement of the bound inhibitor and therefore prevented the possibility to characterize the inhibitor-protein complex by SDS polyacrylamide gel electrophoresis (SPAGE). To obviate this difficulty, it was necessary to bind covalently labeled ATR or CATR to its specific binding protein. This paper describes the synthesis of a photoactive aryl-azido-ATR (N4-azido-2-nitrophenylaminobutyryl-ATR or NAP4-ATR) and its use to label covalently the ADP carrier in mitochondria. It is shown that NAP4-ATR, in the dark, inhibits competitively ADP transport with the same efficiency as ATR and competes with ATR for binding to mitochondria. The nitrene derivative formed upon irradiation of NAP4-ATR binds covalently to mitochondria. By this means, the ATR-binding protein has been characterized by SPAGE; its molecular weight is about 30 000. 2. Materials and methods

Mitochondrial Adaptation to in vivo Polyunsaturated Fatty Acid Deficiency: Increase in Phosphorylation Efficiency

Polyunsaturated fatty acid (PUFA) deficiency affects respiratory rate both in isolated mitochondria and in hepatocytes, an effect that is normally ascribed to major changes in membrane composition causing, in turn, protonophoriclike effects. In this study, we have compared the properties of hepatocytes isolated from PUFA-deficient rats with those from control animals treated with concentrations of the protonophoric uncoupler 2,4-dinitrophenol (DNP). Despite identical respiratory rate and in situ mitochondrial membrane potential (ΔΨ), mitochondrial and cytosolic ATP/ADP–Pi ratios were significantly higher in PUFA-deficient cells than in control cells treated with DNP. We show that PUFA-deficient cells display an increase of phosphorylation efficiency, a higher mitochondrial ATP/ADP–Pi ratio being maintained despite the lower ΔΨ. This is achieved by (1) decreasing mitochondrial Pi accumulation, (2) increasing ATP synthase activity, and (3) by increasing the flux control coefficient of adenine nucleotide translocation. As a consequence, oxidative phosphorylation efficiency was only slightly affected in PUFA-deficient animals as compared to protonophoric uncoupling (DNP). Thus, the energy waste induced by PUFA deficiency on the processes that generate the proton motive force (pmf) is compensated in vivo by powerful adaptive mechanisms that act on the processes that use the pmf to synthesize ATP.