Binding of the Fluorescein Derivative Eosin Y to the Mitochondrial ADP/ATP Carrier:  Characterization of the Adenine Nucleotide Binding Site

Majima, Eiji; Yamaguchi, Nana; Chuman, Hiroshi; Shinohara, Yasuo; Ishida, Mayumi; Goto, Satoru; Terada, Hiroshi

doi:10.1021/bi9710683

Cited by 48 publications

(49 citation statements)

References 36 publications

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“…2 56 was greatly reduced by BKA and this was taken to indicate that BKA binds close to these residues [29,30]. In support of this it was found that Eosin Y, an analogue of EMA that does not possess the thiol-reactive maleimide group, is a high affinity substrate analogue of the ANT whose binding is also inhibited by BKA but not CAT [30,36]. These data are consistent with there being a binding site for adenine nucleotides close to Cys 159 whose affinity for adenine nucleotides is conformationally dependent.…”

Section: Substrate Binding Sites and Their Sensitivity To Thiol Reagentsmentioning

confidence: 89%

“…We have proposed that these cysteines represent obvious candidates for the thiol groups that regulate both CyP-D binding and the inhibitory effects of ADP and membrane potential on the MPTP, and have provided extensive data to support this hypothesis [54,77,91]. Specific modification of Cys 159 within the adenine nucleotide binding site of the ANT with eosin maleimide not only inhibits adenine nucleotide binding and translocation by the ANT [29,36] but also almost totally abolishes the inhibition of the MPTP by ADP [54,77]. Furthermore, we have demonstrated that PAO chemically cross-links Cys 159 with Cys 256 whilst diamide causes a disulphide bond to form between these two cysteine residues through the mediation of oxidised glutathione [77].…”

Section: Thiol Groups On the Ant Play A Critical Role In Pore Openingmentioning

confidence: 97%

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The Adenine Nucleotide Translocase: A Central Component of the Mitochondrial Permeability Transition Pore and Key Player in Cell Death

Halestrap

Brenner²

2003

CMC

450

329

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In addition to its normal function, the adenine nucleotide translocase (ANT) forms the inner membrane channel of the mitochondrial permeability transition pore (MPTP). Binding of cyclophilin-D (CyP-D) to its matrix surface (probably on Pro 61 on loop 1) facilitates a calcium-triggered conformational change converting it from a specific transporter to a non-specific pore. The voltage dependent anion channel (VDAC) binds to the outer face of the ANT, at contact sites between the inner and outer membranes, and together VDAC, ANT and CyP-D probably represent the minimum MPTP configuration. The evidence for this is critically reviewed as is the structure and molecular mechanism of the carrier in its normal physiological mode. This provides helpful insights into MPTP regulation by adenine nucleotides, membrane potential and ANT ligands such as carboxyatractyloside and bongkrekic acid. Oxidative stress activates the MPTP by glutathione-mediated cross-linking of Cys 159 and Cys 256 on matrixfacing loops of the ANT that inhibits ADP binding and enhances CyP-D binding. Molecular modeling of the loop containing the ADP binding site suggests an arrangement of aspartate and glutamate residues that may provide a calcium binding site. There are other proteins that may bind to the ANT, modulating MPTP opening and hence cell death. These included members of the Bax/Bcl-2 family (both oncoproteins and tumor suppressors) and viral proteins. Vpr from HIV-1 can bind to ANT and convert it into a pro-apoptotic pore, whereas vMIA from cytomegalovirus interacts to inhibit opening. Thus the ANT may provide a molecular link between physiopathological mechanisms of infection and the regulation of MPTP function and so represents a potential therapeutic target.

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Section: Substrate Binding Sites and Their Sensitivity To Thiol Reagentsmentioning

confidence: 89%

Section: Thiol Groups On the Ant Play A Critical Role In Pore Openingmentioning

confidence: 97%

The Adenine Nucleotide Translocase: A Central Component of the Mitochondrial Permeability Transition Pore and Key Player in Cell Death

Halestrap

Brenner²

2003

CMC

450

329

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“…In essence the complimentarity between the transported solute and the intermediate (between m-and c-) states of the carrier provides the binding energy for the conformational change to occur. For example, purified ANT in CHAPS detergent adopts the m-state and is unable to bind atractylate unless ADP is added to catalyse the mstate-to-c-state conversion [45]. Thus ADP catalysis of PT pore flicker means that ANT must be in its native state between flickers ; if ANT were in a deformed state (but closed) between flickers, then the binding energy between ADP and ANT would not be available for the conformational change to the c-state to take place, and an open PT pore would not be produced.…”

Section: Adenine Nucleotide Translocase (Ant)mentioning

confidence: 99%

The mitochondrial permeability transition pore and its role in cell death

Crompton

1999

Biochemical Journal

1,810

1,267

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This article reviews the involvement of the mitochondrial permeability transition pore in necrotic and apoptotic cell death. The pore is formed from a complex of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocase and cyclophilin-D (CyP-D) at contact sites between the mitochondrial outer and inner membranes. In vitro, under pseudopathological conditions of oxidative stress, relatively high Ca2+ and low ATP, the complex flickers into an open-pore state allowing free diffusion of low-Mr solutes across the inner membrane. These conditions correspond to those that unfold during tissue ischaemia and reperfusion, suggesting that pore opening may be an important factor in the pathogenesis of necrotic cell death following ischaemia/reperfusion. Evidence that the pore does open during ischaemia/reperfusion is discussed. There are also strong indications that the VDAC-adenine nucleotide translocase-CyP-D complex can recruit a number of other proteins, including Bax, and that the complex is utilized in some capacity during apoptosis. The apoptotic pathway is amplified by the release of apoptogenic proteins from the mitochondrial intermembrane space, including cytochrome c, apoptosis-inducing factor and some procaspases. Current evidence that the pore complex is involved in outer-membrane rupture and release of these proteins during programmed cell death is reviewed, along with indications that transient pore opening may provoke ‘accidental’ apoptosis.

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“…The conformational changes induced by nucleotide binding and stabilizing the nucleotide-Ancp complex are probably not mediated by movements around Ser-147. This residue is located in TMH3, which is directly linked to matrix loop m2, which was supposed to be the primary nucleotide binding site from the matrix space (43,44). However, as can be drawn from exchange measurements, Ser-147 participates in the conformational changes necessary to translocation although less importantly than Pro-43.…”

Section: Discussionmentioning

confidence: 99%

The Transmembrane Prolines of the Mitochondrial ADP/ATP Carrier Are Involved in Nucleotide Binding and Transport and Its Biogenesis

Babot

Blancard

Pélosi

et al. 2012

Journal of Biological Chemistry

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Background:The role of prolines in transmembrane helices (TMH) of mitochondrial ADP/ATP carrier is investigated. Results: They play the role of hinges during nucleotide transport and biogenesis. Conclusion: Pro-kinks in TMH allow carrier plasticity and biogenesis. Significance: TMH proline mutations induce deleterious effects in ADP/ATP carrier import and in mitochondrial biogenesis.The mitochondrial ADP/ATP carrier (Ancp) is a paradigm of the mitochondrial carrier family, which allows cross-talk between mitochondria, where cell energy is mainly produced, and cytosol, where cell energy is mainly consumed. The members of this family share numerous structural and functional characteristics. Resolution of the atomic structure of the bovine Ancp, in a complex with one of its specific inhibitors, revealed interesting features and suggested the involvement of some particular residues in the movements of the protein to perform translocation of nucleotides from one side of the membrane to the other. They correspond to three prolines located in the oddnumbered transmembrane helices (TMH), Pro-27, Pro-132, and Pro-229. The corresponding residues of the yeast Ancp (Pro-43, Ser-147, and Pro-247) were mutated into alanine or leucine, one at a time and analysis of the various mutants evidenced a crucial role of Pro-43 and Pro-247 during nucleotide transport. Beside, replacement of Ser-147 with proline does not inactivate Ancp and this is discussed in view of the conservation of the three prolines at equivalent positions in the Ancp sequences. These prolines belong to the signature sequences of the mitochondrial carriers and we propose they play a dual role in the mitochondrial ADP/ATP carrier function and biogenesis. Unexpectedly their mutations cause more general effects on mitochondrial biogenesis and morphology, as evidenced by measurements of respiratory rates, cytochrome contents, and also clearly highlighted by fluorescence microscopy.The mitochondrial ADP/ATP carrier (Ancp), 3 localized in the mitochondrial inner membrane (MIM), is a key energetic link between cytosol and mitochondria as it provides the mitochondrial ATP synthase with its substrate, ADP or ATP, according to the cell physiological conditions, in exchange for the reaction product. Ancp is the paradigm of the mitochondrial carrier family (MCF) and its atomic structure was solved at high resolution in 2003 (1). It has been since used for homology modeling of other members of the MCF (2-6).Although the three-dimensional structure of the bovine Anc1p (BfAnc1p) is that of an inhibited carrier, we could learn a lot from it. However, it is now necessary to go further to get insights into the precise mechanism of the nucleotide exchange. Ancp is very specifically and efficiently inhibited by two classes of inhibitors whose representative members are carboxyatractyloside (CATR) for one class, and bongkrekic acid (BA) for the other one. CATR binds only to the cytosolic side of the ADP/ATP carrier, whereas BA binds only to the matrix side. The extensive use of thes...

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Binding of the Fluorescein Derivative Eosin Y to the Mitochondrial ADP/ATP Carrier: Characterization of the Adenine Nucleotide Binding Site

Cited by 48 publications

References 36 publications

The Adenine Nucleotide Translocase: A Central Component of the Mitochondrial Permeability Transition Pore and Key Player in Cell Death

The Adenine Nucleotide Translocase: A Central Component of the Mitochondrial Permeability Transition Pore and Key Player in Cell Death

The mitochondrial permeability transition pore and its role in cell death

The Transmembrane Prolines of the Mitochondrial ADP/ATP Carrier Are Involved in Nucleotide Binding and Transport and Its Biogenesis

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