The mitochondrial ATP / ADP carrier: Interaction with detergents and purification by a novel procedure

doi:10.1016/0005-2728(94)90010-8

Biochimica et Biophysica Acta (BBA) - Bioenergetics

Volume 1187, Issue 3, 27 September 1994, Pages 360-367

https://doi.org/10.1016/0005-2728(94)90010-8 Get rights and content

Abstract

The interaction of several classes of detergents with mitochondrial ATP / ADP carrier (AAC) was studied. The detergents that were best suited for solubilization of active AAC differed in several physico-chemical properties, but contained relatively rigid or planar hydrophobic (sub)moieties. Based on specific binding of AAC to Blue Sepharose, a novel method for the purification of the AAC was developed. The new method gave AAC samples which were devoid of non-essential lipids and allowed to purify AAC isoenzymes from several species and tissues to a significantly higher degree of purity than that achieved up to now. Western blot analysis of purified AACs with an antiserum against chicken heart AAC confirmed that immunological variability is more important between tissues than between species. In contrast to liver and kidney AACs, brain AAC displayed similar antigenic properties to heart AAC.

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Cited by (14)

The mitochondrial phosphate carrier interacts with cyclophilin D and may play a key role in the permeability transition
2008, Journal of Biological Chemistry
Citation Excerpt :
Thereafter, the purification followed the procedure described by Smith et al. (36) with anion and cation exchange chromatography performed on an AKTA prime system (GE Healthcare UK Ltd, Little Chalfont, HP7 9NA, UK) using 1 ml of RESOURCE™ Q and RESOURCE™ S columns, respectively. An Antibody Raised against Rat Liver ANT Recognizes a Protein Other Than ANT—In previous studies that investigated whether the MPTP could be detected in proteoliposomes containing reconstituted ANT (37), we used ANT purified from rat liver mitochondria according to the method developed by Rojo and Wallimann (38). We also used this preparation to raise an antibody against the whole ANT that reacted strongly with an inner membrane protein of 30 kDa as expected for the ANT (18).
The mitochondrial permeability transition pore (MPTP) plays a key role in cell death, yet its molecular identity remains uncertain. Although knock-out studies have confirmed critical roles for both cyclophilin-D (CyP-D) and the adenine nucleotide translocase (ANT), given a strong enough stimulus MPTP opening can occur in the absence of either. Here we provide evidence that the mitochondrial phosphate carrier (PiC) may also be a critical component of the MPTP. Phenylarsine oxide (PAO) was found to activate MPTP opening in the presence of carboxyatractyloside (CAT) that prevents ANT binding to immobilized PAO. Only four proteins from solubilized CAT-treated beef heart inner mitochondrial membranes bound to immobilized PAO, one of which was the PiC. GST-CyP-D pull-down and co-immunoprecipitation studies revealed CsA-sensitive binding of PiC to CyP-D; this increased following diamide treatment. Co-immunoprecipitation of the ANT with the PiC was also observed but was insensitive to CsA treatment. N-ethylmaleimide and ubiquinone analogues (UQ₀ and Ro 68-3400) inhibited phosphate transport into rat liver mitochondria with the same concentration dependence as their inhibition of MPTP opening. UQ₀ and Ro 68-3400 also induced the “m” conformation of the ANT, as does NEM, and reduced the binding of both the PiC and ANT to the PAO column. We propose a model for the MPTP in which a calcium-triggered conformational change of the PiC, facilitated by CyP-D, induces pore opening. An interaction of the PiC with the ANT may enable agents that bind to either transporter to modulate pore opening.
Recent progress in elucidating the molecular mechanism of the mitochondrial permeability transition pore
2008, Biochimica et Biophysica Acta - Bioenergetics
The mitochondrial permeability transition pore (MPTP) plays a key role in cell death, especially necrosis, and mediates the injury tissues such as the heart and brain experience following ischaemia and reperfusion. However, the molecular identity of the MPTP remains uncertain. Knockout studies have confirmed a role for cyclophilin-D (CyP-D) in pore opening, probably mediated by its peptidyl–prolyl cis–trans isomerase activity that facilitates a conformational change in an inner membrane protein. However, similar knockout studies have cast doubt on the central role of the adenine nucleotide translocase (ANT), previously regarded as a leading contender for the membrane component that forms the transmembrane channel of the MPTP. Here we review the evidence for and against a role for the ANT in MPTP opening and conclude that it usually plays a regulatory role rather than provide the transmembrane pore component. We suggest that the protein fulfilling the latter role is the mitochondrial phosphate carrier (PiC) and summarise recent evidence in support of this proposal. Our data are consistent with a model for the MPTP in which a calcium-triggered conformational change of the PiC, facilitated by CyP-D, induces pore opening. We propose that this is enhanced by an association of the PiC with the “c” conformation of the ANT. Agents that modulate pore opening may act on either or both the PiC and the ANT.
Bax Is Present as a High Molecular Weight Oligomer/Complex in the Mitochondrial Membrane of Apoptotic Cells
2001, Journal of Biological Chemistry
Citation Excerpt :
Cytosolic samples were centrifuged at 100,000 × g for 5 min and then treated with cross-linker and analyzed as above. Full-length Bax with a His tag at the N terminus was expressed in Escherichia coli and purified as previously described (45). Oligomerization was induced by disrupting the bacteria in buffer containing 1% Triton X-100.
Bax is a Bcl-2 family protein with proapoptotic activity, which has been shown to trigger cytochrome crelease from mitochondria both in vitro and in vivo. In control HeLa cells, Bax is present in the cytosol and weakly associated with mitochondria as a monomer with an apparent molecular mass of 20,000 Da. After treatment of the HeLa cells with the apoptosis inducer staurosporine or UV irradiation, Bax associated with mitochondria is present as two large molecular weight oligomers/complexes of 96,000 and 260,000 Da, which are integrated into the mitochondrial membrane. Bcl-2 prevents Bax oligomerization and insertion into the mitochondrial membrane. The outer mitochondrial membrane protein voltage-dependent anion channel and the inner mitochondrial membrane protein adenosine nucleotide translocator do not coelute with the large molecular weight Bax oligomers/complexes on gel filtration. Bax oligomerization appears to be required for its proapoptotic activity, and the Bax oligomer/complex might constitute the structural entirety of the cytochromec-conducting channel in the outer mitochondrial membrane.
Natural and Azido Fatty Acids Inhibit Phosphate Transport and Activate Fatty Acid Anion Uniport Mediated by the Mitochondrial Phosphate Carrier
2001, Journal of Biological Chemistry
Citation Excerpt :
The labeled mitochondria were either applied to the HTP column, or proteins contained in the HTP pass-through (38) were photolabeled in the detergent micelles with3H-AzHA, using a protocol developed for UCP1 (24, 25). Labeled proteins were further fractionated on blue Sepharose (2 mg/ml of column) using a modified procedure of Rojo and Wallimann (39). BS was prewashed, and the first four elutions (1 ml each) were also performed with 150 mm Na2SO4, 20 mm Na-HEPES, 0.2 mm Tris-EDTA, pH 7.0, containing 0.5% detergent.
The electroneutral P_i uptake via the phosphate carrier (PIC) in rat liver and heart mitochondria is inhibited by fatty acids (FAs), by 12-(4-azido-2-nitrophenylamino)dodecanoic acid (AzDA) and heptylbenzoic acid (∼1 μm doses) and by lauric, palmitic, or 12-azidododecanoic acids (∼0.1 mmdoses). In turn, reconstituted E. coli-expressed yeast PIC mediated anionic FA uniport with a similar pattern leading to FA cycling and H⁺ uniport. The kinetics of P_i/P_i exchange on recombinant PIC in the presence of AzDA better corresponded to a competitive inhibition mechanism. Methanephosphonate was identified as a new PIC substrate. Decanephosphonate, butanephosphonate, 4-nitrophenylphosphate, and other P_i analogs were not translocated and did not inhibit P_i transport. However, methylenediphosphonate and iminodi(methylenephosphonate) inhibited both electroneutral P_i uptake and FA cycling via PIC. AzDA analog 16-(4-azido-2-nitrophenylamino)-[³H₄]-hexadecanoic acid (³H-AzHA) bound upon photoactivation to several mitochondrial proteins, including the 30- and 34-kDa bands. The latter was ascribed to PIC due to its specific elution pattern on Blue Sepharose and Affi-Gel. ³H-AzHA photolabeling of recombinant PIC was prevented by methanephosphonate and diphosphonates and after premodification with 4-azido-2-nitrophenylphosphate. Hence, the demonstrated PIC interaction with monovalent long-chain FA anions, but with divalent phosphonates of short chain only, indicates a pattern distinct from that valid for the mitochondrial uncoupling protein-1.
Characterization of a dCTP transport activity reconstituted from human mitochondria
1999, Journal of Biological Chemistry
A protein fraction of mitochondria from human acute lymphocytic leukemia cells, which could be reconstituted into proteoliposomes to have dCTP transport activity, has been partially purified by hydroxyapatite and blue Sepharose chromatography. The dCTP transport activity in proteoliposomes was time-dependent and could be activated by Ca²⁺ and to a lesser extent by Mg²⁺. None of the other divalent cations tested could activate the transport activity. The K _m value of dCTP in the presence of Ca²⁺ was shown to be 3 μm. dCDP but not dCMP or dCyd could inhibit the transport activity. Other deoxynucleoside triphosphates could also inhibit the uptake of dCTP with the potency dGTP = dATP > TTP. Although ATP could competitively inhibit dCTP uptake with aK _i value of 8 μm, the reconstituted dCTP uptake activity was not sensitive to the ATP/ADP carrier inhibitor atractyloside or the sulfhydryl reagent N-ethylmaleimide. This suggests that the dCTP transport system studied is not the same as the ATP/ADP carrier. In conclusion, these studies describe the first functionally reconstituted mitochondrial carrier that displays an efficient transport activity for dCTP.
Reconstituted adenine nucleotide translocase forms a channel for small molecules comparable to the mitochondrial permeability transition pore
1998, FEBS Letters
Highly purified adenylate translocase (ANT) from rat heart mitochondria was functionally reconstituted as ATP/ADP exchange carrier in asolectin/cardiolipin vesicles. The ANT preparations used were free of porin, cyclophilin D, and Bax as analysed immunologically and by activity measurements. After pre-loading the ANT-containing proteoliposomes with ATP, malate or AMP, a gradual release of the trapped compounds by increasing the external Ca²⁺ concentrations could be demonstrated. N-Methyl-Val-4-cyclosporin did not inhibit the Ca²⁺ dependent release of internal substances from ANT liposomes. This inhibitor was found to be specific for the mitochondrial permeability transition pore (MTP) in intact mitochondria or reconstituted MTP-like protein complexes (e.g. hexokinase, porin, ANT complex). However, ADP in concentrations >20 μM inhibited the liberation of internal compounds, while in contrast, atractyloside (30 μM) and HgCl₂ (5 μM) both induced permeability of the ANT-containing liposomes resulting in a release of trapped substances. These results strongly suggest that ANT itself is capable to adopt a pore-like structure under conditions known to induce the permeability transition in mitochondria.

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Regular paperThe mitochondrial ATP / ADP carrier: Interaction with detergents and purification by a novel procedure

Abstract

Biochim. Biophys. Acta

Curr. Opinion Struct. Biol.

FEBS Lett.

FEBS Lett.

Arch. Biochem. Biophys.

FEBS Lett.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Methods Enzymol.

Anal. Biochem.

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

FEBS Lett.

Biochim. Biophys. Acta

Hoppe-Seyler's Z. Physiol. Chem.

Regular paper
The mitochondrial ATP / ADP carrier: Interaction with detergents and purification by a novel procedure