Heterologous complementation of the Klaac null mutation of Kluyveromyces lactis by the Saccharomyces cerevisiae AAC3 gene encoding the ADP/ATP carrier

The KlAAC gene, encoding the ADP/ATP carrier, has been assumed to be a single gene in Kluyveromyces lactis, an aerobic, petite-negative yeast species. The Klaac null mutation, which causes a respiratory-deficient phenotype, was fully complemented by AAC2, the Saccharomyces cerevisiae major gene for the ADP/ATP carrier and also by AAC1, a gene that is poorly expressed in S. cerevisiae. In this study, we demonstrate that the Klaac null mutation is partially complemented by the ScAAC3 gene, encoding the hypoxic ADP/ATP carrier isoform, whose expression in S. cerevisiae is prevented by oxygen. Once introduced into K. lactis, the AAC3 gene was expressed both under aerobic and under partial anaerobic conditions but did not support the growth of K. lactis under strict anaerobic conditions.     

The molecular basis for relative physiological functionality of the ADP/ATP carrier isoforms in Saccharomyces cerevisiae

AAC2 is one of three paralogs encoding mitochondrial ADP/ATP carriers in the yeast Saccharomyces cerevisiae, and because it is required for respiratory growth it has been the most extensively studied. To comparatively examine the relative functionality of Aac1, Aac2, and Aac3 in vivo, the gene encoding each isoform was expressed from the native AAC2 locus in aac1Delta aac3Delta yeast. Compared to Aac2, Aac1 exhibited reduced capacity to support growth of yeast lacking mitochondrial DNA or of yeast lacking the ATP/Mg-P(i) carrier, both conditions requiring ATP import into the mitochondrial matrix through the ADP/ATP carrier. Sixteen AAC1/AAC2 chimeric genes were constructed and analyzed to determine the key differences between residues or sections of Aac1 and Aac2. On the basis of the growth rate differences of yeast expressing different chimeras, the C1 and M2 loops of the ADP/ATP carriers contain divergent residues that are responsible for the difference(s) between Aac1 and Aac2. One chimeric gene construct supported growth on nonfermentable carbon sources but failed to support growth of yeast lacking mitochondrial DNA. We identified nine independent intragenic mutations in this chimeric gene that suppressed the growth phenotype of yeast lacking mitochondrial DNA, identifying regions of the carrier important for nucleotide exchange activities.     

A third ADP/ATP translocator gene in yeast

The op1 mutation in yeast is known to be due to a defect in the mitochondrial ADP/ATP translocator. Sequencing of the gene AAC2 revealed that the mutation resulted from a single base change that caused a replacement of arginine 97 by a histidine. The gene encoding AAC2 was also cloned and sequenced from an op1 revertant capable of growth on glycerol as a sole carbon source. Sequence analysis indicates that the reverted gene underwent rearrangement in which a portion of an unknown gene was used to repair the mutation. An oligonucleotide complementary to this insert was used to clone a previously unrecognized gene encoding ADP/ATP translocator in yeast. The newly discovered gene, AAC3, is homologous with the previously known genes AAC1 and AAC2. Gene disruption experiments suggest that AAC2 encodes the majority of the translocator. Expression of AAC1 and AAC2 required derepressed conditions whereas expression of AAC3 occurred almost exclusively under anaerobic conditions. Both the op1 mutant and the strain that contains an interrupted AAC2 were able to grow under anaerobic conditions, suggesting that AAC3 can replace the gene product of AAC2. Indeed, when cloned into multicopy plasmid, AAC3 was able to replace the disrupted AAC2 in the JLY-73 strain. The concomitant disruption of the AAC2 and AAC3, however, results in arrest of cell growth under conditions of low oxygen tension. The discovery of a third gene encoding ADP/ATP translocator helps to clarify certain characteristics of op1 mutants which could not be resolved in the past.     

ADP/ATP translocator is essential only for anaerobic growth of yeast Saccharomyces cerevisiae

All three genes (AAC1, AAC2 and AAC3) encoding the mitochondrial ADP/ATP translocator, were inactivated in a haploid yeast strain by a gene disruption technique. The triple mutant was still able to grow on fermentable carbon sources but only in the presence of oxygen. Under aerobic conditions neither translocator-protein nor carrier-mediated transport was detected in all mutants in which the AAC2 gene was disrupted. It was further shown that a functional AAC genes product is essential only for anaerobic growth of Saccharomyces cerevisiae but not for growth under derepressed conditions. Under anaerobic conditions a non-detectable amount of AAC3 gene product is sufficient to ensure the cell growth and multiplication.     

Structure-function studies of adenine nucleotide transport in mitochondria. I. Construction and genetic analysis of yeast mutants encoding the ADP/ATP carrier protein of mitochondria

The gene encoding the major ADP/ATP carrier in yeast AAC2 (pet9; Lawson, J., and Douglas, M. (1988) J. Biol. Chem. 263, 14812-14818) has been disrupted (delta AAC2) by itself and in combination with a disruption of a second translocator gene AAC1 (delta AAC1). Disruption of AAC2 like the pet9 mutation renders yeast unable to grow on a nonfermentable carbon source. The AAC1 AAC2 double disruption exhibits a phenotype identical to the AAC2. This provides the host strain for the analysis of point mutations in the AAC protein. We have initiated this structure-function analysis by characterizing and confirming that the pet9 mutation is a G to A transition resulting in an arginine to histidine change at position 96. Site-directed replacements at Arg96 confirm its essential function for growth on a nonfermentable carbon source. These data also suggest that in the absence of functional AAC1 and AAC2 gene products, adenine nucleotide transport across the mitochondrial inner membrane must occur by an as yet unidentified translocator or translocation mechanism or that within these cells separate intra- and extramitochondrial adenine nucleotide pools can exist to support growth.     

Multiple origins of hydrogenosomes: functional and phylogenetic evidence from the ADP/ATP carrier of the anaerobic chytrid Neocallimastix sp

A mitochondrial-type ADP/ATP carrier (AAC) has been identified in the hydrogenosomes of the anaerobic chytridiomycete fungus Neocallimastix sp. L2. Biochemical and immunocytochemical studies revealed that this ADP/ATP carrier is an integral component of hydrogenosomal membranes. Expression of the corresponding cDNA in Escherichia coli confers the ability on the bacterial host to incorporate ADP at significantly higher rates than ATP--similar to isolated mitochondria of yeast and animals. Phylogenetic analysis of this AAC gene (hdgaac) confirmed with high statistical support that the hydrogenosomal ADP/ATP carrier of Neocallimastix sp. L2 belongs to the family of veritable mitochondrial-type AACs. Hydrogenosome-bearing anaerobic ciliates possess clearly distinct mitochondrial-type AACs, whereas the potential hydrogenosomal carrier Hmp31 of the anaerobic flagellate Trichomonas vaginalis and its homologue from Trichomonas gallinae do not belong to this family of proteins. Also, phylogenetic analysis of genes encoding mitochondrial-type chaperonin 60 proteins (HSP 60) supports the conclusion that the hydrogenosomes of anaerobic chytrids and anaerobic ciliates had independent origins, although both of them arose from mitochondria.     

The Saccharomyces cerevisiae ADP/ATP carrier-iso 1 cytochrome c fusion protein: one-step purification and functional analysis in vitro

Genetic expression versus plasmidic overexpression of a functional recombinant fusion protein combining the yeast Saccharomyces cerevisiae mitochondrial ADP/ATP carrier (Anc2p) and the iso-1-cytochrome c (Cyc1p) has been investigated, with the main aim of increasing the polar surface of the carrier to improve its crystallization properties. The gene encoding the his6-tagged fusion protein was expressed in yeast under the control of the regulatory sequences of ScANC2 or under the control of the strong yeast PMA1 promoter. In both cases, the chimeric carrier, Anc2-Cyc1(His6)p, was able to restore growth on a non-fermentable carbon source of a yeast strain devoid of functional ADP/ATP carrier, demonstrating its transport activity. Nevertheless, when the expression vector was used, the level of expression of Anc2-Cyc1(His6)p was no greater than that of the chimeric carrier obtained in yeast mitochondria after homologous recombination. Optimal conditions to extract and to purify Anc2-Cyc1(His6)p were determined. A series of detergents was screened for their ability to extract and to preserve in vitro the chimeric carrier. A rapid, single step purification of Anc2-Cyc1(His6)p was developed, using n-dodecyl-beta-d-maltoside (DoDM) as the best detergent to solubilize the chimeric protein. Carboxyatractyloside- (CATR-) and nucleotide-binding sites were preserved in the purified protein. Moreover, the Cyc1p moiety of Anc2-Cyc1(His6)p-CATR complex solubilized in DoDM was still able to interact in vitro with the cytochrome c oxidase (COX), with the same affinity as yeast Cyc1p. Improved production and purification of Anc2-Cyc1(His6)p-CATR complex opens up new possibilities for the use of this protein in crystallographic approaches to the yeast ADP/ATP carrier. Furthermore, Anc2-Cyc1(His6)p may be an useful molecular tool to investigate in vivo interactions between components of the respiratory chain complexes such as COX and the proteins implicated in ATP biogenesis, such as the ATP/ADP carrier.     

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.     

Functional characterization and purification of a Saccharomyces cerevisiae ADP/ATP carrier-iso 1 cytochrome c fusion protein

A recombinant fusion protein combining the mitochondrial ADP/ATP carrier (Anc2p) and the iso-1-cytochrome c (Cyc1p), both from Saccharomyces cerevisiae, has been genetically elaborated with the aim of increasing the polar surface area of the carrier to facilitate its crystallization. The gene encoding the his-tagged fusion protein was expressed in yeast under the control of the regulatory sequences of ScANC2. The chimeric carrier, Anc2-Cyc1(His6)p, was able to restore growth on a non-fermentable carbon source of a yeast strain devoid of functional ADP/ATP carrier, which demonstrated its transport activity. The kinetic exchange properties of Anc2-Cyc1(His6)p and the wild type his-tagged carrier Anc2(His6)p were very similar. However, Anc2-Cyc1(His6)p restored cell growth less efficiently than Anc2(His6)p which correlates with the lower amount found in mitochondria. Purification of Anc2-Cyc1(His6)p in complex with carboxyatractyloside (CATR), a high affinity inhibitor of ADP/ATP transport, was achieved by combining ion-exchange chromatography and ion-metal affinity chromatography in the presence of LAPAO, an aminoxide detergent. As characterized by absorption in the visible range, heme was found to be present in isolated Anc2-Cyc1(His6)p, giving the protein a red color. Large-scale purification of Anc2-Cyc1(His6)p-CATR complex opens up novel possibilities for the use of crystallographic approaches to the yeast ADP/ATP carrier.     

Kinetics of electrogenic transport by the ADP/ATP carrier.

The electrogenic transport of ATP and ADP by the mitochondrial ADP/ATP carrier (AAC) was investigated by recording transient currents with two different techniques for performing concentration jump experiments: 1) the fast fluid injection method: AAC-containing proteoliposomes were adsorbed to a solid supported membrane (SSM), and the carrier was activated via ATP or ADP concentration jumps. 2) BLM (black lipid membrane) technique: proteoliposomes were adsorbed to a planar lipid bilayer, while the carrier was activated via the photolysis of caged ATP or caged ADP with a UV laser pulse. Two transport modes of the AAC were investigated, ATP(ex)-0(in) and ADP(ex)-0(in). Liposomes not loaded with nucleotides allowed half-cycles of the ADP/ATP exchange to be studied. Under these conditions the AAC transports ADP and ATP electrogenically. Mg(2+) inhibits the nucleotide transport, and the specific inhibitors carboxyatractylate (CAT) and bongkrekate (BKA) prevent the binding of the substrate. The evaluation of the transient currents yielded rate constants of 160 s(-1) for ATP and >/=400 s(-1) for ADP translocation. The function of the carrier is approximately symmetrical, i.e., the kinetic properties are similar in the inside-out and right-side-out orientations. The assumption from previous investigations, that the deprotonated nucleotides are exclusively transported by the AAC, is supported by further experimental evidence. In addition, caged ATP and caged ADP bind to the carrier with similar affinities as the free nucleotides. An inhibitory effect of anions (200-300 mM) was observed, which can be explained as a competitive effect at the binding site. The results are summarized in a transport model.     

Mitochondrial import of the ADP/ATP carrier protein in Saccharomyces cerevisiae. Sequences required for receptor binding and membrane translocation

The ADP/ATP carrier of yeast (309 amino acids) is an abundant transmembrane protein of the mitochondrial inner membrane whose import involves well-defined steps (Pfanner, N., and Neupert, W. (1987) J. Biol. Chem. 262, 7528-7536). Analysis of the in vitro import of gene fusion products containing ADP/ATP carrier (AAC) sequences at the amino terminus and mouse dihydrofolate reductase (DHFR) at the carboxyl terminus indicates that the first 72 amino acids of the soluble carrier protein, a hydrophilic region of the protein, are not by themselves sufficient for initial binding to the AAC receptor on the mitochondrial surface. However, an AAC-DHFR gene fusion containing the first 111 residues of the ADP/ATP carrier protein exhibited binding to mitochondria at low temperature (2 degrees C) and internalization at 25 degrees C to a mitochondrial space protected from proteinase K in the same manner as the wild-type ADP/ATP carrier protein. The AAC-DHFR protein, in contrast to the wild-type AAC protein imported into mitochondria under optimal conditions, remained extractable at alkaline pH and appeared to be blocked at an intermediate step in the AAC import pathway. Based on its extraction properties, this AAC-DHFR hybrid is proposed to be associated with a proteinaceous component of the import apparatus within mitochondria. These data indicate that the import determinants for the AAC protein are not located at its extreme amino terminus and that protein determinants distal to the first 111 residues of the carrier may be necessary to move the protein beyond the alkali-extractable step in the biogenesis of a functional AAC protein.     

家蚕微孢子虫ADP/ATP转运蛋白的原核表达及间接免疫荧光定位分析

【目的】家蚕微孢子虫Nosema bombycis ADP/ATP转运蛋白可能参与搬运宿主细胞的能量。本研究克隆家蚕微孢子虫ADP/ATP转运蛋白基因,并进行原核表达、抗体制备及间接免疫荧光定位,为控制和防治家蚕微粒子病提供理论基础。【方法】通过同源序列比对鉴定家蚕微孢子虫N. bombycis ADP/ATP转运蛋白序列,采用生物合成的方法将编码3段面向膜内侧肽段的核酸序列拼接合成,在其两端引入BglⅡ和SalⅠ酶切位点,克隆至pUC57载体并测序,再亚克隆至含有二氢叶酸还原酶（dihydrofolate reductase,DHFR）标签的表达载体pQE40中,然后利用BamHⅠ和SalⅠ酶切获得含有DHFR标签的重组序列,并连接至pET30a（+）载体中进行诱导表达。通过SDS-PAGE、镍柱亲和层析和免疫印迹法鉴定表达蛋白,利用间接免疫荧光对ADP/ATP转运蛋白的分布进行检测。【结果】家蚕微孢子虫的ADP/ATP转运蛋白编码序列（GenBank登录号为EOB13854.1）全长1 524 bp,编码蛋白含有507个氨基酸残基,预测分子质量为59 kDa,等电点为9.35。具有12个跨膜结构域和TLC结构域,其中TLC结构域含有4个功能保守位点。与蜜蜂微孢子虫的ADP/ATP转运蛋白比较,氨基酸序列一致性达30%。系统进化分析表明微孢子虫ADP/ATP转运蛋白聚为一类,具有共同的起源。成功构建了NbADP/ATP-△TM-DHFR-pET30a原核表达重组质粒,目的基因获得表达,其融合蛋白分子量约为37 kDa,纯化重组蛋白并制备了多克隆抗体。免疫印迹分析表明,成熟微孢子虫中表达ADP/ATP转运蛋白;间接免疫荧光定位结果显示,家蚕微孢子虫孢子ADP/ATP转运蛋白定位于孢子质膜上。【结论】本研究将为阻断微孢子虫能量来源,达到控制和防治家蚕微粒子病提供新的思路。     

Projection structure of the atractyloside-inhibited mitochondrial ADP/ATP carrier of Saccharomyces cerevisiae

ADP/ATP carriers in the inner mitochondrial membrane catalyze the exchange of cytosolic ADP for ATP synthesized in the mitochondrial matrix by ATP synthase and thereby replenish the eukaryotic cell with metabolic energy. The yeast ADP/ATP carrier (AAC3) was overexpressed, inhibited by atractyloside, purified, and reconstituted into two-dimensional crystals. Images of frozen hydrated crystals were recorded by electron microscopy, and a projection structure was calculated to 8-A resolution. The AAC3 molecule has pseudo 3-fold symmetry in agreement with the 3-fold sequence repeats that are typical of members of the mitochondrial carrier family. The density distribution is consistent with a bundle of six transmembrane alpha-helices with two or three short alpha-helical extensions closing the central pore on the matrix side. The AAC3 molecules in the crystal are arranged in symmetrical homo-dimers, but the translocation pore for adenine nucleotides lies in the center of the molecule and not along the dyad axis of the dimer.     

The substrate specificity of the human ADP/ATP carrier AAC1

Abstract

The mitochondrial ADP/ATP carrier imports ADP from the cytosol into the mitochondrial matrix for its conversion to ATP by ATP synthase and exports ATP out of the mitochondrion to replenish the eukaryotic cell with chemical energy. Here the substrate specificity of the human mitochondrial ADP/ATP carrier AAC1 was determined by two different approaches. In the first the protein was functionally expressed in Escherichia coli membranes as a fusion protein with maltose binding protein and the effect of excess of unlabeled compounds on the uptake of [³²P]-ATP was measured. In the second approach the protein was expressed in the cytoplasmic membrane of Lactococcus lactis. The uptake of [¹⁴C]-ADP in whole cells was measured in the presence of excess of unlabeled compounds and in fused membrane vesicles loaded with unlabeled compounds to demonstrate their transport. A large number of nucleotides were tested, but only ADP and ATP are suitable substrates for human AAC1, demonstrating a very narrow specificity. Next we tried to understand the molecular basis of this specificity by carrying out molecular-dynamics simulations with selected nucleotides, which were placed at the entrance of the central cavity. The binding of the phosphate groups of guanine and adenine nucleotides is similar, yet there is a low probability for the base moiety to be bound, likely to be rooted in the greater polarity of guanine compared to adenine. AMP is unlikely to engage fully with all contact points of the substrate binding site, suggesting that it cannot trigger translocation.     

Separate genes encode functionally equivalent ADP/ATP carrier proteins in Saccharomyces cerevisiae. Isolation and analysis of AAC2

Genetic and biochemical analysis of Saccharomyces cerevisiae containing a disruption of the nuclear gene (AAC1) encoding the mitochondrial ADP/ATP carrier has revealed a second gene for this protein. The second gene, designated AAC2, has been isolated by genetic complementation and sequenced. AAC2 contains a 954-base pair open reading frame coding for a protein of 318 amino acids which is highly homologous to the AAC1 gene product except that it is nine amino acids longer at the NH2 terminus. The two yeast genes are highly conserved at the level of DNA and protein and share identity with the ADP/ATP carriers from other organisms. Both genes complement an ADP/ATP carrier defect (op1 or pet9). However, the newly isolated gene AAC2 need be present only in one or two copies while the previously isolated AAC1 gene must be present in multiple copies to support growth dependent on a functional carrier protein. This gene dosage-dependent complementation combined with the high degree of conservation suggest that these two functionally equivalent genes may be differentially expressed.     

Molecular cloning of a cDNA for a human ADP/ATP carrier which is growth-regulated

We have identified in a human cDNA library a clone (hp2F1) whose cognate RNA is growth-regulated. The insert has been sequenced and the nucleotide sequence shows a strong homology to the nucleotide sequences of the ADP/ATP carrier cDNA and gene, respectively, isolated from Neurospora crassa and Saccharomyces cerevisiae. The putative amino acid sequence of hp2F1 shows an 87% homology to the amino acid sequence of the ADP/ATP carrier from beef heart mitochondria. We conclude that the insert of hp2F1 contains the full coding sequence of a human ADP/ATP carrier. The steady-state RNA levels of the ADP/ATP carrier are growth-regulated. They increase when quiescent cells are stimulated by serum, platelet-derived growth factor, or epidermal growth factor, but not by platelet-poor plasma or insulin. RNA levels of the ADP/ATP carrier decrease instead when growing HL-60 cells are induced to differentiate by either phorbol esters or retinoic acid.     

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.     

Four mutations in transmembrane domains of the mitochondrial ADP/ATP carrier increase resistance to bongkrekic acid

Two distinct conformations of the mitochondrial ADP/ATP carrier involved in the adenine nucleotide transport are called BA and CATR conformations, as they were distinguished by binding of specific inhibitors bongkrekic acid (BA) and carboxyatractyloside (CATR), respectively. To find out which amino acids are implicated in the transition between these two conformations, which occurs during transport, mutants of the Saccharomyces cerevisiae ADP/ATP carrier Anc2p responsible for resistance of yeast cells to BA were identified and characterized after in vivo chemical or UV mutagenesis. Only four different mutations could be identified in spite of a large number of mutants analyzed. They are located in the Anc2p transmembrane segments I (G30S), II (Y97C), III (L142S), and VI (G298S), and are independently enabling growth of cells in the presence of BA. The variant and wild-type Anc2p were produced practically to the same level in mitochondria, as evidenced by immunochemical analysis and by atractyloside binding experiments. ADP/ATP exchange mediated by Anc2p variants in isolated mitochondria was more efficient than that of the wild-type Anc2p in the presence of BA, confirming that BA resistance of the mutant cells was linked to the functional properties of the modified ADP/ATP carrier. These results suggest that resistance to BA is caused by alternate conformation of Anc2p due to appearance of Ser or Cys at specific positions. Different interactions of these residues with other amino acids and/or BA could prevent formation of stable inactive Anc2p BA complex.     

Substrate-induced fluorescence changes of the isolated ADP/ATP carrier protein in solution

Fluorescence studies were carried out on a purified preparation of the ADP/ATP carrier protein solubilized in 3-laurylamido-N-N-dimethylpropylaminoxide. The intrinsic fluorescence of the protein was modified upon addition of ADP and ATP and specific inhibitory ligands (carboxyatractyloside and bongkrekic acid). The fluorescence was transitorily enhanced by micromolar concentrations of ADP or ATP. The rise in fluorescence lasted for 10 sec at 25°C. It was suppressed by carboxyatractyloside and on the contrary enhanced by bongkrekic acid. These data were interpreted as reflecting conformational changes probably related to the functioning of the ADP/ATP carrier. Mg⁺⁺ inhibited the ADP- or ATP-induced rise in fluorescence, indicating that the free forms (and not the Mg⁺⁺ complexed forms) of ADP and ATP are the true substrates for the ADP/ATP carrier.     

Two distinct regions of the yeast mitochondrial ADP/ATP carrier are photolabeled by a new ADP analogue: 2-azido-3'-O-naphthoyl-[beta-32P]ADP. Identification of the binding segments by mass spectrometry

A novel photoactivatable radioactive ADP derivative, namely, 2-azido-3'-O-naphthoyl-[beta-(32)P]ADP (2-azido-N-[(32)P]ADP), was synthesized with the aim at mapping the substrate binding site(s) of the yeast mitochondrial ADP/ATP carrier. It was used with mitochondria isolated from genetically modified strains of Saccharomyces cerevisiae, producing the native or the His-tagged Anc2p isoform of the carrier. In darkness, 2-azido-N-[(32)P]ADP was reversibly bound to the carrier in mitochondria, without being transported. Upon photoirradiation, only the ADP/ATP carrier was covalently radiolabeled among all mitochondrial proteins. Specificity of labeling was demonstrated since carboxyatractyloside (CATR), a potent inhibitor of ADP/ATP transport, totally prevented the incorporation of the photoprobe. To localize the radioactive region(s), the purified photolabeled carrier was submitted to CNBr or hydroxylamine cleavage. The resulting fragments were characterized and identified by SDS-PAGE, Western blotting, amino acid sequencing, and MALDI-MS and ESI-MS analyses. Two short photolabeled distinct segments, eight and nine residues long, were identified: S183-R191, located in the central part of the ADP/ATP carrier; and I311-K318, belonging to its C-terminal end. Plausible models of organization of the nucleotide binding site(s) of the carrier involving the two regions specifically labeled by 2-azido-N-[(32)P]ADP are proposed.