Adenine Nucleotide Transport In Mitochondria Research Articles

Mitochondrial temperature homeostasis resists external metabolic stresses

Based on studies with a fluorescent reporter dye, Mito Thermo Yellow (MTY), and the genetically encoded gTEMP ratiometric fluorescent temperature indicator targeted to mitochondria, the temperature of active mitochondria in four mammalian and one insect cell line was estimated to be up to 15°C above that of the external environment to which the cells were exposed. High mitochondrial temperature was maintained in the face of a variety of metabolic stresses, including substrate starvation or modification, decreased ATP demand due to inhibition of cytosolic protein synthesis, inhibition of the mitochondrial adenine nucleotide transporter and, if an auxiliary pathway for electron transfer was available via the alternative oxidase, even respiratory poisons acting downstream of oxidative phosphorylation (OXPHOS) complex I. We propose that the high temperature of active mitochondria is an inescapable consequence of the biochemistry of OXPHOS and is homeostatically maintained as a primary feature of mitochondrial metabolism.

Mitochondrial temperature homeostasis resists external metabolic stresses.

Based on studies with a fluorescent reporter dye, Mito Thermo Yellow (MTY), and the genetically encoded gTEMP ratiometric fluorescent temperature indicator targeted to mitochondria, the temperature of active mitochondria in four mammalian and one insect cell line was estimated to be up to 15°C above that of the external environment to which the cells were exposed. High mitochondrial temperature was maintained in the face of a variety of metabolic stresses, including substrate starvation or modification, decreased ATP demand due to inhibition of cytosolic protein synthesis, inhibition of the mitochondrial adenine nucleotide transporter and, if an auxiliary pathway for electron transfer was available via the alternative oxidase, even respiratory poisons acting downstream of oxidative phosphorylation (OXPHOS) complex I. We propose that the high temperature of active mitochondria is an inescapable consequence of the biochemistry of OXPHOS and is homeostatically maintained as a primary feature of mitochondrial metabolism.

Identification of differentially expressed genes in two grape varieties cultivated in semi-arid and temperate regions from West-Bank, Palestine

Plants respond to stress conditions by altering genetic pathways. In this study, we aimed to identify and analyze differentially expressed genes in leaves of two grape varieties (genotypes) that were grown in Palestine either in a semi-arid region with a prolonged drought and high temperature stress or in a temperate region with moderate stress levels. In total, twelve transcripts with altered expression patterns, either by stress or genotype, were identified with the differential display RT-PCR (DDRT-PCR) technique and validated via quantitative real-time PCR (qRT-PCR). Eight transcripts represent genes that are down-regulated by stress in the leaves of at least one variety, among of which are members of the DEAD-box RNA helicase, Haloacid Dehalogenase (HAD) hydrolase, kinesin-like, and mitochondrial Adenine nucleotide transporter (ANT) gene families. Two genes encoding for members of the GDSL Lipase/Esterase and Multiprotein Bridging Factor (MBF) gene families were found to be up-regulated in stressed leaves. Two transcripts coding for a NAC-domain containing protein and a WD-repeat containing protein, respectively, were found to be non-responsive to those abiotic stresses but are differentially expressed in a genotype-dependent manner.

Absence of Ca2+-induced mitochondrial permeability transition but presence of bongkrekate-sensitive nucleotide exchange in C. crangon and P. serratus

Mitochondria from the embryos of brine shrimp (Artemia franciscana) do not undergo Ca2+-induced permeability transition in the presence of a profound Ca2+ uptake capacity. Furthermore, this crustacean is the only organism known to exhibit bongkrekate-insensitive mitochondrial adenine nucleotide exchange, prompting the conjecture that refractoriness to bongkrekate and absence of Ca2+-induced permeability transition are somehow related phenomena. Here we report that mitochondria isolated from two other crustaceans, brown shrimp (Crangon crangon) and common prawn (Palaemon serratus) exhibited bongkrekate-sensitive mitochondrial adenine nucleotide transport, but lacked a Ca2+-induced permeability transition. Ca2+ uptake capacity was robust in the absence of adenine nucleotides in both crustaceans, unaffected by either bongkrekate or cyclosporin A. Transmission electron microscopy images of Ca2+-loaded mitochondria showed needle-like formations of electron-dense material strikingly similar to those observed in mitochondria from the hepatopancreas of blue crab (Callinectes sapidus) and the embryos of Artemia franciscana. Alignment analysis of the partial coding sequences of the adenine nucleotide translocase (ANT) expressed in Crangon crangon and Palaemon serratus versus the complete sequence expressed in Artemia franciscana reappraised the possibility of the 208-214 amino acid region for conferring sensitivity to bongkrekate. However, our findings suggest that the ability to undergo Ca2+-induced mitochondrial permeability transition and the sensitivity of adenine nucleotide translocase to bongkrekate are not necessarily related phenomena.

Application of Flow Cytometry to Determine Differential Redistribution of Cytochrome c and Smac/DIABLO from Mitochondria during Cell Death Signaling

Mitochondrially mediated apoptosis is characterized by redistribution of proteins from mitochondria to cytoplasm following permeabilization of the outer mitochondrial membrane. We applied flow cytometry to quantify simultaneously the redistribution of two apoptogenic proteins, cytochrome c (cyt c) and Smac/DIABLO (Smac). Mammalian cells were treated with digitonin that selectively permeabilizes the plasma membrane. Following fixation, treated cells were infused successively with primary and secondary antibodies (the latter fluorescently tagged) enabling independent detection of cyt c and Smac. Digitonin-treated cells that retain cyt c or Smac in mitochondria generate strong fluorescence signals in flow cytometry. Cells in which cyt c or Smac have transited the outer mitochondrial membrane show greatly reduced fluorescence because the proteins are lost from the digitonin-permeabilized cells. Quantitative flow cytometry revealed that in 143B TK- cells treated with staurosporine, cyt c and Smac exit mitochondria asymmetrically, with cyt c redistribution preceding that of Smac. However, in HeLa cells likewise treated, cyt c and Smac exit mitochondria concurrently. Under other conditions of apoptotic induction, for example, 143B TK- cells treated with MT-21 (an apoptotic inducer that binds to the mitochondrial adenine nucleotide transporter), redistribution of Smac precedes that of cyt c. The various patterns of redistribution of these proteins were confirmed by immunocytochemical analysis and confocal microscopy. We conclude that flow cytometry can be employed effectively to quantify simultaneously the redistribution of cyt c and Smac from mitochondria to the cytosol. Moreover, differential redistribution of cyt c and Smac occurs under various conditions, thereby reflecting constraints on availability of these proteins to exit mitochondria after permeabilization of the outer membrane.

Absence of Ca2+-Induced Mitochondrial Permeability Transition but Presence of Bongkrekate-Sensitive Nucleotide Exchange in C. crangon and P. serratus

Mitochondria from the embryos of brine shrimp (Artemia franciscana) do not undergo Ca2+-induced permeability transition in the presence of a profound Ca2+ uptake capacity. Furthermore, this crustacean is the only organism known to exhibit bongkrekate-insensitive mitochondrial adenine nucleotide exchange, prompting the conjecture that refractoriness to bongkrekate and absence of Ca2+-induced permeability transition are somehow related phenomena. Here we report that mitochondria isolated from two other crustaceans, brown shrimp (Crangon crangon) and common prawn (Palaemon serratus) exhibited bongkrekate-sensitive mitochondrial adenine nucleotide transport, but lacked a Ca2+-induced permeability transition. Ca2+ uptake capacity was robust in the absence of adenine nucleotides in both crustaceans, unaffected by either bongkrekate or cyclosporin A. Transmission electron microscopy images of Ca2+-loaded mitochondria showed needle-like formations of electron-dense material strikingly similar to those observed in mitochondria from the hepatopancreas of blue crab (Callinectes sapidus) and the embryos of Artemia franciscana. Alignment analysis of the partial coding sequences of the adenine nucleotide translocase (ANT) expressed in Crangon crangon and Palaemon serratus versus the complete sequence expressed in Artemia franciscana reappraised the possibility of the 208-214 amino acid region for conferring sensitivity to bongkrekate. However, our findings suggest that the ability to undergo Ca2+-induced mitochondrial permeability transition and the sensitivity of adenine nucleotide translocase to bongkrekate are not necessarily related phenomena.

Abstract 1197: The role of mitochondrial permeability transition pore complex proteins VDAC1, ANT, and cyclophilin D in prednisolone-induced apoptosis in B-Lineage acute lymphoblastic leukemia (ALL)

Abstract The loss of mitochondrial membrane permeability is central to apoptosis. The mitochondrial permeability transition pore (PTP) complex, comprising of at least voltage-dependent anion channel 1 (VDAC1), adenine nucleotide transporter (ANT) and cyclophilin D (CyD), mediates the permeability of the mitochondrial membranes. We investigated the involvement of VDAC1, ANT and CyD in prednisolone (PRED)-induced apoptosis in 2 ALL cell lines: Sup-B15 (sensitive) and REH (resistant). After 24h exposure to PRED (10.0µg/ml), VDAC1 protein was consistently upregulated only in PRED-sensitive cell line, but remained unchanged in the resistant one. Co-administration of non-specific VDAC1 inhibitor, 4, 4′-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) with PRED, blocked PRED-induced apoptosis and caspase-3 activity only in sensitive cell line. Surprisingly, complete VDAC1 silencing using siRNA (Dharmacon) failed to abrogate PRED-induced apoptosis, suggesting that VDAC1 is not critical. We next investigated the other members of the PTP complex i.e., ANT and CyD. Co-administration of PRED and bongkrekic acid (BA) a specific inhibitor of ANT, also resulted in a dose-dependent inhibition of apoptosis and the caspase-3 activity. The combination of DIDS and BA, even at reduced doses by 60% and 96% respectively, is highly synergistic in inhibiting PRED-induced apoptosis and surprisingly reduced VDAC1 upregulation in PRED-induced apoptosis, unlike each agent alone. PRED did not alter the level of CyD protein and administration of specific CyD inhibitor, cyclosporine A (CsA), did not affect PRED-induced apoptosis. We conclude that ANT protein is central in the mitochondrial PTP mediated apoptosis during PRED treatment of ALL; VDAC1 and CyD are dispensable. ANT is upstream to VDAC1 and it upregulates VDAC1 during PTP activation in PRED-induced apoptosis. Our investigation provides the basis for looking at the role of mitochondrial PTP complex proteins especially ANT in PRED treatment in childhood ALL. This may provide new avenues to reverse resistance to therapy in patients with relapsed ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1197.

Distribution, pharmacological characterization and function of the 18 kDa translocator protein in rat small intestine

The TSPO (18 kDa translocator protein) is a mitochondrial transmembrane protein involved in cholesterol transport in organs that synthesize steroids and bile salts. Different natural and synthetic high-affinity TSPO ligands have been characterized through their ability to stimulate cholesterol transport, but also to stimulate other physiological functions including cell proliferation, apoptosis and calcium-dependent transepithelial ion secretion. Here, we investigate the localization and functions of TSPO in the small intestine. TSPO was present in enterocyte mitochondria but not in rat intestinal goblet cells. Enterocyte cytoplasm also contained the endogenous TSPO ligand, polypeptide DBI (diazepam-binding inhibitor). Whereas intestinal TSPO had high affinity for the synthetic ligand PK 11195, the pharmacological profile of TSPO in the duodenum was distinct from the jejunum and ileum. Specifically, benzodiazepine Ro5-4864 and protoporphyrin IX showed 5-13-fold lower affinity for duodenal TSPO. The mRNA and protein ratios of TSPO to other mitochondrial membrane proteins VDAC (voltage-dependent anion channel) and ANT (adenine nucleotide transporter) were significantly different. PK 11195 stimulated calcium-dependent chloride secretion in the duodenum and calcium-dependent chloride absorption in the ileum, but did not affect jejunum ion transport. The functional differences in subpopulations of TSPO in different regions of the intestine could be related to structural organization of mitochondrial protein complexes that mediate the ability of TSPO to modulate either chloride secretion or absorption in the duodenum and ileum respectively.

Elevated zinc induces endothelial apoptosis via disruption of glutathione metabolism: role of the ADP translocator

Zinc is the second-most abundant transition metal within cells and an essential micronutrient. Although adequate zinc is essential for cellular function, intracellular free zinc (Zn(2+)) is tightly controlled, as sustained increases in free Zn(2+) levels can directly contribute to apoptotic endothelial cell death. Moreover, exposure of endothelial cells to acute nitrosative and/or oxidative stress induces a rapid rise of Zn(2+) with mitochondrial dysfunction and the initiation of apoptosis. This apoptotic induction can be mimicked through addition of exogenous ZnCl(2) and mitigated by zinc-chelation strategies, indicating Zn(2+)-dependent mechanisms in this process. However, the molecular mechanisms of Zn(2+)-mediated mitochondrial dysfunction are unknown. Here we report that free Zn(2+) disrupts cellular redox status through inhibition of glutathione reductase, and induces apoptosis by redox-mediated inhibition of the mitochondrial adenine nucleotide transporter (ANT). Inhibition of ANT causes increased mitochondrial oxidation, loss of ADP uptake, mitochondrial translocation of bax, and apoptosis. Interestingly, pre-incubation with glutathione ethyl ester protects endothelial cells from these observed effects. We conclude that key mechanisms of Zn(2+)-mediated apoptotic induction include disruption of cellular glutathione homeostasis leading to ANT inhibition and decreases in mitochondrial ATP synthesis. These pathways could represent novel therapeutic targets during acute oxidative or nitrosative stress in cells and tissues.

Cardioprotection and altered mitochondrial adenine nucleotide transport

It is becoming increasingly clear that mitochondrial dysfunction is critically important in myocardial ischemic injury, and that cardioprotective mechanisms must ultimately prevent or attenuate mitochondrial damage. Mitochondria are also essential for energy production, and therefore prevention of mitochondrial injury must not compromise oxidative phosphorylation during reperfusion. This review will focus on one mitochondrial mechanism of cardioprotection involving inhibition of adenine nucleotide transport across the outer mitochondria membrane under de-energized conditions. This slows ATP hydrolysis by the mitochondria, and would be expected to lower mitochondrial membrane potential during ischemia, to inhibit calcium uptake during ischemia, and potentially to reduce free radical generation during early reperfusion. Two interventions that similarly inhibit mitochondrial adenine nucleotide transport are Bcl-2 overexpression and GSK inhibition. A possible final common mechanism shared by both of these interventions is discussed.

Identification and Characterization of ADNT1, a Novel Mitochondrial Adenine Nucleotide Transporter from Arabidopsis

Despite the fundamental importance and high level of compartmentation of mitochondrial nucleotide metabolism in plants, our knowledge concerning the transport of nucleotides across intracellular membranes remains far from complete. Study of a previously uncharacterized Arabidopsis (Arabidopsis thaliana) gene (At4g01100) revealed it to be a novel adenine nucleotide transporter, designated ADNT1, belonging to the mitochondrial carrier family. The ADNT1 gene shows broad expression at the organ level. Green fluorescent protein-based cell biological analysis demonstrated targeting of ADNT1 to mitochondria. While analysis of the expression of beta-glucuronidase fusion proteins suggested that it was expressed across a broad range of tissue types, it was most highly expressed in root tips. Direct transport assays with recombinant and reconstituted ADNT1 were utilized to demonstrate that this protein displays a relatively narrow substrate specificity largely confined to adenylates and their closest analogs. ATP uptake was markedly inhibited by the presence of other adenylates and general inhibitors of mitochondrial transport but not by bongkrekate or carboxyatractyloside, inhibitors of the previously characterized ADP/ATP carrier. Furthermore, the kinetics are substantially different from those of this carrier, with ADNT1 preferring AMP to ADP. Finally, isolation and characterization of a T-DNA insertional knockout mutant of ADNT1, alongside complementation and antisense approaches, demonstrated that although deficiency of this transporter did not seem to greatly alter photosynthetic metabolism, it did result in reduced root growth and respiration. These findings are discussed in the context of a potential function for ADNT1 in the provision of the energy required to support growth in heterotrophic plant tissues.

Differential expression pattern of the four mitochondrial adenine nucleotide transporter ant genes and their roles during the development of Caenorhabditis elegans

The adenine nucleotide transporter (ANT) mediates exchange of cytosolic ADP and mitochondrial ATP. Although most species contain more than one ANT family member, it is not known whether their roles in developmental processes are redundant or specific. Here, we show that the Caenorhabditis elegans genome encodes four candidate ant genes (ant-1.1, ant-1.2, ant-1.3, and ant-1.4). We have investigated their spatiotemporal expression patterns and discovered that, whereas ANT-1.1 is a ubiquitously expressed mitochondrial protein, the other three ANT proteins show a restricted range of cell type expression. Moreover, only the disruption of ant-1.1 function, through RNA interference (RNAi), gives a mutant phenotype. Most of the ant-1.1(RNAi) mutant embryos arrest before the morphogenesis stage. Furthermore, ant-1.1 is also required postembryonically because RNAi mutants exhibit small size and life-span extension. Our results suggest that ant-1.1 is the only ant gene strictly required for embryonic and postembryonic development in C. elegans.

1,1‐bis(3′‐indolyl)‐1‐(p‐substituted phenyl)methanes decrease mitochondrial membrane potential and induce apoptosis in endometrial and other cancer cell lines

1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes, containing p-t-butyl (DIM-C-pPhtBu) and phenyl (DIM-C-pPhC(6)H(5)) substituents, are peroxisome proliferator-activated receptor gamma (PPARgamma) agonists; however, DIM-C-pPhtBu-induced growth inhibition and cell death in human HEC1A endometrial cancer cells is PPARgamma-independent. DIM-C-pPhtBu decreased mitochondrial membrane potential (MMP) and promoted the release of cytochrome c and caspase activation and nuclear uptake of endonuclease G leading to apoptosis of HEC1A cells. DIM-C-pPhtBu specifically targeted the mitochondrial permeability transition pore complex (PTPC) because the DIM-C-pPhtBu-induced pro-apoptotic responses were inhibited by atractyloside (Atra), a compound that specifically interacts with the inner mitochondrial membrane adenine nucleotide transport (ANT) proteins. At the dose of Atra used in this study (300 microM), this compound alone did not alter the PTPC but inhibited the mitochondriotoxic effects of DIM-C-pPhtBu. DIM-C-pPhtBu/DIM-C-pPhC(6)H(5) and Atra also differentially affected the ability of eosin-5-maleimide (EMA) to alkylate Cys160 in the ANT protein and Atra, but not DIM-C-pPhtBu, inhibited the exchange of ATP/ADP in isolated mitochondria suggesting that these pharmacophores act on different sites on the ANT protein. Results of this study show that the receptor-independent proapoptotic activity of DIM-C-pPhtBu and DIM-C-pPhC(6)H(5) were related to novel mitochondriotoxic activities involving inner mitochondrial ANT proteins.

Mechanisms of impaired mitochondrial energy metabolism in acute and chronic neurodegenerative disorders

Altered mitochondrial energy metabolism contributes to the pathophysiology of acute brain injury caused by ischemia, trauma, and neurotoxins and by chronic neurodegenerative disorders such as Parkinson's and Huntington's diseases. Although much evidence supports that the electron transport chain dysfunction in these metabolic abnormalities has both genetic and intracellular environmental causes, alternative mechanisms are being explored. These include direct, reversible inhibition of cytochrome oxidase by nitric oxide, release of mitochondrial cytochrome c, oxidative inhibition of mitochondrial matrix dehydrogenases and adenine nucleotide transport, the availability of NAD for dehydrogenase reactions, respiratory uncoupling by activities such as that of the permeability transition pore, and altered mitochondrial structure and intracellular trafficking. This review focuses on the catabolism of neuronal NAD and the release of neuronal mitochondrial NAD as important contributors to metabolic dysfunction. In addition, the relationship between apoptotic signaling cascades and disruption of mitochondrial energy metabolism is considered in light of the fine balance between apoptotic and necrotic neural cell death.

Prevention of the ischemia-induced decrease in mitochondrial Tom20 content by ischemic preconditioning

Preserved mitochondrial function (respiration, calcium handling) and integrity (cytochrome c release) is central for cell survival following ischemia/reperfusion. Mitochondrial function also requires import of proteins from the cytosol via the translocase of the outer and inner membrane (TOM and TIM complexes). Since mitochondrial function following ischemia/reperfusion is better preserved by ischemic preconditioning (IP), we now investigated whether expression of parts of the import machinery is affected by ischemia/reperfusion without or with IP in vivo. We analyzed the mitochondrial content of the presequence receptor Tom20, the pore forming unit Tom40 and Tim23. Goettinger minipigs were subjected to 90 min of low-flow ischemia without or with preconditioning by 10 min ischemia and 15 min reperfusion. Mitochondria were isolated from the ischemic or preconditioned anterior wall of the left ventricle and from the control posterior wall. Infarct size was significantly reduced by IP (20.1 ± 1.6% of area at risk (non-preconditioned) vs. 6.5 ± 2.5% of area at risk (IP)). Using Western blot analysis, the ratio of Tom20 (normalized to Ponceau S) between mitochondria isolated from the anterior ischemic and posterior control wall was reduced (0.72 ± 0.11, a.u., n = 8), whereas the mitochondrial Tom20 content was preserved by IP (1.17 ± 0.16 a.u., n = 7, P < 0.05). The mitochondrial Tom40, Tim23 and adenine nucleotide transporter (ANT) contents were not significantly different between non-preconditioned and preconditioned myocardium. The preservation of the mitochondrial Tom20 protein level may contribute to the improved mitochondrial function after IP.

Cysteine-scanning Mutagenesis of Serotonin Transporter Intracellular Loop 2 Suggests an α-Helical Conformation

Like other proteins involved in neurotransmitter transport, serotonin transporter (SERT) activity is regulated by multiple intracellular signal transduction pathways. The second intracellular loop (IL2) of SERT contains consensus sequences for cGMP-dependent protein kinase and protein kinase C. A 24-residue region of SERT including IL2, from Ile-270 through Ser-293, was analyzed by cysteine-scanning mutagenesis and chemical modification. 2-(Aminoethyl)methanethiosulfonate hydrobromide (MTSEA) failed to inhibit serotonin transport or binding of the cocaine analog 2beta-carbomethoxy-3beta-(4-[125I]iodophenyl)tropane (beta-CIT) in intact cells expressing these mutants, but it inactivated beta-CIT binding in membrane preparations. From the pattern of sensitivity, IL2 appears to extend from Trp-271 through Ile-290, a significantly longer region than that initially predicted by hydropathy analysis. Six mutants reacted with MTSEA much faster than the others, and the pattern of the more reactive mutations suggested that IL2 is in an alpha-helical conformation. Some of the mutants had significantly elevated transport rates, suggesting a possible mechanism for the regulation of SERT activity.

A custom microarray analysis of gene expression during programmed cell death in Arabidopsis thaliana.

Programmed cell death (PCD) is a form of cellular suicide requiring active gene expression, and occurs in both animals and plants. While the cascade of events and the genes that control PCD have been extensively studied in animals, we remain largely ignorant about the similar process in plant cells. Many of the key proteins of animal cell death such as the Bcl-2 family and the caspase family of proteases do not appear to be conserved in plants, suggesting that plants may employ unique mechanisms to execute PCD. To identify genetic elements of PCD in plants, we monitored changes in transcript levels of approximately 100 selected genes during cell death in an Arabidopsis cell suspension culture using a cDNA microarray. PCD was induced in the cell cultures by two independent means (heat treatment or by allowing the cultures to senesce) to allow the distinction to be drawn between changes in gene expression that are related to PCD and those that are specific to a particular treatment. We argue that genes whose expression is altered during PCD induced by two different means may be generally involved in all types of PCD. We show that certain oxidative stress-related genes, including CSD1, CSD3, and GPX, in addition to cysteine proteinases, some transcription factors, and HR-related genes may serve as markers of a core plant cell death programme. Additionally we observe a down-regulation of the mitochondrial adenine nucleotide transporter and suggest that this may be an early event in the execution of plant PCD.

Structure-function studies of adenine nucleotide transport in mitochondria. II. Biochemical analysis of distinct AAC1 and AAC2 proteins in yeast.

AAC1 and AAC2 genes in yeast each encode functional ADP/ATP carrier (AAC) proteins of the mitochondrial inner membrane. In the present study, mitochondria harboring distinct AAC proteins and the pet9 Arg96 to HIS mutant (Lawson, J., Gawaz, M., Klingenberg, M., and Douglas, M. G. (1990) J. Biol. Chem. 265, 14195-14201) protein have been characterized. In addition, properties of the different AAC proteins have been defined following reconstitution into proteoliposomes. Deletion of AAC2 but not AAC1 causes a major reduction in the mitochondrial cytochrome content and respiration, and this level remains low even when the level of AAC1 protein is increased to 20% that of the AAC2 gene product. In reconstitution studies, the rate of nucleotide transport by isolated AAC1 protein is approximately 40% that of the AAC2 protein. Thus, the lack of mitochondrial-dependent growth supported by the AAC1 gene product alone may be due to the combination of low abundance and reduced activity. Surprisingly, analysis of the Arg96 to His mutant protein revealed binding and transport activities similar to the functional AAC1 and AAC2 gene products. These observations are discussed in relation to a molecular analysis of this highly conserved small transporter and its function in conjunction with other proteins in the mitochondrial membrane.

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.

Structure-function studies of adenine nucleotide transport in mitochondria. II. Biochemical analysis of distinct AAC1 and AAC2 proteins in yeast.

Structure-function studies of adenine nucleotide transport in mitochondria. II. Biochemical analysis of distinct AAC1 and AAC2 proteins in yeast.