Adenine Nucleotide Transporters

The mitochondrial adenine nucleotide transporter is presently the subject of some particular interest. The transporter has not yet been isolated, although encouraging progress has been made in this direction by Shertzer and Racker, who have recently reported the isolation of a partially purified beef heart mitochondrial adenine nucleotide transporter. 

The kinetic and binding parameters of the yeast transporter have been studied in some detail in our laboratory. Adenine nucleotide uptake has been studied in mitochondria isolated from cells grown anaerobically and aerobically under both catabolite repressed or derepressed conditions. The specific inhibitors, atractylate and bongkrekate, have been used to examine the number and nature of the adenine nucleotide binding sites present in the mitochondria of cells grown under the various conditions. Atractylate is a competitive inhibitor of the transporter, while bongkrekate increases the affinity of the transporter for adenine nucleotides so that subsequent dissociation is prevented and the flux of nucleotides across the membrane is inhibited. 

The physiological changes resulting from the different growth conditions did not significantly alter the kinetic and binding constants of adenine nucleotide uptake. In all cases the uptake was resolved into an atractylate-sensitive component which was specific for ADP or ATP, with a of 0.5 ixu (due to a typographical error, this K n value was reported as 5.0 /iM in Haslam et a/. ), and an atractylate-insensitive activity which was nonspecific and could not be saturated. 

We have recently reported that adenine nucleotide uptake of mitochondria prepared from a cytoplasmic petite strain and from cells grown in the presence of erythromycin was not sensitive to atractylate inhibition. It was inferred that a product of mitochondrial protein synthesis was required for atractylate sensitivity. Kolarov and Klingenberg have 

It is worth noting that certain nuclear mutations profoundly affect the properties of the transporter. Examination of one such mutant, selected for its resistance to bongkrekate, showed that the transporter had only one atractylate-sensitive, adenine nucleotide binding site, with a K, of 4 /xm and a of 5.5 /xM. Bongkrekate resistance was observed in vitro, since high levels of bongkrekate only slightly reduced the dissociation constant (3 /xm). 

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Figure 12-11. Combination of phosphate transporter ( ) with the adenine nucleotide transporter ((2)) in ATP synthesis. The H+ZP, symport shown is equivalent to the P /OH antiport shown in Figure 12-10. Four protons are taken into the mitochondrion for each ATP exported. However, one less proton would be taken in when ATP is used inside the mitochondrion.

The chemi-osmotic theory of oxidative phosphorylation has been reviewed,74 a model for mitochondrial oxidative phosphorylation in which a membrane potential or proton gradient might transmit energy from an oxidation step to ATP synthesis has been proposed,76 and adenine nucleotide transport in mitochondria has been reviewed.76... 

The adenine nucleotide transporter is known as a translocase - it transports ADP into and ATP out of the mitochondrion in such a way that, when one molecule of ADP is transported in, one molecule of ATP is transported out... 

According to the methanochondrion concept ATP is synthesized by A/iH across internal membranes and ATP is transported from the organelle into the cytoplasm via an ATP/ADP translocator (for a cartoon see ref [162]). Thus, the observed uncoupler insensitivity of ATP synthesis might be explained on the assumption that the internal membranes are not accessible to these compounds. However, upon reinvestigation of the electron microscopic data and the adenine nucleotide transport this explanation could be ruled out the internal membranes which had been described in the literature for Methanobacterium thermoautotrophicum were found to be artefacts of fixation [163] (see also ref [164]). The adenine nucleotide transport could be explained by a tight and... 

Not all the transporters discussed above are present in aU types of mitochondria the set of activities present in mitochondria depends on the functional needs of the cells from which the mitochondria are isolated. The adenine nucleotide and phosphate transporters are present in all mitochondria thus far studied. This reflects the fact that the major function of mitochondria is the synthesis of ATP. Even in the rare instances (e.g., brown fat mitochondria [55] and mitochondria in anaerobically growing yeast [56]) where the major function is not ATP synthesis, mitochondria normally have active adenine nucleotide transport. The pyruvate transporter also appears to be ubiquitous. The carnitine transporter has been studied in liver [57], heart [35] and sperm [58] and is probably present in all mitochondria which use long-chain fatty acids. 

Although a few subunits of mitochondrial membrane proteins are coded by mitochondrial DNA and synthesized in the mitochondrial matrix, most membrane proteins including the adenine nucleotide carrier are coded by nuclear genes and synthesized on cytoplasmic ribosomes [80,81], Chloramphenicol, an inhibitor of mitochondrial protein synthesis, does not inhibit incorporation of radioactive leucine into the carrier in growing Neurospora crassa, but cycloheximide, an inhibitor of cytoplasmic protein synthesis, does inhibit leucine incorporation [78]. Also, a yeast nuclear respiratory mutant has been shown to cause a defect in adenine nucleotide transport [81], and the nuclear gene responsible for coding the carrier in yeast is currently being cloned for further studies [82]. 

The complete 297 amino acid sequence of the adenine nucleotide transporter has been determined [185] and deductions have been made about the two-dimensional structure in the plane perpendicular to the membrane. Some of these deductions have been made using computer analyses of the amino acid sequence [186], Using a computer program to locate sequence homologies, three domains have been identified within the sequence with significant sequence overlap. The three repeating areas are characterized by a conserved region centered on a cysteine residue found in three... 

Shortzer, H. G-, and Racker, E. (1976). Reconstitution and characterization of the adenine nucleotide transporter derived from bovine heart mitochondria. /. EwJ. Ckem. 251, 2446-2452. 

Fig. 16. Mitochondrial import of cholesterol. The StAR protein is the major cholesterol (CHOL) carrier bringing the lipid to import sites. The StAR protein is phosphorylated by cyclic AMP-dependent protein kinase (PKA) that is recruited to the mitochondria by the protein PAP7. PAP7 is a binding partner of the peripheral benzodiazepine receptor/translocator protein (TSPO), which forms a complex with the voltage-dependent anion channel (VDAC) and an adenine nucleotide transporter (ANT) at contact sites between the inner and outer mitochondrial membranes. The multiprotein complex constitutes a cholesterol transporter that moves cholesterol from StAR to the inner mitochondrial membrane where the side-chain cleavage enzyme (CYP-11 Al) converts it to pregnenolone (PREG).

A key event in preventing apoptosis is thus the retention of cytochrome c within mitochondria. The permeability transition pore complex is formed between the inner and outer mitochondrial membranes and is reported to control protein release from the intermembrane space. The permeabihty transition pore complex comprises the adenine nucleotide transporter, the voltage-dependent anion channel and possibly other proteins such as the benzodiazepine receptor and cyclophilin D [65]. Thus, cells possess specialised systems and processes for retaining cytochrome c within mitochondria to ensure survival, as well as systems that can rapidly mobilise this molecule when the apoptotic pathway is triggered. 

Recently attempts have been made to ascertain whether adenine nucleotide transport across the mitochondrial membrane in intact cells can be a rate-determining factor for extramitochondrial ATP-utilising processes. 

According to Holian et al. [53], however, respiration in tightly coupled mitochondria is controlled by the extramitochondrial ATP/ADP XP ratio (the phosphate potential) rather than by ATP/ADP. Since Pj is not involved in ADP transport it was concluded that ADP transport could not be rate-limiting. However, since under flux conditions both Pj and ADP have to enter to allow exit of ATP, it will be impossible to uncouple the effect of P, and adenine nucleotide transport. This... 

Inhibition of adenine nucleotide transport by fatty acyl-CoA esters. 

Purification, cloning, and initial characterization. J. Biol Chem. 274,27553-27561. Sharer, J. D., Shern, J. F., Van Valkenburgh, H., Wallace, D. C., and Kahn, R. A. (2002). ARL2 and BART enter mitochondria and bind the adenine nucleotide transporter. Mol. Biol Cell 13, 71-83. 

Azido-ADP Adenine nucleotide transport system in mitochondria Photoinduced inhibition of nucleotide exchange 55... 

Most ATP-requiring reactions occur in the cytosol and produce ADP and orthophosphate. Since most ATP is formed by mitochondrial oxidative phosphorylation (in appropriate cells) from ADP and orthophosphate, these molecules must traverse the inner membrane. ATP and ADP are translocated by the specific adenine-nucleotide-transport system. This antiport system is widely distributed in the membrane and exchanges one mitochondrial ATP for one cytoplasmic ADP. The carrier selectively binds and transports ADP inwards and ATP outwards. The phosphate enters the mitochondrion via a different antiport system, the phosphate carrier, which exchanges it for a hydroxyl ion. 

Freshly isolated, intact mitochondria contain considerable amounts of adenine nucleotides which are resistant to removal by repeated washings with isotonic sucrose. This indicates that these compounds are in a compartment—presumably within the inner mitochondrial membrane—which is inaccessible to the sucrose solution. When exogenous adenine nucleotide is added to the mitochondria, there is a rapid exchange with endogenous adenine nucleotides with no net increase in the concentration of adenine nucleotides in the mitochondria. ADP exchanges most rapidly, followed by ATP and then by AMP, which is relatively impermeable. It is the inner mitochondrial membrane through which the adenine nucleotides do not permeate and which contains the specific adenine-nucleotide transporting system. The movement of ATP across the inner mitochondrial membrane (and hence out of the mitochondria) depends directly on the translocation of ADP in the presence of adenylate kinase in the outer compartment of the mitochondria. 

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