ATP, the principal energy currency of the cell, fuels most biosynthetic reactions in the cytoplasm by its hydrolysis into ADP and inorganic phosphate. Because resynthesis of ATP occurs in the mitochondrial matrix, ATP is exported into the cytoplasm while ADP is imported into the matrix. The exchange is accomplished by a single protein, the ADP/ATP carrier. Here we have solved

http://biomol.it/unictbiolmol-lab/figure_didattica/P06.Nature426.39.pdf

Structure of Mitochondrial Adp/ATP Carrier in Complex with Carboxyatractyloside

The mitochondrial carriers (MCs) shuttle a variety of metabolites across the inner mitochondrial membrane (i.m.m.). In man they are encoded by the SLC25 genes. Some MCs have isoforms encoded by different SLC25 genes, whereas the phosphate carrier has two variants arising from an alternative splicing of SLC25A3. Six MCs have been sequenced after purification, and many more have been identified from their transport and kinetic properties following heterologous over-expression and reconstitution into liposomes. All MCs of known function belong to the same protein family, since their polypeptide chains consist of three tandemly related sequences of about 100 amino acids, and the repeats of the different carriers are homologous. They probably function as homodimers, each monomer being folded in the membrane into six transmembrane segments. The functional information obtained in studies with mitochondria and/or the reconstituted system has helped to gain an insight into the physiological role of the MCs in cell metabolism, as have tissue distribution, the use of knock-out mice (and/or yeast) and over-expression in human cell lines (or yeast) of individual carriers and isoforms. At the same time, the cloning and functional identification of many SLC25 genes has made it possible (i) to identify the genes (and their defects) responsible for some diseases, e.g. Stanley syndrome and Amish microcephaly, and (ii) where the genes were already known, to characterize the function of the gene products and hence understand the molecular basis and the symptoms of the diseases, e.g. hyperornithinaemia, hyperammonaemia and homocitrullinuria (HHH) syndrome and type II citrullinemia. It is likely that further extension and functional characterization of the SLC25 gene family will elucidate other diseases caused by MC deficiency.

The mitochondrial transporter family (SLC25): physiological and pathological implications.

Biological significance of phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) in mammalian cells.

The ADP and ATP transport in mitochondria and its carrier

Nucleotide metabolism operates in all living organisms, embodies an evolutionarily ancient and indispensable complex of metabolic pathways and is of utmost importance for plant metabolism and development. In plants, nucleotides can be synthesized de novo from 5-phosphoribosyl-1-pyrophosphate and simple molecules (e.g., CO2, amino acids, and tetrahydrofolate), or be derived from preformed nucleosides and nucleobases via salvage reactions. Nucleotides are degraded to simple metabolites, and this process permits the recycling of phosphate, nitrogen, and carbon into central metabolic pools. Despite extensive biochemical knowledge about purine and pyrimidine metabolism, comprehensive studies of the regulation of this metabolism in plants are only starting to emerge. Here we review progress in molecular aspects and recent studies on the regulation and manipulation of nucleotide metabolism in plants.

Pyrimidine and Purine Biosynthesis and Degradation in Plants

The mitochondrial ADP/ATP carrier: Structural, physiological and pathological aspects

Fluorometric Titration of the Mitochondrial ADP/ATP Carrier Protein in Muscle Homogenate with Atractyloside Derivatives

Tryptophanyl substitution of the Saccharomyces cerevisiae adenine nucleotide carrier (Anc2p isoform) was not deleterious for the transport activity or the folding of the carrier [preceding paper by Le Saux et al. (1996) Biochemistry 35, 16116-16124]. Conformational changes of the isolated wild-type and Trp-substituted Anc2p variants, induced upon binding of specific substrates [adenosine triphosphate (ATP) or diphosphate (ADP)] or inhibitors [carboxyatractyloside (CATR) or bongkrekic acid (BA)], were studied by measurement of intrinsic fluorescence. Titration of CATR and BA binding sites ended in the same number of sites, namely, 6-7 nmol/mg of wild-type and variant Anc2p. Isolated Anc2p in detergent presented similar emission spectra, suggesting that all tryptophanyl residues were in environments of similar hydrophobicity. Trp87 and Trp126 contributed largely and to a similar extent to the fluorescence enhancement observed in response to ATP binding, while Trp235 contributed negatively and to a small extent to the fluorescence change. Both Trp126 and Trp235, and to a lower extent Trp87, participate in the CATR-induced fluorescence decrease of Anc2p. Responses to BA binding were observed only in the presence of ATP; they consisted of a further fluorescence increase of the Anc2p.ATP complex, which was mainly due to Trp87 and Trp126, Trp235 being much less responsive. The different fluorescence responses of the three Trp residues of Anc2 variants to ATP, CATR, and BA are in agreement with distinct binding sites for these ligands and distinct conformations of the carrier protein recognizing specifically CATR or BA. A mechanistic model is proposed to interpret the transitions between the different conformational states of Anc2p.

Conformational changes of the yeast mitochondrial adenosine diphosphate/adenosine triphosphate carrier studied through its intrinsic fluorescence. 2. Assignment of tryptophanyl residues of the carrier to the responses to specific ligands

Adenine nucleotide transport in mitochondria is mediated by a specific carrier protein located in the inner mitochondrial membrane. Chemical, immunochemical, enzymatic, fluorometric and genetic approaches have been used to investigate conformational changes of the ADP/ATP carrier that are thought to participate in the transport process.

A. Le Saux

Papers

The mitochondrial ADP/ATP carrier: Structural, physiological and pathological aspects

Fluorometric Titration of the Mitochondrial ADP/ATP Carrier Protein in Muscle Homogenate with Atractyloside Derivatives

Conformational changes of the yeast mitochondrial adenosine diphosphate/adenosine triphosphate carrier studied through its intrinsic fluorescence. 2. Assignment of tryptophanyl residues of the carrier to the responses to specific ligands

Conformational changes of the beef heart and yeast mitochondrial ADP/ATP carrier