Chemical, immunological, enzymatic, and genetic approaches to studying the arrangement of the peptide chain of the ADP/ATP carrier in the mitochondrial membrane

Abstract

In the process of oxidative phosphorylation, the exchange of cytosolic ADP3- against mitochondrial ATP4- across the inner mitochondrial membrane is mediated by a specific carrier protein. Two different conformations for this carrier have been demonstrated on the basis of interactions with specific inhibitors, namely carboxyatractyloside (CATR) and bongkrekic acid (BA). The two conformations, referred to as CATR and BA conformations, are interconvertible, provided that ADP or ATP are present. The functional ADP/ATP carrier is probably organized as a tetramer. In the presence of CATR or BA the tetramer is split into two dimers combined with either of the two inhibitors. The amino acid sequence of the beef heart carrier monomer (297 residues) contains three repeats of about 100 residues each. Experimental results obtained through different approaches, including photolabeling, immunochemistry, and limited proteolysis, can be interpreted on the basis of a model with five or six transmembrane alpha helices per carrier monomer. Two mobile regions involved in the binding of nucleotides and accessible to proteolytic enzymes have been identified. Each of them may be visualized as consisting of two pairs of short amphipathic alpha helices, which can be juxtaposed to form hydrophilic channels facilitating the nucleotide transport. Mutagenesis in yeast is currently being used to detect strategic amino acids in ADP/ATP transport.

A Novel Adenine Nucleotide Translocase Inhibitor, MT-21, Induces Cytochrome c Release by a Mitochondrial Permeability Transition-independent Mechanism

“…In contrast to PhAsO that binds to the -SH moieties directly, the effect of MT-21 was abrogated by pretreatment with bongkrekic acid. It is known that bongkrekic acid fixes ANT conformation in the m-state (11). Moreover, when mitochondria were pretreated with physiological ligands of ANT such as ADP or ATP, the effect of MT-21 was also canceled (data not shown).…”

“…In c-state a hydrophilic loop of the ANT, which is the nucleotide-binding site, faces toward cytoplasmic side of the inner membrane, and in m-state it is exposed to the matrix side (8,9). Bongkrekic acid inhibits PT pore opening and the release of cytochrome c via binding to ANT and fixing its conformation in the m-state (10,11). Conversely, a different ANT inhibitor, atractyloside, induces PT pore opening and cytochrome c release by changing the ANT conformation to its c-state (12,13).…”

Topology of the mitochondrial cAMP‐dependent protein kinase and its substrates

“…The inhibitory effect exerted by carboxyatractyloside on mitochondrial protein phosphorylation, promoted by cAMP and butyryl-cAMP, results from inhibition of the entry of [7-a2P]ATP into the matrix mediated by the adenine translocator [10]. The inhibition is, in fact, observed also when phosphorylation of the mitochondrial proteins is stimulated by butyryl-cAMP, the lipophilic analog of cAMP which freely permeates the mitochondrial membrane.…”

Yeast Mitochondrial ADP/ATP Carriers Are Monomeric in Detergents as Demonstrated by Differential Affinity Purification

“…7 Cooperativity between protomers has been invoked as an integral part of the transport mechanism, 3,5,[28][29][30] but then substantial structural interactions between protomers are expected to allow for the specific recognition of interacting monomers and for the relay of structural changes. One would expect that such interactions would be partially maintained in the relatively short incubation periods employed here, but no dimers were detected even in the presence of substrates.…”