The substrate specificity of the human ADP/ATP carrier AAC1.

TitleThe substrate specificity of the human ADP/ATP carrier AAC1.
Publication TypeJournal Article
Year of Publication2013
AuthorsMifsud, J, Ravaud, S, Krammer, E-M, Chipot, C, Kunji, ERS, Pebay-Peyroula, E, Dehez, F
JournalMol Membr Biol
Volume30
Issue2
Pagination160-8
Date Published2013 Mar
ISSN1464-5203
KeywordsAdenine Nucleotides, Adenosine Diphosphate, Adenosine Triphosphate, Arylamine N-Acetyltransferase, Binding Sites, Biological Transport, Cell Membrane, Escherichia coli, Guanine, Humans, Isoenzymes, Lactococcus lactis, Mitochondria, Mitochondrial ADP, ATP Translocases, Molecular Dynamics Simulation, Protein Transport, Substrate Specificity
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 [(32)P]-ATP was measured. In the second approach the protein was expressed in the cytoplasmic membrane of Lactococcus lactis. The uptake of [(14)C]-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.

DOI10.3109/09687688.2012.745175
Alternate JournalMol. Membr. Biol.
Citation Key10.3109/09687688.2012.745175
PubMed ID23173940
Grant ListMC_U105663139 / / Medical Research Council / United Kingdom