The substrate specificity of the human ADP/ATP carrier AAC1

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 [³²P]-ATP was measured. In the second approach the protein was expressed in the cytoplasmic membrane of Lactococcus lactis. The uptake of [¹⁴C]-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.

Keywords::

• Mitochondrial carrier
• nucleotide transport activity
• molecular-dynamics simulations

Acknowledgements

The collaboration was funded by the European Community's Seventh Framework Programme FP7/2007–2013 under grant agreement no. HEALTH-F4-2007-201924, EDICT Consortium. The work was also supported by the Agence Nationale de la Recherche (ANR Trans-MIT and MIT-2M projects). We thank P. Machillot for his technical help in the uptake experiments, N. Cherradi and O. Filhol-Cochet for access to the radioactivity room (CEA/iRTSV). The Grand Equipement National de Calcul Informatique and Centre Informatique National de l’Enseignement Supérieur are gratefully acknowledged for provision of computer time.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.