Mechanism of the ANT-mediated transport of fatty acid anions across the inner mitochondrial membrane

The additional protonophoric function of the mitochondrial adenine nucleotide translocase (ANT1) is now recognized. However, the molecular mechanism remains controversial. Fatty acid (FA) cycling hypothesis postulates that FAs transport protons across the inner mitochondrial membrane to the matrix by a flip-flop, whereas ANT1 facilitates the translocation of FA anions (FA ^- ) back to the intermembrane space. By a combined approach involving measurements of current through the planar lipid bilayers reconstituted with recombinant ANT1, site-directed mutagenesis and molecular dynamics simulations, we show that FA ^- is initially caught by R59 on the matrix side of ANT1, then moves along the positively charged protein-lipid interface, and binds to R79, where it is protonated in the hydrated cavity in the presence of D134. R79 is crucial for the competitive binding of ANT1 substrates (ATP and ADP) and inhibitors (carboxyatractyloside, bongkrekic acid). The binding sites are well-conserved in mitochondrial SLC25 members, implying a general transporting mechanism for FA anions.