tRNA-triggered ATP hydrolysis and generation of membrane potential by the Leishmania mitochondrial tRNA import complex

Translocation of tRNAs across mitochondrial membranes is a receptor-mediated active transport process requiring ATP. A large tRNA import complex from the inner membrane of Leishmania mitochondria catalyzes translocation into phospholipid vesicles. In this reconstituted system, the import substrate tRNA(Tyr)(GUA) specifically stimulated hydrolysis of ATP within the vesicles, with the subsequent generation of a membrane potential by pumping out of protons, as shown by the protonophore-sensitive uptake of the potential-sensitive dye rhodamine 123. Generation of membrane potential was dependent on ATP hydrolysis, and inhibited by oligomycin, recalling the proton-translocation mechanism of the respiratory F-1-F-0-ATPase. For translocation of tRNA, ATP could be replaced by low pH of the medium, but proton-dependent import was resistant to oligomycin. Moreover, ATP hydrolysis, generation of membrane potential and tRNA uptake were inhibited by carboxyatractyloside, a specific inhibitor of mitochondrial ATP-ADP translocase, implying an ATP requirement within the vesicles. These observations imply a gating mechanism in which tRNA, on binding to its receptor, triggers the energetic activation of the complex, leading to the opening of import channels.